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Sample records for infecting pseudomonas putida evidence

  1. Diabetic foot gangrene patient with multi-drug resistant Pseudomonas putida infection in Karawaci District, Indonesia

    Directory of Open Access Journals (Sweden)

    Nata Pratama Hardjo Lugito

    2015-01-01

    Full Text Available Pseudomonas putida is a rod-shaped, non fermenting Gram-negative organism frequently found in the environment that utilizes aerobic metabolism, previously thought to be of low pathogenicity. It had been reported as cause of skin and soft tissue infection, especially in immunocompromised patients. A female green grocer, 51 year-old came to internal medicine out-patient clinic with gangrene and osteomyelitis on her 1 st , 2 nd and 3 rd digit and wound on the sole of the right foot since 1 month prior. The patient had history of uncontrolled diabetes since a year ago. She was given ceftriaxone 2 grams b.i.d, metronidazole 500 mg t.i.d empirically and then amikacin 250 mg b.i.d, followed by amputation of the digits and wound debridement. The microorganism′s culture from pus revealed multi drug resistant Pseudomonas putida. She recovered well after antibiotics and surgery.

  2. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida--evidence for close relationship to the T7 group

    International Nuclear Information System (INIS)

    Kovalyova, Irina V.; Kropinski, Andrew M.

    2003-01-01

    The genome of the lytic Pseudomonas putida bacteriophage gh-1 is linear double-stranded DNA containing 37,359 bp with 216-bp direct terminal repeats. Like other members of the T7 group, the gh-1 genome contains regions of high homology to T7 interspersed with nonhomologous regions that contain small open reading frames of unknown function. The genome shares 31 genes in common with other members of the T7 group, including RNA polymerase, and an additional 12 unique putative genes. A major difference between gh-1 and other members of this group is the absence of any open reading frames between the left direct terminal repeat and gene 1. Sequence analysis of the gh-1 genome also revealed the presence of 10 putative phage promoters with a consensus sequence similar to the promoters of T3 and phiYeO3-12 (consensus: TAAAAACCCTCACTRTGGCHSCM). P. putida mutants resistant to gh-1 were demonstrated to have an altered lipopolysaccharide structure, indicating that members of this group use lipopolysaccharide as their cellular receptor

  3. Methylmercury degradation by Pseudomonas putida V1.

    Science.gov (United States)

    Cabral, Lucélia; Yu, Ri-Qing; Crane, Sharron; Giovanella, Patricia; Barkay, Tamar; Camargo, Flávio A O

    2016-08-01

    Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages.

    Directory of Open Access Journals (Sweden)

    Damian J Magill

    Full Text Available We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group.

  5. Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages.

    Science.gov (United States)

    Magill, Damian J; Krylov, Victor N; Shaburova, Olga V; McGrath, John W; Allen, Christopher C R; Quinn, John P; Kulakov, Leonid A

    2017-01-01

    We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group.

  6. Ethylene glycol metabolism by Pseudomonas putida.

    Science.gov (United States)

    Mückschel, Björn; Simon, Oliver; Klebensberger, Janosch; Graf, Nadja; Rosche, Bettina; Altenbuchner, Josef; Pfannstiel, Jens; Huber, Armin; Hauer, Bernhard

    2012-12-01

    In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

  7. Advances of naphthalene degradation in Pseudomonas putida ND6

    Science.gov (United States)

    Song, Fu; Shi, Yifei; Jia, Shiru; Tan, Zhilei; Zhao, Huabing

    2018-03-01

    Naphthalene is one of the most common and simple polycyclic aromatic hydrocarbons. Degradation of naphthalene has been greatly concerned due to its economic, free-pollution and its fine effect in Pseudomonas putida ND6. This review summarizes the development history of naphthalene degradation, the research progress of naphthalene degrading gene and naphthalene degradation pathway of Pseudomonas putida ND6, and the researching path of this strain. Although the study of naphthalene degradation is not consummate in Pseudomonas putida ND6, there is a potential capability for Pseudomonas putida ND6 to degrade the naphthalene in the further research.

  8. Genomic characterisation of clinical and environmental Pseudomonas putida group strains and determination of their role in the transfer of antimicrobial resistance genes to Pseudomonas aeruginosa.

    Science.gov (United States)

    Peter, Silke; Oberhettinger, Philipp; Schuele, Leonard; Dinkelacker, Ariane; Vogel, Wichard; Dörfel, Daniela; Bezdan, Daniela; Ossowski, Stephan; Marschal, Matthias; Liese, Jan; Willmann, Matthias

    2017-11-10

    Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM . These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. These data

  9. Identification and control of a Pseudomonas spp (P. fulva and P. putida) bloodstream infection outbreak in a teaching hospital in Beijing, China.

    Science.gov (United States)

    Liu, Yingmei; Liu, Kun; Yu, Xiaomin; Li, Binbin; Cao, Bin

    2014-06-01

    An outbreak of bacteremia caused by Pseudomonas spp (P. fulva and P. putida) was first identified in our hospital in the summer of 2010 and reoccurred in the following year. Based on the epidemiological data collected in these 2 years, we initiated an investigation on the source of the outbreak. The aim of this study was to report the results of the investigation, as well as the intervention strategies that resulted in successful control of the outbreak. An infection control team was set up consisting of infectious disease specialists, microbiologists, infection control practitioners, and head nurses. The microbiology and medical records of case-patients with P. fulva or P. putida bloodstream infections were reviewed. Environmental samples and intravenous (IV) solutions from the wards and the pharmacy center were collected for culturing. The molecular characteristics of the bacterial isolates were studied by pulsed-field gel electrophoresis (PFGE). Strict infection control strategies were implemented. A total of 20 case-patients from five inpatient wards were identified during three summer seasons from 2010 to 2012. Nineteen of them recovered with proper antibiotics. Unfortunately one died from complications of heart failure. A total of 19 isolates of P. fulva and four of P. putida were identified, of which 20 were from blood, two from environmental surface samples from the hospital pharmacy, and one from an in-use compounded solution from a case-patient in the cardiology ward. Molecular analysis revealed that the P. fulva isolated from the in-use compounded solution (5% glucose solution containing insulin, isosorbide dinitrate, and potassium magnesium aspartate) and the environmental samples had the same PFGE type as the clinical isolates. The investigation identified that contaminated IV solution was the source of the P. fulva bacteremia, which prompted us to implement intensified control measures that resulted in successful control of the outbreak. Copyright

  10. Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies

    OpenAIRE

    Ballerstedt, Hendrik; Volkers, Rita J. M.; Mars, Astrid E.; Hallsworth, John E.; Santos, Vitor A. Martins dos; Puchalka, Ja?ek; van Duuren, Joost; Eggink, Gerrit; Timmis, Ken N.; de Bont, Jan A. M.; Wery, Jan

    2007-01-01

    Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type...

  11. PutidaNET: Interactome database service and network analysis of Pseudomonas putida KT2440

    OpenAIRE

    Park, Seong-Jin; Choi, Jong-Soon; Kim, Byoung-Chul; Jho, Seong-Woong; Ryu, Jea-Woon; Park, Daeui; Lee, Kyung-A; Bhak, Jong; Kim, Seung Il

    2009-01-01

    Abstract Background Pseudomonas putida KT2440 (P. putida KT2440) is a highly versatile saprophytic soil bacterium. It is a certified bio-safety host for transferring foreign genes. Therefore, the bacterium is used as a model organism for genetic and physiological studies and for the development of biotechnological applications. In order to provide a more systematic application of the organism, we have constructed a protein-protein interaction (PPI) network analysis system of P. putida KT2440....

  12. Evidence of the Participation of Remote Residues in the Catalytic Activity of Co-Type Nitrile Hydratase from Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    Brodkin, Heather R.; Novak, Walter R.P.; Milne, Amy C.; D’Aquino, J. Alejandro; Karabacak, N.M.; Goldberg, Ilana G.; Agar, Jeffrey N.; Payne, Mark S.; Petsko, Gregory A.; Ondrechen, Mary Jo; Ringe, Dagmar (Brandeis); (E.I. DuPont); (NEU)

    2011-09-28

    Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. To improve our understanding of the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, and three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues ({alpha}-Asp164, {beta}-Glu56, and {beta}-His147) and one predicted third-shell residue ({beta}-His71) have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues ({alpha}-Glu168) and the three residues not predicted ({alpha}-Arg170, {alpha}-Tyr171, and {beta}-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.

  13. Characterization of starvation-induced dispersion in Pseudomonas putida biofilms

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Sternberg, Claus

    2005-01-01

    that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm...

  14. Assessing carbon source-dependent phenotypic variability in Pseudomonas putida

    DEFF Research Database (Denmark)

    Nikel, Pablo Ivan; de Lorenzo, Victor

    2018-01-01

    The soil bacterium Pseudomonas putida is rapidly becoming a platform of choice for applications that require a microbial host highly resistant to different types of stresses and elevated rates of reducing power regeneration. P. putida is capable of growing in a wide variety of carbon sources...

  15. Mutants of Pseudomonas putida affected in poly-3-hydroxyalkanoate synthesis

    NARCIS (Netherlands)

    Ren, Q; Kessler, B; van der Leij, F; Witholt, B.

    The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene (phaC) were isolated via N-methyl-N'-nitro-N-nitrosoguanidine

  16. Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: Implications for transcriptomics studies

    NARCIS (Netherlands)

    Ballerstedt, H.; Volkers, R.J.M.; Mars, A.E.; Hallsworth, J.E.; Santos, V.A.M.D.; Puchalka, J.; Duuren, J. van; Eggink, G.; Timmis, K.N.; Bont, J.A.M. de; Wery, J.

    2007-01-01

    Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for

  17. Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida

    OpenAIRE

    Vandenbergh, Peter A.; Wright, Ann M.

    1983-01-01

    An organism identified as Pseudomonas putida was found to utilize citronellol or geraniol as the sole carbon and energy source. The ability to degrade these acyclic isoprenols was associated with pSRQ50, a 50-megadalton transmissible plasmid.

  18. Selection of hyperadherent mutants in Pseudomonas putida biofilms

    DEFF Research Database (Denmark)

    Yousef-Coronado, Fatima; Soriano, María Isabel; Yang, Liang

    2011-01-01

    transposon Pseudomonas putida KT2440 mutants showing increased biofilm formation, and the detailed characterization of one of them. This mutant exhibits a complex phenotype, including altered colony morphology, increased production of extracellular polymeric substances and enhanced swarming motility, along...

  19. Benzoate transport in Pseudomonas putida CSV86.

    Science.gov (United States)

    Choudhary, Alpa; Purohit, Hemant; Phale, Prashant S

    2017-07-03

    Pseudomonas putida strain CSV86 metabolizes variety of aromatic compounds as the sole carbon source. Genome analysis revealed the presence of genes encoding putative transporters for benzoate, p-hydroxybenzoate, phenylacetate, p-hydroxyphenylacetate and vanillate. Bioinformatic analysis revealed that benzoate transport and metabolism genes are clustered at the ben locus as benK-catA-benE-benF. Protein topology prediction suggests that BenK (aromatic acid-H+ symporter of major facilitator superfamily) has 12 transmembrane α-helices with the conserved motif LADRXGRKX in loop 2, while BenE (benzoate-H+ symporter protein) has 11 predicted transmembrane α-helices. benF and catA encode benzoate specific porin, OprD and catechol 1,2-dioxygenase, respectively. Biochemical studies suggest that benzoate was transported by an inducible and active process. Inhibition (90%-100%) in the presence of dinitrophenol suggests that the energy for the transport process is derived from the proton motive force. The maximum rate of benzoate transport was 484 pmole min-1 mg-1 cells with an affinity constant, Kmof 4.5 μM. Transcriptional analysis of the benzoate and glucose-grown cells showed inducible expression of benF, benK and benE, suggesting that besides outer membrane porin, both inner membrane transporters probably contribute for the benzoate transport in P. putida strain CSV86. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

    DEFF Research Database (Denmark)

    Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa

    2011-01-01

    We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P....... putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable...... exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however...

  1. Analysis of the core genome and pangenome of Pseudomonas putida.

    Science.gov (United States)

    Udaondo, Zulema; Molina, Lázaro; Segura, Ana; Duque, Estrella; Ramos, Juan L

    2016-10-01

    Pseudomonas putida are strict aerobes that proliferate in a range of temperate niches and are of interest for environmental applications due to their capacity to degrade pollutants and ability to promote plant growth. Furthermore solvent-tolerant strains are useful for biosynthesis of added-value chemicals. We present a comprehensive comparative analysis of nine strains and the first characterization of the Pseudomonas putida pangenome. The core genome of P. putida comprises approximately 3386 genes. The most abundant genes within the core genome are those that encode nutrient transporters. Other conserved genes include those for central carbon metabolism through the Entner-Doudoroff pathway, the pentose phosphate cycle, arginine and proline metabolism, and pathways for degradation of aromatic chemicals. Genes that encode transporters, enzymes and regulators for amino acid metabolism (synthesis and degradation) are all part of the core genome, as well as various electron transporters, which enable aerobic metabolism under different oxygen regimes. Within the core genome are 30 genes for flagella biosynthesis and 12 key genes for biofilm formation. Pseudomonas putida strains share 85% of the coding regions with Pseudomonas aeruginosa; however, in P. putida, virulence factors such as exotoxins and type III secretion systems are absent. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

    Directory of Open Access Journals (Sweden)

    Kivisaar Maia

    2010-04-01

    Full Text Available Abstract Background We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. Results By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. Conclusions ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as

  3. The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria.

    Science.gov (United States)

    Putrins, Marta; Ilves, Heili; Lilje, Liisa; Kivisaar, Maia; Hõrak, Rita

    2010-04-14

    We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this

  4. Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

    Directory of Open Access Journals (Sweden)

    Palloma Rodrigues Marinho

    2009-08-01

    Full Text Available Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3 isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

  5. Biological production of monoethanolamine by engineered Pseudomonas putida S12

    NARCIS (Netherlands)

    Foti, M.J.; Médici, R.; Ruijssenaars, H.J.

    2013-01-01

    Pseudomonas putida S12 was engineered for the production of monoethanolamine (MEA) from glucose via the decarboxylation of the central metabolite l-serine, which is catalyzed by the enzyme l-serine decarboxylase (SDC).The host was first evaluated for its tolerance towards MEA as well as its

  6. Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

    NARCIS (Netherlands)

    Hermes, H.F.M.; Croes, L.M.; Peeters, W.P.H.; Peters, P.J.H.; Dijkhuizen, L.

    1993-01-01

    The metabolism of the natural amino acid L-valine, the unnatural amino acids D-valine, and D-, L-phenylglycine (D-, L-PG), and the unnatural amino acid amides D-, L-phenylglycine amide (D, L-PG-NH2) and L-valine amide (L-Val-NH2) was studied in Pseudomonas putida ATCC 12633. The organism possessed

  7. Pseudomonas putida KT2440 as a host for biochemicals production

    DEFF Research Database (Denmark)

    Calero Valdayo, Patricia

    in industry.This thesis aims at contributing to developing and characterizing tools for the use of alternative hosts organisms with high tolerance towards toxic compounds, such as Pseudomonas putida. The thesis also focuses on identifying target compounds that may be relevant to produce in this strain...

  8. Isolation and characterization of Pseudomonas putida WLY for ...

    African Journals Online (AJOL)

    Using the BMM medium containing 100 mg/L of reactive brilliant red X-3B, a decolorizing bacterium with higher decolorization activity was isolated and it showed a decolorization zone of 10 mm; this decolorizing bacterium was identified as Pseudomonas putida WLY based on physiological and biochemical characteristics ...

  9. The sigma(54) regulon (sigmulon) of Pseudomonas putida

    DEFF Research Database (Denmark)

    Cases, I.; Ussery, David; de Lorenzo, V.

    2003-01-01

    , the sigma(54) regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae. Here we present the analysis of the sigma(54) regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440. We have developed an improved method for the prediction...

  10. Engineering the Soil Bacterium Pseudomonas putida for Arsenic Methylation

    OpenAIRE

    Chen, Jian; Qin, Jie; Zhu, Yong-Guan; de Lorenzo, Víctor; Rosen, Barry P.

    2013-01-01

    Accumulation of arsenic has potential health risks through consumption of food. Here, we inserted the arsenite [As(III)] S-adenosylmethionine methyltransferase (ArsM) gene into the chromosome of Pseudomonas putida KT2440. Recombinant bacteria methylate inorganic arsenic into less toxic organoarsenicals. This has the potential for bioremediation of environmental arsenic and reducing arsenic contamination in food.

  11. Characterization of starvation-induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

    DEFF Research Database (Denmark)

    Gjermansen, M.; Nilsson, M.; Yang, Liang

    2010-01-01

    P>Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface...... proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane-associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild-type, P. putida lapA and P. putida lapAG mutants displayed decreased surface...... adhesion and were deficient in subsequent biofilm formation, suggesting that LapG affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c-di-GMP level via induction of an EAL domain protein led to dispersal of P. putida...

  12. The Pseudomonas putida T6SS is a plant warden against phytopathogens.

    Science.gov (United States)

    Bernal, Patricia; Allsopp, Luke P; Filloux, Alain; Llamas, María A

    2017-04-01

    Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector-immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.

  13. Responses of KT2440 Pseudomonas putida to mild water stress

    DEFF Research Database (Denmark)

    Gülez, Gamze

    betydningen af flagellær motilitet og produktion af EPS under matric stess undersøgt ved brug af den såkaldte Porous Surface Model (PSM), som genererer væske-filmens effekter ved kontrol af !m. Det blev vist, at flagellær motilitet var begrænset under matric stress; Pseudomonas putida KT2440 kolonier udviste...... bakterievækst og målbare genudtryksniveauer har vi vist, at PPSM kan anvendes til at studere bakteriers adfærd under et bredt spektrum af matric potentials. Endelig blev den fuldstændige genudtryknings-dynamik for Pseudomonas putida KT2440 under matric stress klarlagt. Vi identificerede de forskelligt udtrykte...

  14. Cotransport of TiO2 nanoparticles and Pseudomonas putida in porous media

    Science.gov (United States)

    Zaharis, Ioannis; Manariotis, Ioannis D.; Chrysikopoulos, Constantinos V.

    2015-04-01

    The scope of this study was to investigate the cotransport of Pseudomonas putida and TiO2 nanoparticles (NPs) in porous media. Flowthrough experiments were conducted in glass columns with diameter of 2.5 cm and length of 30 cm, packed with 2-mm diameter spherical glass beads. Anatase TiO2 NPs solutions were prepared in distilled water of at two different concentrations: 5 and 50 mg/L. The concentration of P. putida solutions varied from 105 to 109 cfu/mL. Initially, transport experiments were conducted separately for P. putida and TiO2 NPs. Subsequently, TiO2 and P. putida cotransport experiments were conducted. The concentration of TiO2 NPs was measured by a fluorescence spectrophotometer and P. putida concentration was determined by plate counts on agar plates and optical density measurements. All experiments were conducted with two different flow rates: 1 and 2 mL/min. The transport experiments with P. putida exhibited similar transport behavior with the tracer (NaBr) indicating that there was not considerable retention. The mass recovery of P. putida was close to 100% in all of the transport experiments conducted. However, the transport experiments with TiO2 NPs suggested that a significant portion of the NPs was retained in the column. Based on the cotransport experimental data, it is evident that the transport of P. putida was not significantly affected by the presence of TiO2. It should be noted that the mass recovery of NPs in the transport and costransport experiments was between 40 and 60%.

  15. Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

    OpenAIRE

    Paliwal, Vasundhara; Raju, Sajan C.; Modak, Arnab; Phale, Prashant S.; Purohit, Hemant J.

    2014-01-01

    Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Mo...

  16. Dynamics of development and dispersal in sessile microbial communities: examples from Pseudomonas aeruginosa and Pseudomonas putida model biofilms

    DEFF Research Database (Denmark)

    Klausen, M.; Gjermansen, Morten; Kreft, J.-U.

    2006-01-01

    Surface-associated microbial communities in many cases display dynamic developmental patterns. Model biofilms formed by Pseudomonas aeruginosa and Pseudomonas putida in laboratory flow-chamber setups represent examples of such behaviour. Dependent on the experimental conditions the bacteria...

  17. Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

    NARCIS (Netherlands)

    Verhoef, S.; Ruijssenaars, H.J.; Bont, J.A.M.de; Wery, J.

    2007-01-01

    Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia

  18. Genome-wide identification of tolerance mechanisms towards p-coumaric acid in Pseudomonas putida

    DEFF Research Database (Denmark)

    Calero, Patricia; Jensen, Sheila I.; Bojanovič, Klara

    2017-01-01

    The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 towards eleven toxic chemical compounds was investigated. P. putida was found to be significantly more...

  19. Small Rna Regulatory Networks In Pseudomonas Putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; Long, Katherine

    2015-01-01

    chemicals and has a potential to be used as an efficient cell factory for various products. P. putida KT2240 is a genome-sequenced strain and a well characterized pseudomonad. Our major aim is to identify small RNA molecules (sRNAs) and their regulatory networks. A previous study has identified 37 s...... and harvested in different growth phases, as well as osmotic, membrane and oxidative stress conditions. RNA sequencing data has been analysed with the open source software system Rockhopper, and it has revealed over 180 putative sRNAs. Most of them (86%) seem to be novel and uncharacterized. The majority...

  20. Efficient recombinant production of prodigiosin in Pseudomonas putida.

    Science.gov (United States)

    Domröse, Andreas; Klein, Andreas S; Hage-Hülsmann, Jennifer; Thies, Stephan; Svensson, Vera; Classen, Thomas; Pietruszka, Jörg; Jaeger, Karl-Erich; Drepper, Thomas; Loeschcke, Anita

    2015-01-01

    Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

  1. Efficient recombinant production of prodigiosin in Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Andreas eDomröse

    2015-09-01

    Full Text Available Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20 °C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

  2. Sorption of Pseudomonas putida onto differently structured kaolinite minerals

    Science.gov (United States)

    Vasiliadou, I. A.; Papoulis, D.; Chrysikopoulos, C.; Panagiotaras, D.; Karakosta, E.; Fardis, M.; Papavassiliou, G.

    2010-12-01

    The presence of bio-colloids (e.g. bacteria and viruses) in the subsurface could be attributed to the release of particles from septic tanks, broken sewer lines or from artificial recharge with treated municipal wastewater. Bio-colloid transport in the subsurface is significantly affected by sorption onto the solid matrix. Bio-colloid attachment onto mobile or suspended in the aqueous phase soil particles (e.g. clay or other minerals) also may influence their fate and transport in the subsurface. The present study focuses on the investigation of Pseudomonas (Ps.) putida sorption onto well (KGa-1) and poorly (KGa-2) crystallized kaolinite minerals. Batch experiments were carried out to determine the sorption isotherms of Ps. putida onto both types of kaolinite particles. The sorption process of Ps. putida onto KGa-1 and KGa-2 is adequately described by a Langmuir isotherm. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy as well as Nuclear Magnetic Resonance were employed to study the sorption mechanisms of Ps. putida. Experimental results indicated that KGa-2 presented higher affinity and sorption capacity than KGa-1. It was shown that electrostatic interactions and structural disorders can influence the sorption capacity of clay particles.

  3. Redundancy in putrescine catabolism in solvent tolerant Pseudomonas putida S12.

    Science.gov (United States)

    Bandounas, Luaine; Ballerstedt, Hendrik; de Winde, Johannes H; Ruijssenaars, Harald J

    2011-06-10

    Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines have also been implicated in stressed cells. Previous transcriptomics results of P. putida strains producing an aromatic compound, or being exposed to the solvent toluene, indicated differentially expressed genes involved in polyamine transport and metabolism. Therefore, the metabolism of the polyamine, putrescine was investigated in P. putida S12, as no putrescine degradation pathways have been described for this strain. Via transcriptome analysis various, often redundant, putrescine-induced genes were identified as being potentially involved in putrescine catabolism via oxidative deamination and transamination. A series of knockout mutants were constructed in which up to six of these genes were sequentially deleted, and although putrescine degradation was affected in some of these mutants, complete elimination of putrescine degradation in P. putida S12 was not achieved. Evidence was found for the presence of an alternative pathway for putrescine degradation involving γ-glutamylation. The occurrence of multiple putrescine degradation routes in the solvent-tolerant P. putida S12 is indicative of the importance of controlling polyamine homeostasis, as well as of the high metabolic flexibility exhibited by this microorganism. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440

    OpenAIRE

    Simone eSchmitz; Salome eNies; Nick eWierckx; Lars M Blank; Miriam A. Rosenbaum

    2015-01-01

    Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under ox...

  5. Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group.

    Science.gov (United States)

    Yonezuka, Kenta; Shimodaira, Jun; Tabata, Michiro; Ohji, Shoko; Hosoyama, Akira; Kasai, Daisuke; Yamazoe, Atsushi; Fujita, Nobuyuki; Ezaki, Takayuki; Fukuda, Masao

    2017-03-17

    Pseudomonas putida is well-known for degradation activities for a variety of compounds and its infections have been reported. Thus, P. putida includes both clinical and nonclinical isolates. To date, no reports have examined the phylogenetic relationship between clinical and nonclinical isolates of the P. putida group. In this study, fifty-nine strains of P. putida group containing twenty-six clinical, and thirty-three nonclinical, isolates, were subjected to phylogenetic and taxonomic analyses based on 16S rRNA gene sequences and nine housekeeping gene sequences, including argS, dnaN, dnaQ, era, gltA, gyrB, ppnK, rpoB, and rpoD, to obtain insights into the diversity of species in this group. More than 97.6% similarity was observed among the 16S rRNA gene sequences of all the strains examined, indicating that the resolution of 16S rRNA gene sequences is inadequate. Phylogenetic analysis based on the individual housekeeping genes listed above improved the resolution of the phylogenetic trees, which are different from each other. Multilocus sequence analysis (MLSA) based on the concatenated sequences of the nine genes significantly improved the resolution of the phylogenetic tree, and yielded approximately the same results as average nucleotide identity (ANI) analysis, suggesting its high reliability. ANI analysis classified the fifty-nine strains into twenty-six species containing seventeen singletons and nine strain clusters based on the 95% threshold. It also indicated the mixed distribution of clinical and nonclinical isolates in the six clusters, suggesting that the genomic difference between clinical and nonclinical isolates of the P. putida group is subtle. The P. putida type strain NBRC 14164 T is a singleton that is independently located from the P. putida strains distributed among the six clusters, suggesting that the classification of these strains and the differentiation of species in the P. putida group should be re-examined. This study greatly expands

  6. Specific Gene Loci of Clinical Pseudomonas putida Isolates

    Science.gov (United States)

    Molina, Lázaro; Udaondo, Zulema; Duque, Estrella; Fernández, Matilde; Bernal, Patricia; Roca, Amalia; de la Torre, Jesús; Ramos, Juan Luis

    2016-01-01

    Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host’s immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria. PMID:26820467

  7. Swimming pattern of Pseudomonas putida - navigating with stops and reversals

    Science.gov (United States)

    Hintsche, Marius; Waljor, Veronika; Alirezaeizanjani, Zahra; Theves, Matthias; Beta, Carsten

    Bacterial swimming strategies depend on factors such as the chemical and physical environment, as well as the flagellation pattern of a species. For some bacteria the motility pattern and the underlying flagellar dynamics are well known, such as the classical run-and-tumble behavior of E. coli. Here we study the swimming motility and chemotactic behavior of the polar, multi-flagellated soil dwelling bacterium Pseudomonas putida. Compared to E. coli, its motility pattern is more diverse. In addition to different speed levels, P. putida exhibits two types of reorientation events, stops and reversals, the occurrence of which is modulated according to the growth conditions. We also analyzed the swimming pattern in the presence of chemical gradients. Using benzoate as a chemoattractant, we measured key motility parameters in order to characterize P. putida's chemotaxis strategy and to quantify the directional bias in its random walk. Our results indicate a change in the reversal frequency depending on changes in the chemoattractant concentration consistent with the classical scenario of temporal sensing. DFG.

  8. Specific Gene Loci of Clinical Pseudomonas putida Isolates.

    Directory of Open Access Journals (Sweden)

    Lázaro Molina

    Full Text Available Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.

  9. Genome features of Pseudomonas putida LS46, a novel polyhydroxyalkanoate producer and its comparison with other P. putida strains

    Science.gov (United States)

    2014-01-01

    A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation

  10. Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12

    NARCIS (Netherlands)

    Volkers, R.J.M.; Jong, A.L. de; Hulst, A.G.; Baar, B.L.M. van; Bont, J.A.M. de; Wery, J.

    2006-01-01

    The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach

  11. Draft Genome Sequence of the Model Naphthalene-Utilizing Organism Pseudomonas putida OUS82

    DEFF Research Database (Denmark)

    Tay, Martin; Roizman, Dan; Cohen, Yehuda

    2014-01-01

    Pseudomonas putida OUS82 was isolated from petrol- and oil-contaminated soil in 1992, and ever since, it has been used as a model organism to study the microbial assimilation of naphthalene and phenanthrene. Here, we report the 6.7-Mb draft genome sequence of P. putida OUS82 and analyze its...

  12. Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida.

    Science.gov (United States)

    Li, Shi Yan; Dong, Cui Ling; Wang, Shen Yu; Ye, Hai Mu; Chen, Guo-Qiang

    2011-04-01

    Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ(Ac) cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ(A.c)) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T(g)), one melting temperature (T(m)) and one cool crystallization temperature (T(c)). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young's modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community. © Springer-Verlag 2010

  13. Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida.

    Science.gov (United States)

    Pradeep, Vijayalakshmi; Subbaiah, Usha Malavalli

    2015-01-01

    The present study was undertaken with the objective of studying repeated batch and continuous degradation of chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) using Ca-alginate immobilized cells of Pseudomonas putida isolated from an agricultural soil, and to study the genes and enzymes involved in degradation. The study was carried out to reduce the toxicity of chlorpyrifos by degrading it to less toxic metabolites. Long-term stability of pesticide degradation was studied during repeated batch degradation of chlorpyrifos, which was carried out over a period of 50 days. Immobilized cells were able to show 65% degradation of chlorpyrifos at the end of the 50th cycle with a cell leakage of 112 × 10(3) cfu mL(-1). During continuous treatment, 100% degradation was observed at 100 mL h(-1) flow rate with 2% chlorpyrifos, and with 10% concentration of chlorpyrifos 98% and 80% degradation was recorded at 20 mL h(-1) and 100 mL h(-1) flow rate respectively. The products of degradation detected by liquid chromatography-mass spectrometry analysis were 3,5,6-trichloro-2-pyridinol and chlorpyrifos oxon. Plasmid curing experiments with ethidium bromide indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. The results of Polymerase chain reaction indicate that a ~890-bp product expected for mpd gene was present in Ps. putida. Enzymatic degradation studies indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. The study indicates that immobilized cells of Ps. putida have the potential to be used in bioremediation of water contaminated with chlorpyrifos.

  14. Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

    NARCIS (Netherlands)

    Kruijt, M.; Tran, H.; Raaijmakers, J.M.

    2009-01-01

    Aims: Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially

  15. Toluene diffusion and reaction in unsaturated Pseudomonas putida biofilms

    Energy Technology Data Exchange (ETDEWEB)

    Holden, P.A.; Hunt, J.R.; Firestone, M.K. [Univ. of California, Berkeley, CA (United States)

    1997-12-20

    Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, the authors have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. They experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, the authors measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 {times} 10{sup {minus}7} cm{sup 2}/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Their studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems.

  16. Biofilm formation-defective mutants in Pseudomonas putida.

    Science.gov (United States)

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Nosocomial Pseudomonas putida Meningitis: A Case Report and Literature Review

    Directory of Open Access Journals (Sweden)

    Fahmi Yousef Khan

    2017-03-01

    Full Text Available We report a case of Pseudomonas putida meningitis in a 26-year-old Nepalese man who was admitted to Hamad General Hospital with epidermoid cyst for drainage. Ommaya reservoir was placed into the cyst for drainage and externalventricular drainage (EVD was performed. After four days, the patient was transferred to the ward in stable condition. His weakness resolved partially and headache severity decreased. After three days, the patient developed fever and headache severity increased with deterioration of consciousness level. Cerebrospinal fluid (CSF through EVD showed 2 200 leucocytes/µL, protein level of 295 mg/dL, and glucose level of < 1.8 mg/dL. Meropenem was started on the patient. Aspirate from Ommaya reservoir and CSF showed gram-negative rods and cultures yielded P. putida sensitive to cefepime, gentamycin, ciprofloxacin, and amikacin, but resistant to meropenem and piperacillin-tazobactam. EVD was replaced and the patient received cefepime and ciprofloxacin for 21 days after which he improved and was discharged with right sided residual weakness.

  18. Investigation of plasmid-induced growth defect in Pseudomonas putida.

    Science.gov (United States)

    Mi, Jia; Sydow, Anne; Schempp, Florence; Becher, Daniela; Schewe, Hendrik; Schrader, Jens; Buchhaupt, Markus

    2016-08-10

    Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

    2006-01-01

    and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli Yhj......H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

  20. Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment.

    Directory of Open Access Journals (Sweden)

    Susanna K Remold

    Full Text Available By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively. Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.

  1. Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment.

    Science.gov (United States)

    Remold, Susanna K; Purdy-Gibson, Megan E; France, Michael T; Hundley, Thomas C

    2015-01-01

    By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively). Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.

  2. Structural characterization of pyoverdines produced by Pseudomonas putida KT2440 and Pseudomonas taiwanensis VLB120.

    Science.gov (United States)

    Baune, Matthias; Qi, Yulin; Scholz, Karen; Volmer, Dietrich A; Hayen, Heiko

    2017-08-01

    The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.

  3. The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance.

    Science.gov (United States)

    Isken, S; De Bont, J A

    2000-11-01

    The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps.

  4. Synergism of Lumbricus rubellus and Pseudomonas putida Pf-20 in Inducing Resistance to Cucumber Mosaic Virus

    Directory of Open Access Journals (Sweden)

    WIWIEK SRI WAHYUNI

    2006-09-01

    Full Text Available Both Lumbricus rubellus and Pseudomonas putida decompose soil organic matters. The population of P. putida Pf-20 increased if L. rubellus was introduced to the cucumber growth medium. The process of organic decomposition was much better if the medium was introduced with both L. rubellus and P. putida Pf-20, compared to the medium contained only either one of those organisms. The activity of L. rubellus may serve to provide nutrients for both the cucumber and P. putida. The role of P. putida to reduce disease severity was increased if L. rubellus was introduced to the growth medium. The synergism of these two organisms, reduced either the level of disease severity to CMV-48 and C/N ratio of medium, but increased the content of available phosphor and potassium.

  5. Pseudomonas putida CSV86: a candidate genome for genetic bioaugmentation.

    Directory of Open Access Journals (Sweden)

    Vasundhara Paliwal

    Full Text Available Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA(Gly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.

  6. Mechanisms of resistance to chloramphenicol in Pseudomonas putida KT2440.

    Science.gov (United States)

    Fernández, Matilde; Conde, Susana; de la Torre, Jesús; Molina-Santiago, Carlos; Ramos, Juan-Luis; Duque, Estrella

    2012-02-01

    Pseudomonas putida KT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 μg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of the pqq genes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.

  7. Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

    Science.gov (United States)

    Paliwal, Vasundhara; Raju, Sajan C.; Modak, Arnab; Phale, Prashant S.; Purohit, Hemant J.

    2014-01-01

    Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNAGly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation. PMID:24475028

  8. Pseudomonas putida CSV86: a candidate genome for genetic bioaugmentation.

    Science.gov (United States)

    Paliwal, Vasundhara; Raju, Sajan C; Modak, Arnab; Phale, Prashant S; Purohit, Hemant J

    2014-01-01

    Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA(Gly), integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.

  9. Effect of Pseudomonas putida on Growth and Anthocyanin Pigment in Two Poinsettia (Euphorbia pulcherrima) Cultivars

    OpenAIRE

    Ramon Zulueta-Rodriguez; Miguel Victor Cordoba-Matson; Luis Guillermo Hernandez-Montiel; Bernardo Murillo-Amador; Edgar Rueda-Puente; Liliana Lara

    2014-01-01

    Pseudomonas putida is plant growth promoting rhizobacteria (PGPR) that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble) using three strains of P. putida, as well as a mixture of the three (MIX). Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PG...

  10. Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    Wu X.; van der Lelie D.; Monchy, S.; Taghavi, S.; Zhu, W.; Ramos, J.

    2011-03-01

    Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands.

  11. Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida.

    Science.gov (United States)

    Wu, Xiao; Monchy, Sébastien; Taghavi, Safiyh; Zhu, Wei; Ramos, Juan; van der Lelie, Daniel

    2011-03-01

    Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands. Journal compilation © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

  12. Identification of two secondary acyltransferases of lipid A in Pseudomonas putida KT2442.

    Science.gov (United States)

    Zhu, L; Li, Y; Wang, J; Wang, X

    2017-08-01

    Identify the secondary acyltransferases of lipid A in Pseudomonas putida. Two homologues of Escherichia coli acyltransferase LpxL, encoded by PP_0063 and PP_1735, exist in P. putida. Pseudomonas putida mutant strains KWZ001 and KWZ002 were constructed by deleting the genes PP_0063 and PP_1735 respectively. Lipid A species were isolated from P. putidaKT2442, KWZ001 and KWZ002, respectively, and analysed by using electrospray ionization/mass spectrometry (ESI/MS). The results suggest that both PP_0063 and PP_1735 encode secondary acyltransferase of lipid A in P. putida. To further study the site-specificity of these two acyltransferases, PP_0063, PP_1735 and Escherichia coli lpxL were overexpressed in KWZ001 and KWZ002 respectively. Lipid A species isolated from these recombinant strains were analysed by using ESI/MS, and the results suggest that the acyltransferase encoded by PP_0063 catalyses the addition of the 2-OH-C 12:0 chain at the 2-position and the acyltransferase encoded by PP_1735 catalyses the addition of the C 12:0 chain to the 2'-position of lipid A in P. putida. The two acyltransferases encoded by PP_0063 and PP_1735, respectively, are responsible for the site-specific additions of the two secondary acyl chains at the 2- and 2'-positions of lipid A in P. putida. Understanding the lipid A structure variation in P. putida might provide new clues for the survival of P. putida under various stress conditions. © 2017 The Society for Applied Microbiology.

  13. Effect of ZnO Nanostructured Thin Films on Pseudomonas Putida Cell Division

    Science.gov (United States)

    Ivanova, I.; Lukanov, A.; Angelov, O.; Popova, R.; Nichev, H.; Mikli, V.; Dimova-Malinovska, Doriana; Dushkin, C.

    In this report we study the interaction between the bacteria Pseudomonas putida and nanostructured ZnO and ZnO:H thin films prepared by magnetron sputtering of a ZnO target. The nanostructured ZnO and ZnO:H thin films possess some biological-active properties when in contact with bacteria. Our experimental data show that these films have no destructive effect on the cell division of Pseudomonas putida in poor liquid medium and can be applied in biosensor devices.

  14. Physical forces shape group identity of swimming Pseudomonas putida cells

    Directory of Open Access Journals (Sweden)

    David Rodriguez-Espeso

    2016-09-01

    Full Text Available The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving towards each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e. intolerance to mix in time and space with otherwise identical others has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces –not genetic or metabolic

  15. Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

    Science.gov (United States)

    Choi, Chi-Won; Park, Edmond Changkyun; Yun, Sung Ho; Lee, Sang-Yeop; Lee, Yeol Gyun; Hong, Yeonhee; Park, Kyeong Ryang; Kim, Sang-Hyun; Kim, Gun-Hwa; Kim, Seung Il

    2014-10-03

    Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

  16. NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida.

    Science.gov (United States)

    Paris, Ülvi; Mikkel, Katren; Tavita, Kairi; Saumaa, Signe; Teras, Riho; Kivisaar, Maia

    2015-07-01

    Under growth-restricting conditions bacterial populations can rapidly evolve by a process known as stationary-phase mutagenesis. Bacterial nonhomologous end-joining (NHEJ) system which consists of the DNA-end-binding enzyme Ku and the multifunctional DNA ligase LigD has been shown to be important for survival of bacteria especially during quiescent states, such as late stationary-phase populations or sporulation. In this study we provide genetic evidence that NHEJ enzymes participate in stationary-phase mutagenesis in a population of carbon-starved Pseudomonas putida. Both the absence of LigD or Ku resulted in characteristic spectra of stationary-phase mutations that differed from each other and also from the wild-type spectrum. This indicates that LigD and Ku may participate also in mutagenic pathways that are independent from each other. Our results also imply that both phosphoesterase (PE) and polymerase (POL) domains of the LigD protein are involved in the occurrence of mutations in starving P. putida. The participation of both Ku and LigD in the occurrence of stationary-phase mutations was further supported by the results of the analysis of mutation spectra in stationary-phase sigma factor RpoS-minus background. The spectra of mutations identified in the RpoS-minus background were also distinct if LigD or Ku was absent. Interestingly, the effects of the presence of these enzymes on the frequency of occurrence of certain types of mutations were different or even opposite in the RpoS-proficient and deficient backgrounds. These results imply that RpoS affects performance of mutagenic pathways in starving P. putida that utilize LigD and/or Ku. Copyright © 2015. Published by Elsevier B.V.

  17. De novo production of the monoterpenoid geranic acid by metabolically engineered Pseudomonas putida.

    Science.gov (United States)

    Mi, Jia; Becher, Daniela; Lubuta, Patrice; Dany, Sarah; Tusch, Kerstin; Schewe, Hendrik; Buchhaupt, Markus; Schrader, Jens

    2014-12-04

    Production of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest. Unfortunately, antimicrobial activity of most monoterpenoids hampers a wide application of microorganisms for their production. Strains of Pseudomonas putida, a fast growing and metabolically versatile bacterium, often show an outstanding high tolerance towards organic solvents and other toxic compounds. Therefore, Pseudomonas putida constitutes an attractive alternative host in comparison to conventionally used microorganisms. Here, metabolic engineering of solvent tolerant Pseudomonas putida as a novel microbial cell factory for de novo production of monoterpenoids is reported for the first time, exemplified by geranic acid production from glycerol as carbon source. The monoterpenoic acid is an attractive compound for application in the flavor, fragrance, cosmetics and agro industries. A comparison between Escherichia coli, Saccharomyces cerevisiae and Pseudomonas putida concerning the ability to grow in the presence of geranic acid revealed that the pseudomonad bears a superior resilience compared to the conventionally used microbes. Moreover, Pseudomonas putida DSM 12264 wildtype strain efficiently oxidized externally added geraniol to geranic acid with no further degradation. Omitting external dosage of geraniol but functionally expressing geraniol synthase (GES) from Ocimum basilicum, a first proof-of-concept for de novo biosynthesis of 1.35 mg/L geranic acid in P. putida DSM 12264 was achieved. Doubling the amount of glycerol resulted in twice the amount of product. Co-expression of the six genes of the mevalonate pathway from Myxococcus xanthus to establish flux from acetyl-CoA to the universal terpenoid precursor isopentenylpyrophosphate yielded 36 mg/L geranic acid in shake flask experiments. In the bioreactor, the recombinant strain produced 193 mg/L of geranic acid under fed-batch conditions within 48 h. Metabolic engineering turned Pseudomonas

  18. Iron Uptake Analysis in a Set of Clinical Isolates of Pseudomonas putida

    Science.gov (United States)

    Molina, Lázaro; Geoffroy, Valérie A.; Segura, Ana; Udaondo, Zulema; Ramos, Juan-Luis

    2016-01-01

    Pseudomonas putida strains are frequent inhabitants of soil and aquatic niches and they are occasionally isolated from hospital environments. As the available iron sources in human tissues, edaphic, and aquatic niches are different, we have analyzed iron-uptake related genes in different P. putida strains that were isolated from all these environments. We found that these isolates can be grouped into different clades according to the genetics of siderophore biosynthesis and recycling. The pyoverdine locus of the six P. putida clinical isolates that have so far been completely sequenced, are not closely related; three strains (P. putida HB13667, HB3267, and NBRC14164T) are grouped in Clade I and the other three in Clade II, suggesting possible different origins and evolution. In one clinical strain, P. putida HB4184, the production of siderophores is induced under high osmolarity conditions. The pyoverdine locus in this strain is closely related to that of strain P. putida HB001 which was isolated from sandy shore soil of the Yellow Sea in Korean marine sand, suggesting their possible origin, and evolution. The acquisition of two unique TonB-dependent transporters for xenosiderophore acquisition, similar to those existing in the opportunistic pathogen P. aeruginosa PAO, is an interesting adaptation trait of the clinical strain P. putida H8234 that may confer adaptive advantages under low iron availability conditions. PMID:28082966

  19. Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57.

    Science.gov (United States)

    Nitisakulkan, Tisana; Oku, Shota; Kudo, Daizo; Nakashimada, Yutaka; Tajima, Takahisa; Vangnai, Alisa S; Kato, Junichi

    2014-03-01

    In this study, we investigated the ability of Pseudomonas putida toluene dioxygenase to oxidize chloroanilines. Toluene-induced P. putida T57 cells degraded 4-chloroaniline (4CA) more rapidly than toluene-non-induced cells, suggesting that toluene dioxygenase pathway was involved in 4CA degradation. Escherichia coli harboring P. putida T57 genes encoding toluene dioxygenase complex (todC1C2BA) showed 4CA degradation activity, demonstrating that toluene dioxygenase oxidizes 4CA. Thin-layer chromatography (TLC) and mass spectrometry (MS) analyses identified 4-chlorocatechol and 2-amino-5-chlorophenol as reaction products, suggesting that toluene dioxygenase catalyzes both 1,2- and 2,3-dioxygenation of 4CA. A plasmid containing the entire tod operon (todC1C2BADE) was introduced to P. putida T57 to enhance its ability to degrade 4CA. Resulting P. putida T57 (pHK-C1C2BADE) showed 250-fold higher 4CA degradation activity than P. putida T57 parental strain. P. putida T57 (pHK-C1C2BADE) degraded 2-chloroaniline (2CA), 3-chloroaniline (3CA), and 3,4-dichloroaniline (34DCA) as well as 4CA, but not 3,5-dichloroaniline (35DCA). The order of the degradation rate was: 4CA > 3CA > 2CA > 34DCA. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard

    Science.gov (United States)

    Asif, Huma; Studholme, David J.; Khan, Asifullah; Aurongzeb, M.; Khan, Ishtiaq A.; Azim, M. Kamran

    2016-01-01

    Abstract We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches. PMID:27560648

  1. Investigation Of The Primary Transcriptome Of The Production Organism Pseudomonas Putida

    DEFF Research Database (Denmark)

    D'Arrigo, Isotta; Bojanovic, Klara; Long, Katherine

    2015-01-01

    Introduction: Pseudomonas putida is a nonpathogenic, Gram-negative bacterium and an excellent model organism for biotechnological applications. Due to its metabolic versatility, P. putida can grow in different environments including in extreme conditions. It has several genes to degrade xenobiotic...... compounds, a capability that renders the bacterium useful in bioremediation. Finally, P. putida shows a high potential as a cell factory for the production of several compounds. Methods: Here, a differential RNA-sequencing approach (dRNA-seq) is used to gain new insights into the organization of the P....... putida KT2440 transcriptome, in the presence of citrate or glucose as sole carbon source. Results: A total of 7937 putative transcription start sites (TSSs) have been identified. 5’ RACE experiments have been performed to confirm putative TSSs, and 5’ UTR regions have been investigated for conservative...

  2. Novel small protein identification and quantitative proteomic analysis in Pseudomonas putida KT-­2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen

    .This thesis investigated an industrial bacterium, Pseudomonas putida KT-2440, in two aspects. First, the research focused on discovering novel small proteins (s-proteins) in the bacterium. With large-scale approaches for gene identification, groups of novel s-proteins were identified and validated from...... level under different environmental conditions. The results yield insights intothe adaptation of P. putida KT-2440 in different environments.Based on bioinformatic, proteomic and transcriptomic approaches, global gene expression was analyzed on both transcriptional and translational levels. Our research...... for the first time validates novel s-proteins in P. putida KT-2440, and significantly increases the knowledge of P.putida protein expression and environmental adaptation....

  3. Comparative genomic and functional analyses: unearthing the diversity and specificity of nematicidal factors in Pseudomonas putida strain 1A00316

    Science.gov (United States)

    Guo, Jing; Jing, Xueping; Peng, Wen-Lei; Nie, Qiyu; Zhai, Yile; Shao, Zongze; Zheng, Longyu; Cai, Minmin; Li, Guangyu; Zuo, Huaiyu; Zhang, Zhitao; Wang, Rui-Ru; Huang, Dian; Cheng, Wanli; Yu, Ziniu; Chen, Ling-Ling; Zhang, Jibin

    2016-01-01

    We isolated Pseudomonas putida (P. putida) strain 1A00316 from Antarctica. This bacterium has a high efficiency against Meloidogyne incognita (M. incognita) in vitro and under greenhouse conditions. The complete genome of P. putida 1A00316 was sequenced using PacBio single molecule real-time (SMRT) technology. A comparative genomic analysis of 16 Pseudomonas strains revealed that although P. putida 1A00316 belonged to P. putida, it was phenotypically more similar to nematicidal Pseudomonas fluorescens (P. fluorescens) strains. We characterized the diversity and specificity of nematicidal factors in P. putida 1A00316 with comparative genomics and functional analysis, and found that P. putida 1A00316 has diverse nematicidal factors including protein alkaline metalloproteinase AprA and two secondary metabolites, hydrogen cyanide and cyclo-(l-isoleucyl-l-proline). We show for the first time that cyclo-(l-isoleucyl-l-proline) exhibit nematicidal activity in P. putida. Interestingly, our study had not detected common nematicidal factors such as 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin in P. putida 1A00316. The results of the present study reveal the diversity and specificity of nematicidal factors in P. putida strain 1A00316. PMID:27384076

  4. Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440

    Science.gov (United States)

    Schmitz, Simone; Nies, Salome; Wierckx, Nick; Blank, Lars M.; Rosenbaum, Miriam A.

    2015-01-01

    Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems (BES). We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 μg/mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 μA/cm2 anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains. PMID:25914687

  5. Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2007-01-01

    The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 degrees C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.

  6. A substrate dependent biological containment systems for Pseudomonas putida based on the Escherichia coli gef gene

    DEFF Research Database (Denmark)

    Jensen, Lars Bogø; Ramos, J. L.; Kaneva, Z.

    1993-01-01

    A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained...... within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway...... operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence...

  7. Carbon source-dependent inhibition of xyl operon expression of the Pseudomonas putida TOL plasmid.

    OpenAIRE

    Holtel, A; Marqués, S; Möhler, I; Jakubzik, U; Timmis, K N

    1994-01-01

    TOL plasmid-encoded degradation of benzyl alcohol by Pseudomonas putida is inhibited by glucose and other compounds related to the main carbohydrate metabolism in Pseudomonas species. We report here that this effect is exerted at the level of expression of the xyl catabolic operons, and two xyl promoters, Pu and Ps, were identified as the primary targets of this inhibition. xyl promoter activation was also inhibited by glucose in the heterologous Escherichia coli system, apparently not howeve...

  8. First detection in Europe of the metallo-β-lactamase IMP-15 in clinical strains of Pseudomonas putida and Pseudomonas aeruginosa.

    Science.gov (United States)

    Gilarranz, R; Juan, C; Castillo-Vera, J; Chamizo, F J; Artiles, F; Álamo, I; Oliver, A

    2013-09-01

    In a prospective study (2009-2011) in healthcare institutions from the Canary Islands (Spain), 6 out of 298 carbapenem non-susceptible Pseudomonas aeruginosa isolates produced a metallo-β-lactamase: four IMP-15, two VIM-2 (including one IMP-15-positive isolate) and one VIM-1. Multilocus sequence typing identified the single VIM-1-producing isolate as clone ST111 and two IMP-15-producing isolates as ST606, but, strikingly, bacterial re-identification revealed that the other three isolates (producing IMP-15 and/or VIM-2) were actually Pseudomonas putida. Further retrospective analysis revealed a very high prevalence (close to 50%) of carbapenem resistance in this environmental species. Hence, we report the simultaneous emergence in hospitals on the Canary Islands of P. putida and P. aeruginosa strains producing IMP-15, a metallo-β-lactamase not previously detected in Europe, and suggest an underestimated role of P. putida as a nosocomial reservoir of worrying transferable resistance determinants. ©2013 The Authors Clinical Microbiology and Infection ©2013 European Society of Clinical Microbiology and Infectious Diseases.

  9. Draft Genome Sequences of Four Hospital-Associated Pseudomonas putida Isolates.

    Science.gov (United States)

    Mustapha, Mustapha M; Marsh, Jane W; Ezeonwuka, Chinelo D; Pasculle, Anthony W; Pacey, Marissa P; Querry, Ashley M; Muto, Carlene A; Harrison, Lee H

    2016-09-29

    We present here the draft genome sequences of four Pseudomonas putida isolates belonging to a single clone suspected for nosocomial transmission between patients and a bronchoscope in a tertiary hospital. The four genome sequences belong to a single lineage but contain differences in their mobile genetic elements. Copyright © 2016 Mustapha et al.

  10. C1 compounds as auxiliary substrate for engineered Pseudomonas putida S12

    NARCIS (Netherlands)

    Koopman, F.W.; De Winde, J.H.; Ruijssenaars, H.J.

    2009-01-01

    The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C1 compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a

  11. Rhizoplane colonisation of peas by Rhizobium leguminosarum bv. viceae and a deleterious Pseudomonas putida

    NARCIS (Netherlands)

    Berggren, I.; Alstrom, S.; Vuurde, van J.W.L.; Martensson, A.M.

    2005-01-01

    Pseudomonas putida strain angstrom 313, a deleterious rhizosphere bacterium, reduced pea nitrogen content when inoculated alone or in combination with Rhizobium leguminosarum bv. viceae on plants in the presence of soil under greenhouse conditions. When plants were grown gnotobiotically in liquid

  12. Redundancy in putrescine catabolism in solvent tolerant Pseudomonas putida S12

    NARCIS (Netherlands)

    Bandounas, L.; Ballerstedt, H.; Winde, J.H. de; Ruijssenaars, H.J.

    2011-01-01

    Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines

  13. Complete genome sequence of the naphthalene-degrading Pseudomonas putida strain ND6.

    Science.gov (United States)

    Li, Shanshan; Zhao, Huabing; Li, Yaxiao; Niu, Shumin; Cai, Baoli

    2012-09-01

    Pseudomonas putida strain ND6 is an efficient naphthalene-degrading bacterium. The complete genome of strain ND6 was sequenced and annotated. The genes encoding the enzymes involved in catechol degradation by the ortho-cleavage pathway were found in the chromosomal sequence, which indicated that strain ND6 is able to metabolize naphthalene by the catechol meta- and ortho-cleavage pathways.

  14. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

    NARCIS (Netherlands)

    Poblete-Castro, I.; Binger, D.; Rodrigues, A.; Becker, J.; Martins Dos Santos, V.A.P.; Wittmann, C.

    2013-01-01

    Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism

  15. Establishment of oxidative D-xylose metabolism in Pseudomonas putida S12

    NARCIS (Netherlands)

    Meijnen, J.P.; Winde, J.H. de; Ruijssenaars, H.J.

    2009-01-01

    The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g

  16. Active efflux systems in the solvent-tolerant bacterium Pseudomonas putida S12

    NARCIS (Netherlands)

    Kieboom, J.

    2002-01-01

    The aim of the research presented in this thesis was to study the molecular mechanisms of organic solvent tolerance in Pseudomonas putida S12. This bacterium is capable of growth at saturated solvent concentrations, which are lethal to normal bacteria. Organic

  17. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    Science.gov (United States)

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  18. Differential transcriptional response to antibiotics by Pseudomonas putida DOT-T1E

    DEFF Research Database (Denmark)

    Molina-Santiago, Carlos; Daddaoua, Abdelali; Gómez Lozano, María

    2015-01-01

    is of critical importance. Pseudomonas putidaDOT-T1E exhibits an impressive array of RND efflux pumps, which confer this microorganism high resistance to organic solvents and antibiotics that would kill most other microorganisms. We have chosen DOT-T1E as a model microbe to study the microbial responses...

  19. Engineering Pseudomonas putida S12 for efficient utilization of D-Xylose and L-Arabinose

    NARCIS (Netherlands)

    Meijnen, J.P.; Winde, J.H. de; Ruijssenaars, H.J.

    2008-01-01

    The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate−1], where CDW is cell dry weight), and the growth rate

  20. Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by Pseudomonas putida Strain 1290

    NARCIS (Netherlands)

    Leveau, J.H.J.; Lindow, S.E.

    2005-01-01

    We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of

  1. Identification and molecular characterization of an efflux system involved in Pseudomonas putida 12 multidrug resistance

    NARCIS (Netherlands)

    Kieboom, J.; Bont, de J.A.M.

    2001-01-01

    The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump,

  2. Ni 2-uptake in Pseudomonas putida strain S4: a possible role of Mg ...

    Indian Academy of Sciences (India)

    Essential metal ion homeostasis is based on regulated uptake of metal ions, both during its scarcity and abundance. Pseudomonas putida strain S4, a multimetal resistant bacterium, was employed to investigate Ni2+ entry into cells. It was observed that Mg2+ regulates the entry of Ni2+ and by this plays a protective role to ...

  3. Complete Genome of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida BIRD-1

    Energy Technology Data Exchange (ETDEWEB)

    Matilla, M.A.; van der Lelie, D.; Pizarro-Tobias, P.; Roca, A.; Fernandez, M.; Duque, E.; Molina, L.; Wu, X.; Gomez, M. J.; Segura, A.; Ramos, J.-L.

    2011-03-01

    We report the complete sequence of the 5.7-Mbp genome of Pseudomonas putida BIRD-1, a metabolically versatile plant growth-promoting rhizobacterium that is highly tolerant to desiccation and capable of solubilizing inorganic phosphate and iron and of synthesizing phytohormones that stimulate seed germination and plant growth.

  4. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.

    2010-01-01

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...

  5. ACTIVE EFFLUX OF ORGANIC SOLVENTS BY PSEUDOMONAS PUTIDA S12 IS INDUCED BY SOLVENTS

    Science.gov (United States)

    Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the...

  6. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...

  7. Purification and Characterization of an L-Aminopeptidase from Pseudomonas putida ATCC 12633

    NARCIS (Netherlands)

    Hermes, H.F.M.; Sonke, T.; Peters, P.J.H.; Balken, J.A.M. van; Kamphuis, J.; Dijkhuizen, L.; Meijer, E.M.

    1993-01-01

    An L-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of D,L-amino acid amide racemates, was purified to homogeneity. The highly L-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent

  8. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

    Directory of Open Access Journals (Sweden)

    Márcia Aiko Shirakawa

    2011-06-01

    Full Text Available The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS, as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

  9. Testosterone 15β-hydroxylation by solvent tolerant Pseudomonas putida S12

    NARCIS (Netherlands)

    Ruijssenaars, H.J.; Sperling, E.M.G.M.; Wiegerinck, P.H.G.; Brands, F.T.L.; Wery, J.; Bont, J.A.M.de

    2007-01-01

    A steroid 15β-hydroxylating whole-cell solvent tolerant biocatalyst was constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12. Testosterone hydroxylation was improved by a factor 16 by co-expressing Fer, a putative Fe-S protein

  10. Physiological response of Pseudomonas putida S12 subjected to reduced water activity.

    NARCIS (Netherlands)

    Kets, E.P.W.; Bont, de J.A.M.; Heipieper, H.J.

    1996-01-01

    The effect of osmotic stress, given as decreased water activity (aw), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced aw was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the

  11. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-03-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers.

  12. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    International Nuclear Information System (INIS)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-01-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers

  13. Benzene, toluene and xylene biodegradation by Pseudomonas putida CCMI 852 Biodegradação de benzeno, tolueno e xileno pela Pseudomonas putida CCMI 852

    Directory of Open Access Journals (Sweden)

    Marcelo Henrique Otenio

    2005-09-01

    Full Text Available A minimal liquid medium containing benzene (B, toluene (T and xylene (X and mixtures thereof, was used to evaluate degradation activity of Pseudomonas putida CCMI 852 containing a TOL plasmid. Experiments were developed with B, T and X (100 mg L-1, with mixtures of BT, BX, and TX (50 + 50 mg L-1 each and BTX (33.3 + 33.3 + 33.3 mg L-1 each, added to 500 mL of medium. After 18 to 24 hours, the inoculum was added and solvent disappearance was determined after 24 to 25 hours by GC. Results showed that P. putida CCMI 852 was able to metabolize T and X, but B was not metabolized. In a BTX mixture, B was not metabolized and T and X degradation rate decreased 50%.Meio mineral líquido contendo benzeno (B ou tolueno (T ou xileno (X a 100 mg L-1 e suas misturas de BT, BX e TX (50 + 50 mg L-1 cada mistura e BTX (33,3 + 33,3 + 33,3 mg L-1 cada mistura foram utilizados para avaliar a atividade de degradação de B, T e X por Pseudomonas putida CCMI 852 contendo um plasmídeo TOL. Após 18 a 24 horas de homogenização da mistura, o inoculo foi adicionado e o decréscimo da concentração dos solventes foi determinado entre 24 e 25 horas por GC. Pseudomonas putida CCMI 852 foi capaz de metabolizar T e X, mas não B. Na mistura BTX, B não foi metabolizado também e a velocidade de degradação de T e X decresceu cerca de 50% comparado com soluções contendo apenas T ou X.

  14. DETECTION OF PHENOL DEGRADING BACTERIA AND PSEUDOMONAS PUTIDA IN ACTIVATED SLUDGE BY POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    H. Movahedyan ، H. Khorsandi ، R. Salehi ، M. Nikaeen

    2009-04-01

    Full Text Available Phenol is one of the organic pollutants in various industrial wastewaters especially petrochemical and oil refining. Biological treatment is one of the considerable choices for removing of phenol present in these wastewaters. Identification of effective microbial species is considered as one of the important priorities for production of the biomass in order to achieve desirable kinetic of biological reactions. Basic purpose of this research is identification of phenol-degrading Pseudomonas Putida in activated sludge by polymerase chain reaction (PCR that has high speed and specificity. In this research, 10 various colonies of phenol-degrading bacteria were isolated from municipal activated sludge and the rate of phenol removal and growth rate of these bacteria were assessed in different concentrations of phenol (200 – 900 mg/L. Confirmation of the largest subunit of multicomponent phenol hydroxylase (LmPH gene and gene coding the N fragment in Pseudomonas Putida-derived methyl phenol operon (DmpN gene through PCR were used for general identification of phenol-degrading bacteria and Pseudomonas Putida, respectively. Presence of a 600 bp (base pairs bond in all of isolated strains indicated that they contain phenol hydroxylase gene. 6 of 10 isolated bacteria were Pseudomonas Putida because they produced a 199 bp PCR product by DmpN primers. According to PCR results in this study, the best phenol-degrading bacteria that can utilize 500 – 600 mg/L phenol completely after 48 hours incubation, belong to Pseudomonas Putida strains. It is clear that use of isolated bacteria can lead to considerable decrease of treatment time as well as promotion of phenol removal rate.

  15. Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system.

    Science.gov (United States)

    Sun, Jun; Wang, Qingzhuo; Jiang, Yu; Wen, Zhiqiang; Yang, Lirong; Wu, Jianping; Yang, Sheng

    2018-03-13

    The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. In this study, we established a fast and convenient CRISPR-Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR-Cas9 system, we also constructed a CRISPR-Cpf1 system, which we validated for genome editing in P. putida KT2440. In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.

  16. Content of cardiolipin of the membrane and sensitivity to cationic surfactants in Pseudomonas putida.

    Science.gov (United States)

    López, G A; Heredia, R M; Boeris, P S; Lucchesi, G I

    2016-10-01

    To establish the role of cardiolipin (CL) of the membrane in response to the presence of tetradecyltrimethylammonium in Pseudomonas putida A (ATCC 12633). Two ORFs of Ps. putida A (ATCC 12633), which in Ps. putida KT2440 encode the putative CL synthase genes cls and cls2, were cloned, sequenced and mutated. Only the double mutant lacking cls and cls2 showed a reduction of the CL content, 83% lower than the amount produced by the wild-type. Accompanying this change was a 40% decrease in the content of unsaturated fatty acid. Consequently, the membrane of the mutant was more rigid than the one of the parental strain, as observed using fluorescence polarization techniques. The mutant strain showed reduced viability in the presence of tetradecyltrimethylammonium. The incorporation of exogenous CL into its membrane relieved sensitivity to the cationic detergent. Pseudomonas Putida cells with low levels of CL die in the presence of tetradecyltrimethylammonium, because they cannot counter the fluidizing effect of the cationic surfactant. The modification in the membrane phospholipids composition allows knowing the adaptation strategy of Ps. putida when these bacteria are exposed to cationic surfactant. © 2016 The Society for Applied Microbiology.

  17. Differential proteomics and physiology of Pseudomonas putida KT2440 under filament-inducing conditions

    Directory of Open Access Journals (Sweden)

    Crabbé Aurélie

    2012-11-01

    Full Text Available Abstract Background Pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. This study assessed whether P. putida filamentation could confer survival advantages. Filamentation of P. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed. Results P. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. Proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein IbpA and recombinase RecA in filament-inducing conditions. Our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of RecA. Conclusions This study provides insights into the altered metabolism of P. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by P. putida as a survival strategy in its hostile, recurrently changing habitat.

  18. Manganese (Mn oxidation increases intracellular Mn in Pseudomonas putida GB-1.

    Directory of Open Access Journals (Sweden)

    Andy Banh

    Full Text Available Bacterial manganese (Mn oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS. Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection.

  19. Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Simone eSchmitz

    2015-04-01

    Full Text Available Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida’s obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems. We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/ L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 µg/ mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 µA/ cm² anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains.

  20. Pseudomonas putida KT2442 as a platform for the biosynthesis of polyhydroxyalkanoates with adjustable monomer contents and compositions

    DEFF Research Database (Denmark)

    Tripathi, Lakshmi; Wu, Lin-Ping; Dechuan, Meng

    2013-01-01

    The β-oxidation weakened Pseudomonas putida were established as a platform for the production of polyhydroxyalkanoates (PHA) with adjustable monomer compositions and micro-structures. When mutant P. putida KTOYO6ΔC (phaPCJA.c) was cultivated on mixtures of sodium butyrate and sodium hexanoate (C4...

  1. Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate from benzoate

    NARCIS (Netherlands)

    Duuren, van J.B.J.H.

    2011-01-01

    Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate from benzoate P. putida KT2440 was used as biocatalyst given its versatile and energetically robust metabolism. Therefore, a mutant was generated and a process developed based on which a life cycle assessment

  2. The Transcriptional Landscape of the Production Organism Pseudomonas putida

    DEFF Research Database (Denmark)

    D'Arrigo, Isotta

    Bacterial cell factories represent a valid alternative to fossil fuel-based production. A promising bacterium that can be optimized as cell factory is Pseudomonas putida. However, its development in bioproduction applications poses some challenges including a clear understanding of the bacterial...... such as riboswitches and small RNAs (sRNAs), metabolic pathways and transporter systems. The results reported here significantly increase knowledge of the P. putida transcriptome, adaptation mechanisms, and reveal novel bacterial features that will aid the design and optimization of the bacterium as a cell factory....

  3. Optimization Production of Biosurfactant by Pseudomonas putida Using Crude Palm Oil (CPO) as Substrate

    Science.gov (United States)

    Suryanti, V.; Handayani, D. S.; Masykur, A.; Lindasari

    2017-07-01

    The production of biosurfactant by Pseudomonas putida has been studied. P. putida FNCC 0071 was grown in the nutrient broth medium supplemented with NaCl and crude palm oil (CPO). The effect of CPO concentration and fermentation time on the biosurfactant production were evaluated. The biosurfactant production was evaluated every 24 h for 10 days by optical density, surface tension and emulsification index. The best culture medium was found to be medium containing 5% v/v of CPO with 5 days of incubation time. The biosurfactant was identified as rhamnolipids.

  4. The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose

    NARCIS (Netherlands)

    Nijkamp, K.; Luijk, N. van; Bont, J.A.M. de; Wery, J.

    2005-01-01

    A Pseudomonas putida S12 strain was constructed that efficiently produced thefine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine

  5. Growth, nitrogen fixation and mineral acquisition of Alnus sieboldiana after inoculation of Frankia together with Gigaspora margarita and Pseudomonas putida.

    Science.gov (United States)

    Takashi Yamanaka; Akio Akama; Ching-Yan Li; Hiroaki. Okabe

    2005-01-01

    The role of tetrapartite associations among Frankia, Gigaspora margarita (an arbuscular mycorrhizal fungus), Pseudomonas putida (rhizobacterium), and Alnus sieboldiana in growth, nitrogen fixation, and mineral acquisition of A. sieboldiana was investigated....

  6. Pseudomonas aeruginosa biofilm infections

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim

    2014-01-01

    the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...

  7. Comparative evaluation of the efficacy of Pseudomonas putida in ...

    African Journals Online (AJOL)

    This study was carried out to compare the efficacy of Рseudomonas putida, contained in the biopreparation «Pseudomin» in the bioremediation of diesel fuel contaminated derno-podzoluivisolic soil of two different horizons. By analyzing the Total Petroleum Hydrocarbons (TPH) content using IR-spectrometry method under ...

  8. Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara

    Bacteria commonly encounter stressful conditions during growth in their natural environments and in industrial biotechnology applications such as the biobased production of chemicals. As the coordinated regulation of gene expression is necessary to adapt to changing environments, bacteria have...... such as sRNAs and riboswitches. Further, the sRNAome during the growth of bacteria was investigatedand compared to the strain without Hfq protein. Hfq has a big impact on sRNAs and gene expression in P. putida, hence many Hfq-associated sRNAs and mRNAs were found. Together, the results reported here...... response of Pseudomonas putida KT2440 in different conditions via identification ofdifferentially expressed mRNAs and sRNAs. P. putida is a soil bacterium with a versatile metabolism and innate stress endurance traits, which makes it suitable as future cell factory for the production of valuable compounds...

  9. Experimental and theoretical study of Pseudomonas putida transport in a three-dimensional model aquifer

    Science.gov (United States)

    Vasiliadou, I. A.; Katzourakis, V. E.; Syngouna, V. I.; Chrysikopoulos, C. V.

    2012-04-01

    This study is focused on the transport of Pseudomonas (P.) putida bacterial cells in a three-dimensional model aquifer. The pilot-scale aquifer consisted of a rectangular glass tank with internal dimensions: 120 cm length, 48 cm width, and 50 cm height, carefully packed with well-characterized quartz sand. The P. putida attachment onto the aquifer sand was determined with batch experiments, and was adequately described by a linear isotherm. Transport experiments with a conservative tracer and P. putida were conducted to characterize the aquifer and to investigate the bacterial behavior during transport in water saturated porous media. A three-dimensional, finite-difference numerical model for bacterial transport in saturated, homogeneous porous media was developed and was used to successfully fit the experimental data. Furthermore, theoretical interaction energy calculations suggested that the extended DLVO theory seems to predict bacteria attachment onto the aquifer sand better than the classical DLVO theory.

  10. The RNA chaperone Hfq enables the environmental stress tolerance super-phenotype of Pseudomonas putida.

    Science.gov (United States)

    Arce-Rodríguez, Alejandro; Calles, Belén; Nikel, Pablo I; de Lorenzo, Víctor

    2016-10-01

    The natural physiological regime of the soil bacterium Pseudomonas putida involves incessant exposure to endogenous metabolic conflicts and environmental physicochemical insults. Yet, the role of assisted small RNA-mRNA pairing in the stress tolerance super-phenotype that is the trademark of this bacterium has not been accredited. We have thoroughly explored the physiological consequences -in particular those related to exogenous stress - of deleting the hfq gene of P. putida, which encodes the major RNA chaperone that promotes sRNA-target mRNA interactions. While the overall trend was a general weakening of every robustness descriptor of the Δhfq strain, growth parameters and production of central metabolic enzymes were comparatively less affected than other qualities that depend directly on energy status (e.g. motility, DNA repair). The overall catalytic vigour of the mutant decreased to putida. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Carbon Source Influences Population Heterogeneity In Pseudomonas Putida Kt2440 Biofilms

    DEFF Research Database (Denmark)

    Juel Christensen, Anne-Mette; Sternberg, Claus; Molin, Søren

    2015-01-01

    Introduction: Pseudomonas putida is well known as a potential cell factory for many different biochemicals. Biofilm-based production can be advantageous for possibly toxic products due to increased chemical tolerance and robustness. Biofilm cells frequently differentiate, which challenges...... the benefits of biofilm-based production, and knowledge about factors driving the heterogeneity is therefore of importance.Methods: Biofilm flow chamber systems connected to confocal laser scanning microscopy were used to study biofilm structures of P. putida KT2440 at different carbon conditions. Subsequent...... plating of mature biofilm allowed for variant selection followed by pheno- and genotypic analysis.Results: Structure and cell differentiation in mature P. putida KT2440 biofilms were highly dependent on the type of carbon source utilised. Low glucose concentrations (0.3 mM – 10 mM) did not alter biofilm...

  12. Expression analysis of the fpr (ferredoxin-NADP+ reductase) gene in Pseudomonas putida KT2440

    International Nuclear Information System (INIS)

    Lee, Yunho; Pena-Llopis, Samuel; Kang, Yoon-Suk; Shin, Hyeon-Dong; Demple, Bruce; Madsen, Eugene L.; Jeon, Che Ok; Park, Woojun

    2006-01-01

    The ferredoxin-NADP + reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440

  13. The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

    Science.gov (United States)

    Sudarsan, Suresh; Dethlefsen, Sarah; Blank, Lars M; Siemann-Herzberg, Martin; Schmid, Andreas

    2014-09-01

    What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. Regulatory tasks of the phosphoenolpyruvate-phosphotransferase system of Pseudomonas putida in central carbon metabolism.

    Science.gov (United States)

    Chavarría, Max; Kleijn, Roelco J; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2012-01-01

    Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. This study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the

  15. Degradation of phenanthrene and pyrene using genetically engineered dioxygenase producing Pseudomonas putida in soil

    Directory of Open Access Journals (Sweden)

    Mardani Gashtasb

    2016-01-01

    Full Text Available Bioremediation use to promote degradation and/or removal of contaminants into nonhazardous or less-hazardous substances from the environment using microbial metabolic ability. Pseudomonas spp. is one of saprotrophic soil bacterium and can be used for biodegradation of polycyclic aromatic hydrocarbons (PAHs but this activity in most species is weak. Phenanthrene and pyrene could associate with a risk of human cancer development in exposed individuals. The aim of the present study was application of genetically engineered P. putida that produce dioxygenase for degradation of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC method. The nahH gene that encoded catechol 2,3-dioxygenase (C23O was cloned into pUC18 and pUC18-nahH recombinant vector was generated and transformed into wild P. putida, successfully. The genetically modified and wild types of P. putida were inoculated in soil and pilot plan was prepared. Finally, degradation of phenanthrene and pyrene by this bacterium in spiked soil were evaluated using HPLC measurement technique. The results were showed elimination of these PAH compounds in spiked soil by engineered P. putida comparing to dishes containing natural soil with normal microbial flora and inoculated autoclaved soil by wild type of P. putida were statistically significant (p0.05 but it was few impact on this process (more than 2%. Additional and verification tests including catalase, oxidase and PCR on isolated bacteria from spiked soil were indicated that engineered P. putida was alive and functional as well as it can affect on phenanthrene and pyrene degradation via nahH gene producing. These findings indicated that genetically engineered P. putida generated in this work via producing C23O enzyme can useful and practical for biodegradation of phenanthrene and pyrene as well as petroleum compounds in polluted environments.

  16. Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7

    International Nuclear Information System (INIS)

    Lee, Kangtaek; Park, Jin-Won; Ahn, Ik-Sung

    2003-01-01

    Addition of a carbon source as a nutrient into soil is believed to enhance in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the subsurface and are capable of degrading contaminants. However, it may inhibit the biodegradation of organic contaminants and result in diauxic growth. The objective of this work is to study the effect of pyruvate as another carbon source on the biodegradation of polynuclear aromatic hydrocarbons (PAHs). In this study, naphthalene was used as a model PAH, ammonium sulfate as a nitrogen source, and oxygen as an electron acceptor. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. From a chemostat culture, the growth kinetics of P. putida G7 on pyruvate was determined. At concentrations of naphthalene and pyruvate giving similar growth rates of P. putida G7, diauxic growth of P. putida G7 was not observed. It is suggested that pyruvate does not inhibit naphthalene biodegradation and can be used as an additional carbon source to stimulate the growth of P. putida G7 that can degrade polynuclear aromatic hydrocarbons

  17. Solvent resistance pumps of Pseudomonas putida S12: Applications in 1-naphthol production and biocatalyst engineering.

    Science.gov (United States)

    Janardhan Garikipati, S V B; Peeples, Tonya L

    2015-09-20

    The solvent resistance capacity of Pseudomonas putida S12 was applied by using the organism as a host for biocatalysis and through cloning and expressing solvent resistant pump genes into Escherichia coli. P. putida S12 expressing toluene ortho mononooxygenase (TOM-Green) was used for 1-naphthol production in a water-organic solvent biphasic system. Application of P. putida S12 improved 1-naphthol production per gram cell dry weight by approximately 42% compared to E. coli. Moreover, P. putida S12 enabled the use of a less expensive solvent, decanol, for 1-naphthol production. The solvent resistant pump (srpABC) genes of P. putida S12 were cloned into a solvent sensitive E. coli strain to transfer solvent tolerance. Recombinant strains bearing srpABC genes in either a low-copy number or a high-copy number plasmid grew in the presence of saturated concentration of toluene. Both of the recombinant strains were more tolerant to 1% v/v of toxic solvents, decanol and hexane, reaching similar cell density as the no-solvent control. Reverse-transcriptase analysis revealed that the srpABC genes were transcribed in engineered strains. The results demonstrate successful transfer of the proton-dependent solvent resistance mechanism and suggest that the engineered strain could serve as more robust biocatalysts in media with organic solvents. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Benzoxazinoids in root exudates of maize attract Pseudomonas putida to the rhizosphere.

    Directory of Open Access Journals (Sweden)

    Andrew L Neal

    Full Text Available Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H-one (DIMBOA, are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.

  19. Toxicity of graphene oxide on growth and metabolism of Pseudomonas putida.

    Science.gov (United States)

    Combarros, R G; Collado, S; Díaz, M

    2016-06-05

    The increasing consumption of graphene derivatives leads to greater presence of these materials in wastewater treatment plants and ecological systems. The toxicity effect of graphene oxide (GO) on the microbial functions involved in the biological wastewater treatment process is studied, using Pseudomonas putida and salicylic acid (SA) as bacterial and pollutant models. A multiparametric flow cytometry (FC) method has been developed to measure the metabolic activity and viability of P. putida in contact with GO. A continuous reduction in the percentages of viable cells and a slight increase, lower than 5%, in the percentages of damaged and dead cells, suggest that P. putida in contact with GO loses the membrane integrity but preserves metabolic activity. The growth of P. putida was strongly inhibited by GO, since 0.05mgmL(-1) of GO reduced the maximum growth by a third, and the inhibition was considerably greater for GO concentrations higher than 0.1mgmL(-1). The specific SA removal rate decreased with GO concentration up to 0.1mgmL(-1) indicating that while GO always reduces the growth of P. putida, for concentrations higher than 0.1mgmL(-1), it also reduces its activity. Similar behaviour is observed using simulated urban and industrial wastewaters, the observed effects being more acute in the industrial wastewaters. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Benzoxazinoids in Root Exudates of Maize Attract Pseudomonas putida to the Rhizosphere

    Science.gov (United States)

    Neal, Andrew L.; Ahmad, Shakoor; Gordon-Weeks, Ruth; Ton, Jurriaan

    2012-01-01

    Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize. PMID:22545111

  1. Draft Genome Sequence of Caprolactam-Degrading Pseudomonas putida Strain SJ3.

    Science.gov (United States)

    Hong, Sung-Jun; Park, Gun-Seok; Khan, Abdur Rahim; Jung, Byung Kown; Park, Yeong-Jun; Yoo, Na-Kyung; Lee, Changhee; Park, Choi Kyu; Shin, Jae-Ho

    2015-07-23

    Pseudomonas putida strain SJ3, which possesses caprolactam-degrading ability, was isolated from dyeing industry wastewater in Daegu, Republic of Korea. Here, we describe the draft genome sequence and annotation of the strain. The 5,596,765-bp-long genome contains 4,293 protein-coding genes and 68 RNA genes with 61.70% G+C content. Copyright © 2015 Hong et al.

  2. Optimierung von Pseudomonas putida für die Produktion niedermolekularer Verbindungen

    OpenAIRE

    Graf, Nadja

    2013-01-01

    Das Gram-negative Bakterium Pseudomonas putida ist aufgrund seines ausgeprägten Stoffwechsels für aliphatische und aromatische Kohlenwasserstoffe in den letzten Jahren zu einem der wichtigsten Wirtsorganismen in der Biotechnologie geworden. Für die gezielte Manipulation seines Genoms stehen gentechnische Methoden zur Verfügung, die jedoch ineffektiv und nur schwer zu handhaben sind. Basierend auf der Toxizität des Antimetaboliten 5-Fluoruracil und der Wirkung der Uracilphosphoribosyl-Transfer...

  3. Kontinuierliche Co-Kultur von Tetrahymena thermophila und Pseudomonas putida in einem Bioreaktor

    OpenAIRE

    Hauptmann, Ulla

    2000-01-01

    Im Rahmen der vorliegenden Arbeit wurde ein mikrobielles Ökosystem in einem kontinuierlich betriebenen gerührten Bioreaktor (CSTR) im Labormaßstab etabliert und seine Wachstumskinetik untersucht. In diesem in vitro Räuber-Beute-System wurden der Ciliat Tetrahymena thermophila und das Bakterium Pseudomonas putida über einen Zeitraum von mehr als 400 Stunden gemeinsam kultiviert. Die Interaktion von Bakterien und Protozoen stellt einen elementaren Prozess in der Ökologie nahezu jedes G...

  4. Gibberellin secreting rhizobacterium, Pseudomonas putida H-2-3 modulates the hormonal and stress physiology of soybean to improve the plant growth under saline and drought conditions.

    Science.gov (United States)

    Kang, Sang-Mo; Radhakrishnan, Ramalingam; Khan, Abdul Latif; Kim, Min-Ji; Park, Jae-Man; Kim, Bo-Ra; Shin, Dong-Hyun; Lee, In-Jung

    2014-11-01

    The physiological changes in tolerant soybean plants under salt and drought stress conditions with Pseudomonas putida H-2-3 were investigated. A bacterial isolate H-2-3 was isolated from soil and identified as Pseudomonas putida H-2-3 by 16S rDNA sequences. The treatment of P. putida H-2-3 significantly increased the length, fresh and dry weight of shoot and chlorophyll content in gibberellins (GAs) deficient mutant Waito-c rice seedlings over the control, it might be the presence of GA1, GA4, GA9 and GA20. The soybean plant growth was retarded in salt (120 mM sodium chloride) and drought (15% polyethylene glycol) stress conditions at 10 days treatments, while P. putida H-2-3 effectively enhanced the shoot length and fresh weight of plants suffered at salt and drought stress. The chlorophyll content was lower in abiotic stress conditions and bacterial inoculant P. putida H-2-3 mitigated the stress effects by an evidence of higher quantity of chlorophyll content in plants exposed to salt and drought. The stress hormonal analysis revealed that individual treatment of P. putida H-2-3, salt and drought significantly enhanced the abscisic acid and salicylic acid content than their control. P. putida H-2-3 applied to salt and drought stressed plants showed a lower level of abscisic acid and salicylic acid and a higher level of jasmonic acid content. Under stress condition induced by salt and drought in plants expressed higher level of total polyphenol, superoxide dismutase and radical scavenging activity and no significant changes in flavonoids. The bio-inoculant, P. putida H-2-3 modulated those antioxidants by declining superoxide dismutase, flavonoids and radical scavenging activity. P. putida H-2-3 induced tolerance against abiotic stress was confirmed by a reduction of Na content in abiotic stressed plants. The results suggest that P. putida H-2-3 application reprograms the chlorophyll, stress hormones and antioxidants expression in abiotic stress affected

  5. Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost.

    Science.gov (United States)

    Ravi, Krithika; García-Hidalgo, Javier; Gorwa-Grauslund, Marie F; Lidén, Gunnar

    2017-06-01

    Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26-0.27 h -1 ) than those on ferulate and vanillate (0.21 and 0.22 h -1 ), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (g CDW  h) -1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.

  6. Cotransport of Pseudomonas putida and kaolinite particles through water-saturated columns packed with glass beads

    Science.gov (United States)

    Vasiliadou, Ioanna A.; Chrysikopoulos, Constantinos V.

    2011-02-01

    This study is focused on Pseudomonas putida bacteria transport in porous media in the presence of suspended kaolinite clay particles. Experiments were performed with bacteria and kaolinite particles separately to determine their individual transport characteristics in water-saturated columns packed with glass beads. The results indicated that the mass recovery of bacteria and clay particles decreased as the pore water velocity decreased. Batch experiments were carried out to investigate the attachment of Pseudomonas putida onto kaolinite particles. The attachment process was adequately described by a Langmuir isotherm. Finally, bacteria and kaolinite particles were injected simultaneously into a packed column in order to investigate their cotransport behavior. The experimental data suggested that the presence of clay particles significantly inhibited the transport of bacteria in water-saturated porous media. The observed reduction of Pseudomonas putida recovery in the column outflow was attributed to bacteria attachment onto kaolinite particles, which were retained onto the solid matrix of the column. A mathematical model was developed to describe the transport of bacteria in the presence of suspended clay particles in one-dimensional water-saturated porous media. Model simulations were in good agreement with the experimental results.

  7. Production of Polyhydroxyalkanoates from Sludge Palm Oil Using Pseudomonas putida S12.

    Science.gov (United States)

    Kang, Du-Kyeong; Lee, Cho-Ryong; Lee, Sun Hee; Bae, Jung-Hoon; Park, Young-Kwon; Rhee, Young Ha; Sung, Bong Hyun; Sohn, Jung-Hoon

    2017-05-28

    Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

  8. Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil.

    Science.gov (United States)

    Frasson, David; Opoku, Michael; Picozzi, Tara; Torossi, Tanja; Balada, Stefanie; Smits, Theo H M; Hilber, Urs

    2017-08-01

    Within the frame of a biotechnological screening, we isolated two Pseudomonas strains from forest soil. 16S rRNA gene sequence analysis indicated that strain CCOS 864T shared 99.8 % similarity with Pseudomonas donghuensis HYST, while strain CCOS 865T shared 99.0 % similarity with Pseudomonas putida DSM 291T and lower similarity with other P. putida group type strains. Based on multilocus sequence analysis, the two strains were genotypically distinct from each other, each forming a separate clade. Strains CCOS 864T and CCOS 865T were Gram-stain-negative, motile and rod-shaped, growing at a temperature range of 4-37 °C. Strain CCOS 864T could be phenotypically distinguished from P. putida group species by the combination of gelatinase-positive reaction and positive growth on N-acetyl-d-glucosamine, p-hydroxyphenylacetic acid and inosine but lack of fluorescein production on King's B medium, while strain CCOS 865T could be distinguished from P. putida group species by the combination of positive growth with saccharic acid and negative growth with p-hydroxyphenylacetic acid and l-pyroglutamic acid. The major polar lipid for both strains was phosphatidylethanolamine; the major quinone was ubiquinone Q-9. DNA-DNA hybridization and average nucleotide identities confirmed the novel species status for the two strains. The DNA G+C contents of CCOS 864T and CCOS 865T were 62.1 and 63.8 mol%, respectively. The phenotypic, phylogenetic and DNA-DNA relatedness data support the suggestion that CCOS 864T and CCOS 865T represent two novel Pseudomonas species. The names Pseudomonas wadenswilerensis sp. nov. (type strain CCOS 864T=LMG 29327T) and Pseudomonas reidholzensis sp. nov. (type strain CCOS 865T=LMG 29328T) are proposed.

  9. Species-specific repetitive extragenic palindromic (REP) sequences in Pseudomonas putida

    Science.gov (United States)

    Aranda-Olmedo, Isabel; Tobes, Raquel; Manzanera, Maximino; Ramos, Juan L.; Marqués, Silvia

    2002-01-01

    Pseudomonas putida KT2440 is a soil bacterium that effectively colonises the roots of many plants and degrades a variety of toxic aromatic compounds. Its genome has recently been sequenced. We describe that a 35 bp sequence with the structure of an imperfect palindrome, originally found repeated three times downstream of the rpoH gene terminator, is detected more than 800 times in the chromosome of this strain. The structure of this DNA segment is analogous to that of the so-called enterobacteriaceae repetitive extragenic palindromic (REP) sequences, although its sequence is different. Computer-assisted analysis of the presence and distribution of this repeated sequence in the P.putida chromosome revealed that in at least 80% of the cases the sequence is extragenic, and in 82% of the cases the distance of this extragenic element to the end of one of the neighbouring genes was <100 bp. This 35 bp element can be found either as a single element, as pairs of elements, or sometimes forming clusters of up to five elements in which they alternate orientation. PCR scanning of chromosomes from different isolates of Pseudomonas sp. strains using oligonucleotides complementary to the most conserved region of this sequence shows that it is only present in isolates of the species P.putida. For this reason we suggest that the P.putida 35 bp element is a distinctive REP sequence in P.putida. This is the first time that REP sequences have been described and characterised in a group of non-enterobacteriaceae. PMID:11937637

  10. Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.

    Science.gov (United States)

    Kruijt, Marco; Tran, Ha; Raaijmakers, Jos M

    2009-08-01

    Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy

  11. Experimental validation of in silico estimated biomass yields of Pseudomonas putida KT2440.

    Science.gov (United States)

    Hintermayer, Sarah Beate; Weuster-Botz, Dirk

    2017-06-01

    Pseudomonas putida is rapidly becoming a microbial cell platform for biotechnological applications. In order to understand genotype-phenotype relationships genome scale models represent helpful tools. However, the validation of in silico predictions of genome scale models is a task that is rarely performed. In this study the theoretical biomass yields of Pseudomonas putida KT2440 were estimated for 57 different carbon sources based on a genome scale stoichiometric model applying flux balance analysis. The batch growth of P. putida KT2440 with six individual carbon sources covering the range of maximal to minimal in silico biomass yields (acetate, glycerol, citrate, succinate, malate and methanol, respectively) was studied in a defined mineral medium in a fully controlled stirred-tank bioreactor on a 3 L scale. The highest growth rate of P. putida KT2440 was measured with succinate as carbon source (0.51 h -1 ). Among the 57 carbon sources tested, glycerol resulted in the highest estimated biomass yield (0.61 molC Biomass molC -1 Glycerol ) which was experimentally confirmed. The comparison of experimental determined biomass yields with a modified version of the model iJP815 showed deviations of only up to 10%. The experimental data generated in this study can also be used in future studies to further improve the genome scale models of P. putida KT2440. Improved models will then help to gain deeper insights in genotype-phenotype relationships. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant Pseudomonas putida GS1.

    Science.gov (United States)

    Mi, Jia; Schewe, Hendrik; Buchhaupt, Markus; Holtmann, Dirk; Schrader, Jens

    2016-07-01

    In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads' superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield YP/S of 79 %. To the authors' knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.

  13. Toxicity Testing of Pristine and Aged Silver Nanoparticles in Real Wastewaters Using Bioluminescent Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Florian Mallevre

    2016-03-01

    Full Text Available Impact of aging on nanoparticle toxicity in real matrices is scarcely investigated due to a lack of suitable methodologies. Herein, the toxicity of pristine and aged silver nanoparticles (Ag NPs to a bioluminescent Pseudomonas putida bioreporter was measured in spiked crude and final wastewater samples (CWs and FWs, respectively collected from four wastewater treatment plants (WWTPs. Results showed lower toxicity of pristine Ag NPs in CWs than in FWs. The effect of the matrix on the eventual Ag NP toxicity was related to multiple physico-chemical parameters (biological oxygen demand (BOD, chemical oxygen demand (COD, total suspended solids (TSS pH, ammonia, sulfide and chloride based on a multivariate analysis. However, no collection site effect was concluded. Aged Ag NPs (up to eight weeks were found less toxic than pristine Ag NPs in CWs; evident increased aggregation and decreased dissolution were associated with aging. However, Ag NPs exhibited consistent toxicity in FWs despite aging; comparable results were obtained in artificial wastewater (AW simulating effluent. The study demonstrates the potency of performing nanoparticle acute toxicity testing in real and complex matrices such as wastewaters using relevant bacterial bioreporters.

  14. Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

    DEFF Research Database (Denmark)

    Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

    2003-01-01

    The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

  15. The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42.

    Science.gov (United States)

    Aparicio, Tomás; Jensen, Sheila I; Nielsen, Alex T; de Lorenzo, Victor; Martínez-García, Esteban

    2016-10-01

    Some strains of the soil bacterium Pseudomonas putida have become in recent years platforms of choice for hosting biotransformations of industrial interest. Despite availability of many genetic tools for this microorganism, genomic editing of the cell factory P. putida EM42 (a derivative of reference strain KT2440) is still a time-consuming endeavor. In this work we have investigated the in vivo activity of the Ssr protein encoded by the open reading frame T1E_1405 from Pseudomonas putida DOT-T1E, a plausible functional homologue of the β protein of the Red recombination system of λ phage of Escherichia coli. A test based on the phenotypes of pyrF mutants of P. putida (the yeast's URA3 ortholog) was developed for quantifying the ability of Ssr to promote invasion of the genomic DNA replication fork by synthetic oligonucleotides. The efficiency of the process was measured by monitoring the inheritance of the changes entered into pyrF by oligonucleotides bearing mutated sequences. Ssr fostered short and long genomic deletions/insertions at considerable frequencies as well as single-base swaps not affected by mismatch repair. These results not only demonstrate the feasibility of recombineering in P. putida, but they also enable a suite of multiplexed genomic manipulations in this biotechnologically important bacterium. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1.

    Science.gov (United States)

    Geszvain, Kati; Smesrud, Logan; Tebo, Bradley M

    2016-07-01

    The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a

  17. Effect of Pseudomonas putida on Growth and Anthocyanin Pigment in Two Poinsettia (Euphorbia pulcherrima Cultivars

    Directory of Open Access Journals (Sweden)

    Ramon Zulueta-Rodriguez

    2014-01-01

    Full Text Available Pseudomonas putida is plant growth promoting rhizobacteria (PGPR that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble using three strains of P. putida, as well as a mixture of the three (MIX. Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4, volume of roots (45 versus 35 cm3, number of leaves (78 versus 58, and area of leaf (1,788 versus 1,331 cm2, except for the number of flowers (8.8 versus 11.6. To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars.

  18. Biosorption of aluminum through the use of non-viable biomass of Pseudomonas putida.

    Science.gov (United States)

    Boeris, Paola Sabrina; Agustín, María Del Rosario; Acevedo, Diego Fernando; Lucchesi, Gloria Inés

    2016-10-20

    Living and non-living biomass of Pseudomonas putida A (ATCC 12633) was used as biosorbent for the removing of Al(3+) from aqueous solutions. The process was stable with time, efficient at pH 4.3 and between 15°C and 42°C. Two isotherms models were applied to describe the interaction between the biosorbent and Al(3+). Non-living biomass of P. putida A (ATCC 12633) was found to be the most efficient at adsorbing Al(3+) with a maximum sorption capacity of 0.55mg Al(3+)/gr adsorbent and with 36×10(5) binding sites of Al(3+)/microorganisms. Infrared spectroscopy analysis shows that the biosorbent present some vibrational band of functional groups that change in presence of Al(3+): hydroxyl, carboxyl and phosphate. Considering that Al(3+) binds to the phosphate group of phosphatidylcholine, non-viable biomass of P. putida PB01 (mutant lacking phosphatidylcholine) was used. Aluminum adsorption of the parental strain was 30 times higher than values registered in P. putida PB01 (36×10(5) sites/microorganism vs 1.2×10(5) sites/microorganism, respectively). This result evidenced that the absence of phosphatidylcholine significantly affected the availability of the binding sites and consequently the efficiency of the biomass to adsorb Al(3+). Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Metal Inhibition of Growth and Manganese Oxidation in Pseudomonas putida GB-1

    Science.gov (United States)

    Pena, J.; Sposito, G.

    2009-12-01

    Biogenic manganese oxides (MnO2) are ubiquitous nanoparticulate minerals that contribute to the adsorption of nutrient and toxicant metals, the oxidative degradation of various organic compounds, and the respiration of metal-reducing bacteria in aquatic and terrestrial environments. The formation of these minerals is catalyzed by a diverse and widely-distributed group of bacteria and fungi, often through the enzymatic oxidation of aqueous Mn(II) to Mn(IV). In metal-impacted ecosystems, toxicant metals may alter the viability and metabolic activity of Mn-oxidizing organisms, thereby limiting the conditions under which biogenic MnO2 can form and diminishing their potential as adsorbent materials. Pseudomonas putida GB-1 (P. putida GB-1) is a model Mn-oxidizing laboratory culture representative of freshwater and soil biofilm-forming bacteria. Manganese oxidation in P. putida GB-1 occurs via two single-electron-transfer reactions, involving a multicopper oxidase enzyme found on the bacterial outer membrane surface. Near the onset of the stationary phase of growth, dark brown MnO2 particles are deposited in a matrix of bacterial cells and extracellular polymeric substances, thus forming heterogeneous biomineral assemblages. In this study, we assessed the influence of various transition metals on microbial growth and manganese oxidation capacity in a P. putida GB-1 culture propagated in a nutrient-rich growth medium. The concentration-response behavior of actively growing P. putida GB-1 cells was investigated for Fe, Co, Ni, Cu and Zn at pH ≈ 6 in the presence and absence of 1 mM Mn. Toxicity parameters such as EC0, EC50 and Hillslope, and EC100 were obtained from the sigmoidal concentration-response curves. The extent of MnO2 formation in the presence of the various metal cations was documented 24, 50, 74 and 104 h after the metal-amended medium was inoculated. Toxicity values were compared to twelve physicochemical properties of the metals tested. Significant

  20. Activity of toluene-degrading Pseudomonas putida in the early growth phase of a biofilm for waste gas treatment

    DEFF Research Database (Denmark)

    Pedersen, A.R.; Møller, S.; Molin, S.

    1997-01-01

    the increase of active biomass and polymers was linear. In order to investigate the toluene degradation, various toluene degraders from the multispecies biofilm were isolated, and a Pseudomonas putida was chosen as a representative of the toluene-degrading population. A specific rRNA oligonucleotide probe...... was used to follow the toluene-degrading P. putida in the multispecies biofilm in the filter by means of number and cellular rRNA content. P. putida appeared to detach from the biofilm during the first three days of growth, after which P. putida was found at a constant level of 10% of the active biomass...... in the biofilm. Based on the rRNA content, the in situ activity was estimated to be reduced to 20% of cells grown at maximum conditions in batch culture. The toluene degraded by P. putida was estimated to be a minor part (11%) of the overall toluene degradation. (C) 1997 John Wiley & Sons, Inc....

  1. Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge

    OpenAIRE

    McCarthy Andrea T; Zhang Bing; VerBerkmoes Nathan C; Thieman Stephanie B; Wickham Gene S; Chourey Karuna; Thompson Dorothea K; Rudisill Matt A; Shah Manesh; Hettich Robert L

    2010-01-01

    Abstract Background Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9...

  2. Comparison of kinetics of active tetracycline uptake and active tetracycline efflux in sensitive and plasmid RP4-containing Pseudomonas putida.

    OpenAIRE

    Hedstrom, R C; Crider, B P; Eagon, R G

    1982-01-01

    Membrane vesicles prepared from tetracycline-sensitive cells of Pseudomonas putida took up tetracycline by an active transport system with an apparent Km of 2.5 mM and a Vmax of 50 nmol min-1 mg protein-1. In contrast, resistance determinant RP4-containing P. putida had an active high-affinity efflux system for tetracycline with a Km of 2.0 to 3.54 microM and a Vmax of 0.15 nmol min-1 mg protein-1. Thus, the efflux system of tetracycline-resistant P. putida(RP4) had an average of 1,000-fold g...

  3. Biofiltration of ethyl acetate by Pseudomonas putida immobilized on walnut shell.

    Science.gov (United States)

    Zare, Hossein; Najafpour, Ghasem; Rahimnejad, Mostafa; Tardast, Ali; Gilani, Saeedeh

    2012-11-01

    A biofilter packed with walnut shells was used to eliminate ethyl acetate from an air stream. The shells treated with NaOH were used as medium for immobilization of Pseudomonas putida PTCC 1694. At an empty bed residence time (EBRT) of 60s, a removal efficiency of 99% was achieved at inlet concentrations lower than 430ppm of ethyl acetate. The removal efficiency decreased below 80% with an increase in inlet concentration of ethyl acetate. When the EBRT was increased to 75 s, the removal efficiency remained above 80% even though the inlet loading rate was increased to 421g/m(3)h. Michaelis-Menten type and zero-order diffusion limited models were employed and the predicted data perfectly matched the experimental data. Thus P. putida immobilized on walnut shell has potential for the removal of ethyl acetate from air streams. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida.

    Science.gov (United States)

    Martin, Collin H; Prather, Kristala L Jones

    2009-01-01

    Hydroxyacids represent an important class of compounds that see application in the production of polyesters, biodegradable plastics and antibiotics, and that serve as useful chiral synthetic building blocks for other fine chemicals and pharmaceuticals. An economical, high-titer method for the production of 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) from the inexpensive and renewable carbon source levulinic acid was developed. These hydroxyvalerates were produced by periodically feeding levulinate to Pseudomonas putida KT2440 expressing a recombinant thioesterase II (tesB) gene from Escherichia coli K12. The titer of 4HV in shake flask culture reached 13.9+/-1.2 g L(-1) from P. putida tesB(+) cultured at 32 degrees C in LB medium periodically supplemented with glucose and levulinate. The highest 3HV titer obtained was 5.3+/-0.1 g L(-1) in M9 minimal medium supplemented with glucose and levulinate.

  5. LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida.

    Science.gov (United States)

    Lahesaare, Andrio; Ainelo, Hanna; Teppo, Annika; Kivisaar, Maia; Heipieper, Hermann J; Teras, Riho

    2016-01-01

    The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida.

  6. Regulation of Hydroxylation and Nitroreduction Pathways during Metabolism of the Neonicotinoid Insecticide Imidacloprid by Pseudomonas putida.

    Science.gov (United States)

    Lu, Tian-Qi; Mao, Shi-Yun; Sun, Shi-Lei; Yang, Wen-Long; Ge, Feng; Dai, Yi-Jun

    2016-06-22

    Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism.

  7. Interspecies cross-talk between co-cultured Pseudomonas putida and Escherichia coli.

    Science.gov (United States)

    Molina-Santiago, Carlos; Udaondo, Zulema; Cordero, Baldo F; Ramos, Juan L

    2017-08-01

    Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be isolated from soil rhizosphere, the surface of vegetables, fresh waters and wastewaters - environments in which they likely co-exist. Despite this, the potential interactions between these microbes have not been studied in detail. To analyse these interactions, we carried out RNA-seq transcriptomic analysis of these microbes as monocultures and as co-cultures. Our results show that co-culture of these microbes significantly alters transcriptional profiles. The most dramatic transcriptional changes in both microorganisms were involved in central carbon metabolism, as well as adhesion to surfaces and the activation of drug efflux pumps. We also found that acetate production was one of the mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida DOT-T1E. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Efficient recombinant production of prodigiosin in Pseudomonas putida

    OpenAIRE

    Domröse, Andreas; Klein, Andreas S.; Hage-Hülsmann, Jennifer; Thies, Stephan; Svensson, Vera; Classen, Thomas; Pietruszka, Jörg; Jaeger, Karl-Erich; Drepper, Thomas; Loeschcke, Anita

    2015-01-01

    Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas p...

  9. Efficient recombinant production of prodigiosin in Pseudomonas putida

    OpenAIRE

    Andreas eDomröse; Andreas Sebastian Klein; Jennifer eHage-Hülsmann; Stephan eThies; Vera eSvensson; Thomas eClassen; Thomas eClassen; Jörg ePietruszka; Jörg ePietruszka; Karl-Erich eJaeger; Karl-Erich eJaeger; Thomas eDrepper; Anita eLoeschcke

    2015-01-01

    Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas pu...

  10. Heavy metal tolerance genes alter cellular thermodynamics in Pseudomonas putida and river Pseudomonas spp. and influence amebal predation.

    Science.gov (United States)

    McTee, Michael R; Gibbons, Sean M; Feris, Kevin; Gordon, Nathan S; Gannon, James E; Ramsey, Philip W

    2013-10-01

    Predation rates were measured for two Acanthamoeba castellanii strains feeding on metal-tolerant and metal-sensitive strains of Pseudomonas putida and compared with cellular thermodynamic data. Predation rates by A. castellanii strain ATCC 30010 correlated with cell volume of the prey. To explore whether this observation could be environmentally relevant, pseudomonad species were isolated from a pristine and a metal-contaminated river and were paired based on phylogenetic and physiological relatedness. Then, cellular thermodynamics and predation rates were measured on the most similar pseudomonad pair. Under cadmium stress, the strain from contaminated river sediments, Pseudomonas sp. CF150, exited metabolic dormancy faster than its pair from pristine sediments, Pseudomonas sp. N9, but consumed available resources less efficiently (more energy was lost as heat). Predation rates by both strains of ameba were greater on Pseudomonas sp. CF150 than on Pseudomonas sp. N9 at the highest cadmium concentration. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. The elicitation of a systemic resistance by Pseudomonas putida BTP1 in tomato involves the stimulation of two lipoxygenase isoforms

    Directory of Open Access Journals (Sweden)

    Thonart Philippe

    2011-02-01

    Full Text Available Abstract Background Some non-pathogenic rhizobacteria called Plant Growth Promoting Rhizobacteria (PGPR possess the capacity to induce in plant defense mechanisms effective against pathogens. Precedent studies showed the ability of Pseudomonas putida BTP1 to induce PGPR-mediated resistance, termed ISR (Induced Systemic Resistance, in different plant species. Despite extensive works, molecular defense mechanisms involved in ISR are less well understood that in the case of pathogen induced systemic acquired resistance. Results We analyzed the activities of phenylalanine ammonia-lyase (PAL and lipoxygenase (LOX, key enzymes of the phenylpropanoid and oxylipin pathways respectively, in tomato treated or not with P. putida BTP1. The bacterial treatment did not stimulate PAL activity and linoleate-consuming LOX activities. Linolenate-consuming LOX activity, on the contrary, was significantly stimulated in P. putida BTP1-inoculated plants before and two days after infection by B. cinerea. This stimulation is due to the increase of transcription level of two isoforms of LOX: TomLoxD and TomLoxF, a newly identified LOX gene. We showed that recombinant TomLOXF preferentially consumes linolenic acid and produces 13-derivative of fatty acids. After challenging with B. cinerea, the increase of transcription of these two LOX genes and higher linolenic acid-consuming LOX activity were associated with a more rapid accumulation of free 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids, two antifungal oxylipins, in bacterized plants. Conclusion In addition to the discovery of a new LOX gene in tomato, this work is the first to show differential induction of LOX isozymes and a more rapid accumulation of 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids in rhizobacteria mediated-induced systemic resistance.

  12. Systemic resistance and lipoxygenase-related defence response induced in tomato by Pseudomonas putida strain BTP1

    Directory of Open Access Journals (Sweden)

    Dommes Jacques

    2008-11-01

    Full Text Available Abstract Background Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes. Results In this study, we showed the protective effect of this bacterium in tomato against Botrytis cinerea. Following treatment by P. putida BTP1, analyses of acid-hydrolyzed leaf extracts showed an accumulation of antifungal material after pathogen infection. The fungitoxic compounds thus mainly accumulate as conjugates from which active aglycones may be liberated through the activity of hydrolytic enzymes. These results suggest that strain BTP1 can elicit systemic phytoalexin accumulation in tomato as one defence mechanism. On another hand, we have shown that key enzymes of the lipoxygenase pathway are stimulated in plants treated with the bacteria as compared with control plants. Interestingly, this stimulation is observed only after pathogen challenge in agreement with the priming concept almost invariably associated with the ISR phenomenon. Conclusion Through the demonstration of phytoalexin accumulation and LOX pathway stimulation in tomato, this work provides new insights into the diversity of defence mechanisms that are inducible by non-pathogenic bacteria in the context of ISR.

  13. Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2008-04-01

    The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

  14. Structural studies of the O-specific polysaccharide from detergent degrading bacteria Pseudomonas putida TSh-18.

    Science.gov (United States)

    Sigida, Elena N; Fedonenko, Yuliya P; Shashkov, Alexander S; Zdorovenko, Evelina L; Ignatov, Vladimir V; Knirel, Yuriy A

    2017-08-07

    An O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of bacteria Pseudomonas putida TSh-18, capable of degrading non-ionogenic technical detergents. The polysaccharide was found to contain a rarely occurring sugar derivative 4,6-dideoxy-4-[(R)-3-hydroxybutanoylamino]-d-galactose [d-Fucp4N(RHb)]. Sugar and methylation analyses, Smith degradation, solvolysis with CF 3 CO 2 H, and 1 H and 13 C NMR spectroscopy enabled elucidation of the following structure of the branched trisaccharide repeating units of the polysaccharide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Biosorption of heavy metals and uranium by starfish and Pseudomonas putida.

    Science.gov (United States)

    Choi, Jaeyoung; Lee, Ju Young; Yang, Jung-Seok

    2009-01-15

    Biosorption of heavy metals and uranium from contaminated wastewaters may represent an innovative purification process. This study investigates the removal ability of unit mass of Pseudomonas putida and starfish for lead, cadmium, and uranium by quantifying the adsorption capacity. The adsorption of heavy metals and uranium by the samples was influenced by pH, and increased with increasing Pb, Cd, and U concentrations. Dead cells adsorbed the largest quantity of all heavy metals than live cells and starfish. The adsorption capacity followed the order: U(VI)>Pb>Cd. The results also suggest that bacterial membrane cells can be used successfully in the treatment of high strength metal-contaminated wastewaters.

  16. Establishment of oxidative D-xylose metabolism in Pseudomonas putida S12.

    Science.gov (United States)

    Meijnen, Jean-Paul; de Winde, Johannes H; Ruijssenaars, Harald J

    2009-05-01

    The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose(-1)) and a maximum growth rate of 0.21 h(-1). Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and alpha-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose.

  17. C(1) compounds as auxiliary substrate for engineered Pseudomonas putida S12.

    Science.gov (United States)

    Koopman, Frank W; de Winde, Johannes H; Ruijssenaars, Harald J

    2009-06-01

    The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C(1) compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.

  18. Probing the proteome response to toluene exposure in the solvent tolerant Pseudomonas putida S12.

    Science.gov (United States)

    Wijte, Dorien; van Baar, Ben L M; Heck, Albert J R; Altelaar, A F Maarten

    2011-02-04

    To enhance target production from biocatalysts, it is necessary to thoroughly understand the molecular mechanisms involved in production, degradation, and, importantly, adaptation to the required environment. One such bacterium with high potential for biocatalysis is the solvent-tolerant bacteria Pseudomonas putida S12, which, among others, is able to degrade organic solvents. For bioconversion of organic solvents to become a successful industrial process, the understanding of the molecular response upon solvent tolerance is essential. Here we performed a quantitative analysis of the P. putida S12 proteome at different stages of adaptation to toluene. Using a stable isotope dimethylation labeling approach we monitored the differential expression of 528 proteins, including often hard-to-detect membrane associate proteins, such as multiple RND-family transporters and ABC transporters of nutrients. Our quantitative proteomics approach revealed the remarkable ability of P. putida S12 to severely change its protein expression profile upon toluene exposure. This proteome response entails a significant increase in energy metabolism and expression of the solvent efflux pump SrpABC, confirming its role in solvent tolerance. Other proteins strongly up-regulated in the presence of toluene include the multidrug efflux membrane protein PP1272 and the cation/acetate symporter ActP and may form interesting alternative targets for improving solvent tolerance.

  19. Pseudomonas putida F1 uses energy taxis to sense hydroxycinnamic acids.

    Science.gov (United States)

    Hughes, Jonathan G; Zhang, Xiangsheng; Parales, Juanito V; Ditty, Jayna L; Parales, Rebecca E

    2017-10-01

    Soil bacteria such as pseudomonads are widely studied due to their diverse metabolic capabilities, particularly the ability to degrade both naturally occurring and xenobiotic aromatic compounds. Chemotaxis, the directed movement of cells in response to chemical gradients, is common in motile soil bacteria and the wide range of chemicals detected often mirrors the metabolic diversity observed. Pseudomonas putida F1 is a soil isolate capable of chemotaxis toward, and degradation of, numerous aromatic compounds. We showed that P. putida F1 is capable of degrading members of a class of naturally occurring aromatic compounds known as hydroxycinnamic acids, which are components of lignin and are ubiquitous in the soil environment. We also demonstrated the ability of P. putida F1 to sense three hydroxycinnamic acids: p-coumaric, caffeic and ferulic acids. The chemotaxis response to hydroxycinnamic acids was induced during growth in the presence of hydroxycinnamic acids and was negatively regulated by HcaR, the repressor of the hydroxycinnamic acid catabolic genes. Chemotaxis to the three hydroxycinnamic acids was dependent on catabolism, as a mutant lacking the gene encoding feruloyl-CoA synthetase (Fcs), which catalyzes the first step in hydroxycinnamic acid degradation, was unable to respond chemotactically toward p-coumaric, caffeic, or ferulic acids. We tested whether an energy taxis mutant could detect hydroxycinnamic acids and determined that hydroxycinnamic acid sensing is mediated by the energy taxis receptor Aer2.

  20. Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes.

    Science.gov (United States)

    Benedetti, Ilaria; de Lorenzo, Víctor; Nikel, Pablo I

    2016-01-01

    Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degradation of c-di-GMP--thus making P. putida to form biofilms at user's will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phoshodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegradation. Upon addition of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and physical forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochemical activity. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2*

    Science.gov (United States)

    Ibrahim, Susan A.; Crack, Jason C.; Rolfe, Matthew D.; Acuña, José Manuel Borrero-de; Thomson, Andrew J.; Le Brun, Nick E.; Schobert, Max; Stapleton, Melanie R.; Green, Jeffrey

    2015-01-01

    The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. PMID:25971977

  2. Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

    Science.gov (United States)

    Calero, Patricia; Jensen, Sheila I.; Bojanovič, Klara; Lennen, Rebecca M.; Koza, Anna

    2017-01-01

    Abstract The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity. PMID:29131301

  3. Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegradation of aromatic compounds.

    Science.gov (United States)

    Nikel, Pablo I; Pérez-Pantoja, Danilo; de Lorenzo, Víctor

    2016-10-01

    The metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP + -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida.

    Science.gov (United States)

    Jiménez-Fernández, Alicia; López-Sánchez, Aroa; Calero, Patricia; Govantes, Fernando

    2015-02-01

    We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

  5. Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

    Science.gov (United States)

    Duque, Estrella; Fernández, Matilde; Molina-Santiago, Carlos; Roca, Amalia; Porcel, Mario; de la Torre, Jesús; Segura, Ana; Plesiat, Patrick; Jeannot, Katy; Ramos, Juan-Luis

    2014-01-01

    Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts. PMID:24465371

  6. Characterization of a Pseudomonas putida rough variant evolved in a mixed species biofilm with Acinetobacter sp. strain C6

    DEFF Research Database (Denmark)

    Hansen, Susse Kirkelund; Haagensen, Janus Anders Juul; Gjermansen, Morten

    2007-01-01

    Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria...... biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter....... putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response...

  7. Entner-Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1.

    Science.gov (United States)

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-08-04

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

  8. Effect of gravity on Pseudomonas putida and kaolinite cotransport in water saturated porous media

    Science.gov (United States)

    Vasiliadou, Ioanna A.; Chrysikopoulos, Constantinos V.

    2013-04-01

    Bacterial transport in porous media can be affected by several factors, such as cell concentration, water velocity, and attachment onto the solid matrix or suspended in the aqueous phase soil particles (e.g. clays). Gravity, also may significantly influence bacterial transport behavior in the subsurface. The present study aims to determine the gravity effect on transport and cotransport of bacteria species Pseudomonas (P.) putida and kaolinite colloid particles in porous media. Transport experiments were conducted under horizontal-, up- and down-flow conditions in water saturated columns packed with glass beads. These different flow modes represent different gravity effects, namely: no-, negative- and positive-gravity effect. Initial experiments were performed with bacteria and kaolinite alone in order to evaluate the effect of gravity on their individual transport characteristics. No significant gravity effect was observed on the transport of individual bacterial cells. In contrary, each different flow mode was found to differently affect kaolinite transport. Compared to the horizontal-flow mode, the kaolinite mass recovery was decreased during the up-flow mode, and increased during the down-flow mode. Finally, P. putida and kaolinite particles were injected simultaneously into the packed column in order to investigate their cotransport behavior under different flow modes. The experimental data indicated that the kaolinite-P. putida cotransport behavior was similar to that observed for the transport of individual kaolinite particles. It was observed that the P. putida mass recovery decreased during down-flow conditions. This phenomenon may be caused by the attachment of bacteria onto kaolinite particles, which were adsorbed onto the solid matrix.

  9. Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase.

    Science.gov (United States)

    Blank, Lars M; Ionidis, Georgios; Ebert, Birgitta E; Bühler, Bruno; Schmid, Andreas

    2008-10-01

    A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of

  10. Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains

    Science.gov (United States)

    Lindmeyer, Martin; Jahn, Michael; Vorpahl, Carsten; Müller, Susann; Schmid, Andreas; Bühler, Bruno

    2015-01-01

    Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed. PMID:26483771

  11. Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

    Science.gov (United States)

    Cheng, Lei; Yin, Sheng; Chen, Min; Sun, Baoguo; Hao, Shuai; Wang, Chengtao

    2016-08-01

    As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13 mg L(-1) after 24 h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas.

  12. Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

    Science.gov (United States)

    Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping

    2011-01-01

    Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121

  13. Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73

    International Nuclear Information System (INIS)

    Niu Guilan; Zhang Junjie; Zhao Shuo; Liu Hong; Boon, Nico; Zhou Ningyi

    2009-01-01

    The strain Pseudomonas putida ZWL73, which metabolizes 4-chloronitrobenzene (4CNB) by a partial-reductive pathway, was inoculated into lab-scale 4CNB-contaminated soil for bioaugmentation purposes in this study. The degradation of 4CNB was clearly stimulated, as indicated with the gradual accumulation of ammonium and chloride. Simultaneously, the diversity and quantity of cultivable heterotrophic bacteria decreased due to 4CNB contamination, while the quantity of 4CNB-resistant bacteria increased. During the bioaugmentation, denaturing gradient gel electrophoresis analysis showed the changes of diversity in dominant populations of intrinsic soil microbiota. The results showed that Alphaproteobacteria and Betaproteobacteria were not distinctly affected, but Actinobacteria were apparently stimulated. In addition, an interesting dynamic within Acidobacteria was observed, as well as an influence on ammonia-oxidizing bacteria population. These combined findings demonstrate that the removal of 4CNB in soils by inoculating strain ZWL73 is feasible, and that specific populations in soils rapidly changed in response to 4CNB contamination and subsequent bioaugmentation. - Pseudomonas putida ZWL73 can accelerate 4CNB removal in lab-scale soils, causing dynamic changes within intrinsic Actinobacteria and Acidobacteria

  14. Degradation of (-)-ephedrine by Pseudomonas putida. Detection of (-)-ephedrine: NAD+-oxidoreductase from Arthrobacter globiformis.

    Science.gov (United States)

    Klamann, E; Lingens, F

    1980-01-01

    A bacterium utilizing the alkaloid (-)-ephedrine as its sole source of carbon was isolated by an enrichment-culture technique from soil supplemented with 4-benzoyl-1,3-oxazolidinon-(2). The bacterium was indentified as Pseudomonas putida by morphological and physiological studies. The following metabolites were isolated from the culture fluid: methylamine, formaldehyde, methylbenzoylcarbinol (2-hydroxy-1-oxo-1 phenylpropane), benzoid acid, pyrocatechol and cis, cis-muconic acid. A pathway for the degradation of (-)-ephedrine by Pseudomonas putida is proposed and compared with the degradative pathway in Arthrobacter globiformis. The enzyme, which is responsible for the first step in the catabolism of (-)-ephedrine could be demonstrated in extracts from Arthrobacter globiformis. This enzyme catalyses the dehydrogenation of (-)-ephedrine yielding phenylacetylcarbinol/methylbenzoylcarbinol and methylamine. It requires NAD+ as cofactor and exhibits optimal activity at pH 11 in 0.1 M glycine/NaOH buffer. The Km value for (-)-ephedrine is 0.02 mM and for NAD+ 0.11 mM, respectively. No remarkable loss of activity is observed following treatment with EDTA. The enzyme has been shown to react with a wide range of ethanolamines. A slight enrichment was obtained by ammonium sulphate precipitation. The name (-)-ephedrine: NAD+-oxidoreductase (deaminating) is proposed.

  15. Mechanisms of solvent resistance mediated by interplay of cellular factors in Pseudomonas putida.

    Science.gov (United States)

    Ramos, Juan-Luis; Sol Cuenca, Maria; Molina-Santiago, Carlos; Segura, Ana; Duque, Estrella; Gómez-García, María R; Udaondo, Zulema; Roca, Amalia

    2015-07-01

    A number of microorganisms have the ability to thrive in the presence of a range of toxic solvents. Tolerance to these chemicals is a multifactorial process, meaning that bacterial cells use a set of physiological and gene expression changes to overcome the damage imparted by these chemicals. This review focuses mainly on issues related to tolerance to aromatic hydrocarbons and butanol in Pseudomonas, although other microorganisms are also discussed. Pseudomonas putida strains contain a circular chromosome of approximately 6 Mbp which encodes about 5300 genes. A combination of physiological and biochemical assays, a genome-wide collection of mutants and several omics approaches have provided useful information to help identify functions involved in solvent tolerance in P. putida. The solvent response involves fine-tuning of lipid fluidity to adjust membrane functions including impermeabilization, activation of a general stress-response system, increased energy generation and induction of specific efflux pumps that extrude solvents to the medium. These responses are modulated at the transcriptional level by local and global regulators as well as by a number of sRNAs whose levels fluctuate with the presence of solvents in the environment. Taken as a whole these regulatory inputs orchestrate the complex network of metabolic responses observed after solvent addition. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Interspecies signalling: Pseudomonas putida efflux pump TtgGHI is activated by indole to increase antibiotic resistance.

    Science.gov (United States)

    Molina-Santiago, Carlos; Daddaoua, Abdelali; Fillet, Sandy; Duque, Estrella; Ramos, Juan-Luis

    2014-05-01

    In Gram-negative bacteria, multidrug efflux pumps are responsible for the extrusion of chemicals that are deleterious for growth. Some of these efflux pumps are induced by endogenously produced effectors, while abiotic or biotic signals induce the expression of other efflux pumps. In Pseudomonas putida, the TtgABC efflux pump is the main antibiotic extrusion system that respond to exogenous antibiotics through the modulation of the expression of this operon mediated by TtgR. The plasmid-encoded TtgGHI efflux pump in P. putida plays a minor role in antibiotic resistance in the parental strain; however, its role is critical in isogenic backgrounds deficient in TtgABC. Expression of ttgGHI is repressed by the TtgV regulator that recognizes indole as an effector, although P. putida does not produce indole itself. Because indole is not produced by Pseudomonas, the indole-dependent antibiotic resistance seems to be part of an antibiotic resistance programme at the community level. Pseudomonas putida recognizes indole added to the medium or produced by Escherichia coli in mixed microbial communities. Transcriptomic analyses revealed that the indole-specific response involves activation of 43 genes and repression of 23 genes. Indole enhances not only the expression of the TtgGHI pump but also a set of genes involved in iron homeostasis, as well as genes for amino acid catabolism. In a ttgABC-deficient P. putida, background ampicillin and other bactericidal compounds lead to cell death. Co-culture of E. coli and P. putida ΔttgABC allowed growth of the P. putida mutant in the presence of ampicillin because of induction of the indole-dependent efflux pump. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  17. Genome sequence of Pseudomonas putida S12, a potential platform strain for industrial production of valuable chemicals.

    Science.gov (United States)

    Tao, Fei; Shen, Yaling; Fan, Ziqi; Tang, Hongzhi; Xu, Ping

    2012-11-01

    Pseudomonas putida strain S12, a well-studied solvent-tolerant bacterium, is considered a platform strain for the production of many chemicals. Here, we present a 6.28-Mb assembly of its genome sequence. We have annotated 32 coding sequences (CDSs) encoding efflux systems of organic compounds and 195 CDSs responsible for the metabolism of aromatic compounds.

  18. Transcriptome analysis of a phenol-producing Pseudomonas putida S12 construct: Genetic and physiological basis for improved production

    NARCIS (Netherlands)

    Wierckx, N.J.P.; Ballerstedt, H.; Bont, J.A.M.de; Winde, J.H.de; Ruijssenaars, H.J.; Wery, J.

    2008-01-01

    The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine

  19. Improved p-hydroxybenzoate production by engineered Pseudomonas putida S12 by using a mixed-substrate feeding strategy

    NARCIS (Netherlands)

    Meijnen, J.P.; Verhoef, S.; Briedjlal, A.A.; De Winde, J.H.; Ruijssenaars, H.J.

    2011-01-01

    The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP

  20. Bioluminescent Bioreporter Pseudomonas putida TVA8 as a Detector of Water Pollution. Operational Conditions and Selectivity of Free Cells Sensor

    Czech Academy of Sciences Publication Activity Database

    Kuncová, Gabriela; Pazlarová, J.; Hlavatá, Alena; Ripp, S.; Sayler, G.S.

    2011-01-01

    Roč. 11, č. 3 (2011), s. 882-887 ISSN 1470-160X R&D Projects: GA MŠk ME 893 Institutional research plan: CEZ:AV0Z40720504 Keywords : whole-cell biosensor * bioluminiscence * pseudomonas putida TVA8 Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.695, year: 2011

  1. Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida

    NARCIS (Netherlands)

    Zaat, S. A.; Slegtenhorst-Eegdeman, K.; Tommassen, J.; Geli, V.; Wijffelman, C. A.; Lugtenberg, B. J.

    1994-01-01

    In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene

  2. Detection and characterization of a bacteriocin, putadicin T01, produced by Pseudomonas putida isolated from hot spring water.

    Science.gov (United States)

    Ghrairi, Taoufik; Braiek, Olfa Ben; Hani, Khaled

    2015-03-01

    Pseudomonas strains isolated from hot spring water were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria. Molecular identification was carried out through specific PCR and 16S RNA sequence analysis. Isolates were identified as Brevundimonas diminuta and Pseudomonas putida, the latter exhibited antimicrobial activity. Pseudomonas putida strains produce an inhibitory substance against other Pseudomonas strains and other species including food-borne pathogens. The BLS was sensitive to the proteolytic action of proteinase K, pronase E and trypsin but resistant to α-amylase, RNase and lipase C, reflecting its proteinaceous nature. The BLS was stable at 100 °C and also after thermal treatment at 121 °C for 15 min. Additionally, it was stable within a wide range of pH (2-10). The substance from P. putida T01 strain was bactericidal to Escherichia coli. SDS-PAGE analysis of the partial purified supernatant of strain T01 revealed a BLS with an approximate molecular mass of 8 kDa. Therefore, the results of this study show that P. putida strain T01 produces a BLS with a higher activity spectrum, which may find application in human medicine and in minimally processed food preservation. © 2014 APMIS. Published by John Wiley & Sons Ltd.

  3. Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

    NARCIS (Netherlands)

    Arif, Muhammad Ilan; Samin, Ghufrana; van Leeuwen, Jan G. E.; Oppentocht, Jantien; Janssen, Dick B.

    A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation

  4. TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12

    NARCIS (Netherlands)

    Volkers, R.J.M.; Ballerstedt, H.; Ruijssenaars, H.; Bont, J.A.M. de; Winde, J.H. de; Wery, J.

    2009-01-01

    Pseudomonas putida S12 is well known for its remarkable solvent tolerance. Transcriptomics analysis of this bacterium grown in toluene-containing chemostats revealed the differential expression of 253 genes. As expected, the genes encoding one of the major solvent tolerance mechanisms, the solvent

  5. Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.

    Science.gov (United States)

    Simon, Oliver; Klaiber, Iris; Huber, Armin; Pfannstiel, Jens

    2014-09-23

    Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a

  6. Regulation of phenylacetic acid uptake is sigma54 dependent in Pseudomonas putida CA-3.

    LENUS (Irish Health Repository)

    O' Leary, Niall D

    2011-10-13

    Abstract Background Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene\\/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. Results One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ54 consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by

  7. Colony morphology and transcriptome profiling of Pseudomonas putida KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials

    DEFF Research Database (Denmark)

    Gülez, Gamze; Altintas, Ali; Fazli, Mustafa

    2014-01-01

    Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect...... of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role...... active to maintain homeostasis. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 wild type and its mutants deficient in synthesis of either alginate or all known EPS. Overall our results support that alginate is an important exopolysaccharide...

  8. Identification, cloning and biochemical characterization of Pseudomonas putida A (ATCC 12633) monooxygenase enzyme necessary for the metabolism of tetradecyltrimethylammonium bromide.

    Science.gov (United States)

    Liffourrena, Andrés S; Lucchesi, Gloria I

    2014-05-01

    This study presents the first report of the purification and characterization of a monooxygenase enzyme from Pseudomonas putida A (ATCC 12633) that is responsible for the oxidation of physiologically relevant quaternary ammonium compounds, the tetradecyltrimethylammonium bromide. The degradation of tetradecyltrimethylammonium bromide by P. putida A (ATCC 12633) is initiated by N-dealkylation and catalysed by tetradecyltrimethylammonium monooxygenase (TTABMO), resulting in the formation of tetradecylalkanal and trimethylamine. Based on sequence analysis, the gene for TTABMO (ttbmo) corresponded to an ORF named PP2033 in the genome of P. putida KT2440. Mutation in ttabmo blocked the utilization of tetradecyltrimethylammonium bromide by Pseudomonas putida A (ATCC 12633) as carbon and nitrogen sources. The enzyme can be highly overexpressed in P. putida Δttabmo-T7 in active form and purified as a hexahistidine fusion protein. Like the native enzyme, the his-TTABMO was found to be a monomer with molecular mass of 40 kDa, the isoelectric point 7.3, that catalyses the breakdown of tetradecyltrimethylammonium bromide and utilized NADPH and FAD as cofactor. The biochemical properties and the analysis of the respective protein sequence revealed that TTABMO represents a typical flavoprotein monooxygenase, which is member of a flavoprotein family that is distinct from Baeyer-Villiger monooxygenases.

  9. Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

    NARCIS (Netherlands)

    Dubern, J.F.; Coppoolse, E.R.; Stiekema, W.J.; Bloemberg, G.V.

    2008-01-01

    Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene

  10. Pseudomonas putida response in membrane bioreactors under salicylic acid-induced stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Collado, Sergio; Rosas, Irene; González, Elena; Gutierrez-Lavin, Antonio; Diaz, Mario, E-mail: mariodiaz@uniovi.es

    2014-02-01

    Highlights: • MBR under feed-induced stress conditions: starvation and changing feeding conditions. • High capacity of MBR to withstand high variations in feed loads. • Slow biofilm formation under starvation conditions during the first days. • Observed growth of P. putida for substrate to microorganism ratio higher than 0.6 g/g. • Maximum specific growth rate and growth yield values of around 37.5 h{sup −1} and 0.5 g/g. - Abstract: Starvation and changing feeding conditions are frequently characteristics of wastewater treatment plants. They are typical causes of unsteady-state operation of biological systems and provoke cellular stress. The response of a membrane bioreactor functioning under feed-induced stress conditions is studied here. In order to simplify and considerably amplify the response to stress and to obtain a reference model, a pure culture of Pseudomonas putida was selected instead of an activated sludge and a sole substrate (salicylic acid) was employed. The system degraded salicylic acid at 100–1100 mg/L with a high level of efficiency, showed rapid acclimation without substrate or product inhibition phenomena and good stability in response to unsteady states caused by feed variations. Under starvation conditions, specific degradation rates of around 15 mg/g h were achieved during the adaptation of the biomass to the new conditions and no biofilm formation was observed during the first days of experimentation using an initial substrate to microorganisms ratio lower than 0.1. When substrate was added to the reactor as pulses resulting in rapidly changing concentrations, P. putida growth was observed only for substrate to microorganism ratios higher than 0.6, with a maximum Y{sub X/S} of 0.5 g/g. Biofilm development under changing feeding conditions was fast, biomass detachment only being significant for biomass concentrations on the membrane surface that were higher than 16 g/m{sup 2}.

  11. Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM.

    Science.gov (United States)

    Peng, Yanjie; He, Yanhui; Wu, Zhansheng; Lu, Jianjiang; Li, Chun

    2014-01-01

    The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K₂HPO₄, 0.19 g/L MnSO₄ and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K₂HPO₄, 0.17 g/L MnSO₄ and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

  12. Kinetics of mercury reduction by Serratia marcescens mercuric reductase expressed by pseudomonas putida strains

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.; Deckwer, W.D. [GBF-Gesellschaft fuer Biotechnologische Forschung mbH, Abteilung TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig (Germany)

    2005-10-01

    Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl{sub 2}, Hg{sub 2}SO{sub 4}, Hg(NO{sub 3}){sub 2} and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

  13. Pseudomonas putida response in membrane bioreactors under salicylic acid-induced stress conditions

    International Nuclear Information System (INIS)

    Collado, Sergio; Rosas, Irene; González, Elena; Gutierrez-Lavin, Antonio; Diaz, Mario

    2014-01-01

    Highlights: • MBR under feed-induced stress conditions: starvation and changing feeding conditions. • High capacity of MBR to withstand high variations in feed loads. • Slow biofilm formation under starvation conditions during the first days. • Observed growth of P. putida for substrate to microorganism ratio higher than 0.6 g/g. • Maximum specific growth rate and growth yield values of around 37.5 h −1 and 0.5 g/g. - Abstract: Starvation and changing feeding conditions are frequently characteristics of wastewater treatment plants. They are typical causes of unsteady-state operation of biological systems and provoke cellular stress. The response of a membrane bioreactor functioning under feed-induced stress conditions is studied here. In order to simplify and considerably amplify the response to stress and to obtain a reference model, a pure culture of Pseudomonas putida was selected instead of an activated sludge and a sole substrate (salicylic acid) was employed. The system degraded salicylic acid at 100–1100 mg/L with a high level of efficiency, showed rapid acclimation without substrate or product inhibition phenomena and good stability in response to unsteady states caused by feed variations. Under starvation conditions, specific degradation rates of around 15 mg/g h were achieved during the adaptation of the biomass to the new conditions and no biofilm formation was observed during the first days of experimentation using an initial substrate to microorganisms ratio lower than 0.1. When substrate was added to the reactor as pulses resulting in rapidly changing concentrations, P. putida growth was observed only for substrate to microorganism ratios higher than 0.6, with a maximum Y X/S of 0.5 g/g. Biofilm development under changing feeding conditions was fast, biomass detachment only being significant for biomass concentrations on the membrane surface that were higher than 16 g/m 2

  14. Evaluating the efficiency of two phase partitioning stirred tank bio-reactor for treating xylene vapors from the airstreamthrough a bed of Pseudomonas Putida

    Directory of Open Access Journals (Sweden)

    F. Golbabaei

    2015-04-01

    Conclusion: Overall, the results of the present research revealed that the application of two phase stirred tank bioreactors (TPPBs containing pure strains of Pseudomonas putida was successful for treatment of air streams with xylene.

  15. Whole-Genome Sequence of Pseudomonas putida Strain UASWS0946, a Highly Ammonia-Tolerant Nitrifying Bacterium Isolated from Sewage Sludge Aerobic Granules

    OpenAIRE

    Crovadore, Julien; Calmin, Gautier; Cochard, Bastien; Chablais, Romain; Grizard, Damien; Berthon, Jean-Yves; Lefort, François

    2015-01-01

    We report here the genome of Pseudomonas putida strain UASWS0946, a highly ammonia-tolerant nitrifying strain isolated from sewage sludge aerobic granules, which displays adequate genetic equipment for soil depollution, sludge treatment, and biological fertilization in agriculture.

  16. Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens.

    Science.gov (United States)

    Ye, Lumeng; Hildebrand, Falk; Dingemans, Jozef; Ballet, Steven; Laus, George; Matthijs, Sandra; Berendsen, Roeland; Cornelis, Pierre

    2014-01-01

    Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors.

  17. The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation

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    Stephanie Karmann

    2017-06-01

    Full Text Available Poly(hydroxyalkanoates (PHAs are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

  18. Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440.

    Science.gov (United States)

    Nikel, Pablo I; Kim, Juhyun; de Lorenzo, Víctor

    2014-01-01

    While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in the shift between customary C sources (e.g. glucose or succinate) to the polyol. Transcriptomic results were contrasted with key enzymatic activities under the same culture conditions. Cognate expression profiles revealed that genes encoding enzymes of the Entner-Doudoroff route and other catabolic pathways, e.g. the gluconate and 2-ketogluconate loops, were significantly downregulated on glycerol. Yet, the compound simultaneously elicited a gluconeogenic response that indicated an efficient channelling of C skeletons back to biomass build-up through the glyoxylate shunt rather than energization of the cells through downwards pathways, i.e. tricarboxylic acid cycle and oxidative phosphorylation. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favouring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other C sources. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Metabolic engineering of Pseudomonas putida KT2440 for the production of para-hydroxy benzoic acid

    Directory of Open Access Journals (Sweden)

    Shiqin Yu

    2016-11-01

    Full Text Available para-hydroxy benzoic acid (PHBA is the key component for preparing parabens, a common preservatives in food, drugs and personal care products, as well as high performance bioplastics such as liquid crystal polymers (LCP. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA or competing for the precursor chorismate (pheA and trpE were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose-4- phosphate and NAPH supply. The best strain achieved a maximum titre of 1.73 g L-1 and a carbon yield of 18.1 % (C-mol C-mol-1 in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase.

  20. Pseudomonas putida-a versatile host for the production of natural products.

    Science.gov (United States)

    Loeschcke, Anita; Thies, Stephan

    2015-08-01

    The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.

  1. The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida.

    Science.gov (United States)

    Escapa, I F; García, J L; Bühler, B; Blank, L M; Prieto, M A

    2012-04-01

    The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  2. A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

    Science.gov (United States)

    Samin, Ghufrana; Pavlova, Martina; Arif, M Irfan; Postema, Christiaan P; Damborsky, Jiri; Janssen, Dick B

    2014-09-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis.

    Science.gov (United States)

    Banerjee, Anirban; Kaul, Praveen; Banerjee, U C

    2006-08-01

    (R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

  4. Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies.

    Science.gov (United States)

    Banerjee, Anirban; Dubey, Sachin; Kaul, Praveen; Barse, Brajesh; Piotrowski, Markus; Banerjee, U C

    2009-01-01

    Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid.

  5. Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance.

    Science.gov (United States)

    Kieboom, J; de Bont, J

    2001-01-01

    The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance.

  6. Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida

    Science.gov (United States)

    Ito, Fumihiro; Tamiya, Takayuki; Ohtsu, Iwao; Fujimura, Makoto; Fukumori, Fumiyasu

    2014-01-01

    Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles. The KTΔclpB mutant showed the same heat shock response (HSR) as the wild-type, both in terms of heat-shock protein (Hsp) synthesis (other than ClpB) and in hsp gene expression; however, the mutant was quite sensitive to high temperatures and was unable to disaggregate into thermo-mediated protein aggregates, indicating that ClpB is important for cell survival after heat stress and essential for solubilization of protein aggregates. On the other hand, the KTΔdnaJ mutant was temperature-sensitive, and formed more protein aggregates (especially of high molecular weight) upon heat stress than did KT. P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ32 activity and quantity, as well as the mRNA levels of hsp genes. PMID:25303383

  7. Bypasses in intracellular glucose metabolism in iron-limited Pseudomonas putida.

    Science.gov (United States)

    Sasnow, Samantha S; Wei, Hua; Aristilde, Ludmilla

    2016-02-01

    Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  8. Organo-mineral interactions in Pseudomonas putida-birnessite assemblages: Impact on mineral reactivity

    Science.gov (United States)

    Simanova, Anna; Kroll, Alexandra; Pena, Jasquelin

    2016-04-01

    The ability of microorganisms to precipitate biogenic birnessite nanoparticles is widely spread in the bacterial and fungal trees of life, with this process accounting largely for the formation of birnessite in nature. Birnessite minerals occur typically as nanoparticles that exhibit significant chemical and structural disorder. Furthermore, the mineral is embedded within a biomass matrix composed of microbial cells and extracellular polymeric substances, where the biomass not only provides reactive surfaces but can mediate electron transfer reactions. The overarching question guiding our research is: How do nanoscale properties and admixing with microbial biomass modify the reactivity of Mn oxide minerals? In this study, we investigate the biomass-birnessite composites of Pseudomonas putida GB-1 biomass and δ-MnO2 nanoparticles. We characterized the structure and composition of the mineral fraction using X-ray diffraction, Mn K-edge X-ray absorption spectroscopy and wet-chemical methods. To characterize the biomass fraction, we employed FTIR spectroscopy and size-exclusion chromatography analysis of the extracellular polymeric substances. Finally, we measured Ni(II) sorption isotherms at pH 6 and Ni K-edge EXAFS spectra to determine the extent and mechanism of Ni sorption in the biomass-mineral composites and in biomass-only and mineral-only systems. This approach provided direct and indirect evidence for the extent of organo-mineral interactions in the composites, as well as a direct measure of sorption reactivity in the composites relative to biomass-only and mineral-only systems. We found that admixing of mineral nanoparticles with biomass reduced the reactivity of the edge sites of birnessite particles towards Ni(II) through the attachment of organic moieties to the mineral particles and/or modification of the assemblage surface charge properties. In addition, the interaction of biomass components with MnO2 particles leads to partial Mn(IV) reduction and

  9. Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates.

    Science.gov (United States)

    Meade, M J; Waddell, R L; Callahan, T M

    2001-10-16

    Triclosan is a broad-spectrum antimicrobial agent that has been incorporated into many household and medical products. Bacteria with high levels of triclosan resistance were isolated from compost, water, and soil samples. Two of these bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, were able to use triclosan as a sole carbon source and clear particulate triclosan from agar. A decrease in triclosan concentration was measured by HPLC within 6 h of inoculation with strain TriRY and 24 h with strain TR1. Bioassays demonstrated that triclosan was inactivated in liquid cultures and/or embedded in plastic by the growth of strain TriRY and strain TR1, permitting the growth of triclosan-sensitive bacteria.

  10. Silver nanotoxicity using a light-emitting biosensor Pseudomonas putida isolated from a wastewater treatment plant.

    Science.gov (United States)

    Dams, R I; Biswas, A; Olesiejuk, A; Fernandes, T; Christofi, N

    2011-11-15

    The effect of silver ions, nano- and micro-particles on a luminescent biosensor bacterium Pseudomonas putida originally isolated from activated sludge was assessed. The bacterium carrying a stable chromosomal copy of the lux operon (luxCDABE) was able to detect toxicity of ionic and particulate silver over short term incubations ranging from 30 to 240 min. The IC(50) values obtained at different time intervals showed that highest toxicity (lowest IC(50)) was obtained after 90 min incubation for all toxicants and this is considered the optimum incubation for testing. The data show that ionic silver is the most toxic followed by nanosilver particles with microsilver particles being least toxic. Release of nanomaterials is likely to have an effect on the activated sludge process as indicated by the study using a common sludge bacterium involved in biodegradation of organic wastes. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    Science.gov (United States)

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

  12. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; D'Arrigo, Isotta; Long, Katherine

    2017-01-01

    intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous......Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification...... of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent...

  13. Assessing storage of stability and mercury reduction of freeze-dried Pseudomonas putida within different types of lyoprotectant

    Science.gov (United States)

    Azoddein, Abdul Aziz Mohd; Nuratri, Yana; Azli, Faten Ahada Mohd; Bustary, Ahmad Bazli

    2017-12-01

    Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose was able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage at 4 °C without vacuum. Polyethylene glycol (PEG) pre-treated freeze dried cells and broth pre-treated freeze dried cells after the freeze-dry process recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introducing freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage of 3 weeks was 17.91 %. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been grown in agar. Result from this study may be beneficial and useful as initial reference before

  14. Competition triggers plasmid-mediated enhancement of substrate utilisation in Pseudomonas putida.

    Directory of Open Access Journals (Sweden)

    Hiren Joshi

    2009-06-01

    Full Text Available Competition between species plays a central role in the activity and structure of communities. Stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. Outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. Survival and proliferation of a particular species is governed by its competitive fitness. Therefore, bacteria must be able to continuously sense their immediate environs for presence of competitors and prevailing conditions. Here we present results of our investigations on a novel competition sensing mechanism in the rhizosphere-inhabiting Pseudomonas putida KT2440, harbouring gfpmut3b-modified Kan(R TOL plasmid. We monitored benzyl alcohol (BA degradation rate, along with GFP expression profiling in mono species and dual species cultures. Interestingly, enhanced plasmid expression (monitored using GFP expression and consequent BA degradation were observed in dual species consortia, irrespective of whether the competitor was a BA degrader (Pseudomonas aeruginosa or a non-degrader (E. coli. Attempts at elucidation of the mechanistic aspects of induction indicated the role of physical interaction, but not of any diffusible compounds emanating from the competitors. This contention is supported by the observation that greater induction took place in presence of increasing number of competitors. Inert microspheres mimicking competitor cell size and concentration did not elicit any significant induction, further suggesting the role of physical cell-cell interaction. Furthermore, it was also established that cell wall compromised competitor had minimal induction capability. We conclude that P. putida harbouring pWW0 experience a competitive stress when grown as dual-species consortium, irrespective of the counterpart being BA degrader or not. The immediate

  15. Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

    Directory of Open Access Journals (Sweden)

    Andreas Domröse

    2017-12-01

    Full Text Available Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for

  16. Pyoverdine synthesis by the Mn(II-oxidizing bacterium Pseudomonas putida GB-1

    Directory of Open Access Journals (Sweden)

    Dorothy Lundquist Parker

    2014-05-01

    Full Text Available When iron-starved, the Mn(II-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1, siderophores that both influence iron uptake and inhibit manganese(II oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs: chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase, coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III.

  17. Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1

    Science.gov (United States)

    Parker, Dorothy L.; Lee, Sung-Woo; Geszvain, Kati; Davis, Richard E.; Gruffaz, Christelle; Meyer, Jean-Marie; Torpey, Justin W.; Tebo, Bradley M.

    2014-01-01

    When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III). PMID:24847318

  18. Isolation of NDM-1-producing multidrug-resistant Pseudomonas putida from a paediatric case of acute gastroenteritis, India

    Directory of Open Access Journals (Sweden)

    D. Bhattacharya

    2015-05-01

    Full Text Available Pseudomonas putida is an uncommon opportunistic pathogen, usually susceptible to antimicrobial agents. Data concerning resistance to antimicrobial agents in clinical P. putida isolates are limited. To the best of our knowledge we report for the first time the isolation of NDM-1-producing multidrug-resistant P. putida from a case of acute gastroenteritis. The isolate showed resistance to a wide range of antimicrobials, including fluoroquinolones, third-generation cephalosporins and carbapenems. The isolate also exhibited multiple mutations in the quinolone resistance determining region and showed the presence of qepA, blaTEM, blaOXA1 and blaOXA7 genes. The present study highlights the importance of looking for the relatively rare aetiological agents in clinical samples that do not yield common pathogens.

  19. A Metabolic Widget Adjusts the Phosphoenolpyruvate-Dependent Fructose Influx in Pseudomonas putida

    Science.gov (United States)

    Chavarría, Max; Goñi-Moreno, Ángel

    2016-01-01

    ABSTRACT Fructose uptake in the soil bacterium Pseudomonas putida occurs through a canonical phosphoenolpyruvate (PEP)-dependent sugar transport system (PTSFru). The logic of the genetic circuit that rules its functioning is puzzling: the transcription of the fruBKA operon, encoding all the components of PTSFru, can escape the repression exerted by the catabolite repressor/activator protein Cra solely in the presence of intracellular fructose-1-P, an agonist formed only when fructose has been already transported. To study this apparently incongruous regulatory architecture, the changes in the transcriptome brought about by a seamless Δcra deletion in P. putida strain KT2440 were inspected under different culture conditions. The few genes found to be upregulated in the cra mutant unexpectedly included PP_3443, encoding a bona fide glyceraldehyde-3-P dehydrogenase. An in silico model was developed to explore emergent properties that could result from such connections between sugar uptake with Cra and PEP. Simulation of fructose transport revealed that sugar uptake called for an extra supply of PEP (obtained through the activity of PP_3443) that was kept (i.e., memorized) even when the carbohydrate disappeared from the medium. This feature was traced to the action of two sequential inverters that connect the availability of exogenous fructose to intracellular PEP levels via Cra/PP_3443. The loss of such memory caused a much longer lag phase in cells shifted from one growth condition to another. The term “metabolic widget” is proposed to describe a merged biochemical and regulatory patch that tailors a given node of the cell molecular network to suit species-specific physiological needs. IMPORTANCE The regulatory nodes that govern metabolic traffic in bacteria often show connectivities that could be deemed unnecessarily complex at a first glance. Being a soil dweller and plant colonizer, Pseudomonas putida frequently encounters fructose in the niches that it

  20. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory

    Science.gov (United States)

    Nogales, Juan; Palsson, Bernhard Ø; Thiele, Ines

    2008-01-01

    Background Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions. Results We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate), not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected. Conclusion Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in

  1. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory.

    Science.gov (United States)

    Nogales, Juan; Palsson, Bernhard Ø; Thiele, Ines

    2008-09-16

    Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions. We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate), not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected. Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in bioremediation and bioplastic

  2. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory

    Directory of Open Access Journals (Sweden)

    Thiele Ines

    2008-09-01

    Full Text Available Abstract Background Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions. Results We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate, not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected. Conclusion Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i a knowledge-base, ii a discovery tool, and iii an engineering platform to explore P

  3. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

    Directory of Open Access Journals (Sweden)

    Martínez-Toledo Ángeles

    2013-01-01

    Full Text Available In situ biosurfactant (rhamnolipid production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus, P. putida addition, and addition of both (P. putida and nutrients. The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019 between TPH concentration (mg/kg and surface tension (mN/m, When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009 was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.

  4. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy

    DEFF Research Database (Denmark)

    Møller, Søren; Pedersen, Anne Rathmann; Poulsen, L.K.

    1996-01-01

    As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe, The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy....... demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending ft om the surface Into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm......, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters fur P. putida showed reduced I RNA content and cell size (relative to that ill a batch culture), indicating that the P. putida population mas not degrading toluene...

  5. The Role of CzcRS Two-Component Systems in the Heavy Metal Resistance of Pseudomonas putida X4

    Directory of Open Access Journals (Sweden)

    Pulin Liu

    2015-07-01

    Full Text Available The role of different czcRS genes in metal resistance and the cross-link between czcRS and czcCBA in Pseudomonas putida X4 were studied to advance understanding of the mechanisms by which P. putida copes with metal stress. Similar to P. putida KT2440, two complete czcRS1 and czcRS2 two-component systems, as well as a czcR3 without the corresponding sensing component were amplified in P. putida X4. The histidine kinase genes czcS1 and czcS2 were inactivated and fused to lacZ by homologous recombination. The lacZ fusion assay revealed that Cd2+ and Zn2+ caused a decrease in the transcription of czcRS1, whereas Cd2+ treatment enhanced the transcription of czcRS2. The mutation of different czcRSs showed that all czcRSs are necessary to facilitate full metal resistance in P. putida X4. A putative gene just downstream of czcR3 is related to metal ion resistance, and its transcription was activated by Zn2+. Data from quantitative real-time polymerase chain reaction (qRT-PCR strongly suggested that czcRSs regulate the expression of czcCBA, and a cross-link exists between different czcRSs.

  6. The Role of CzcRS Two-Component Systems in the Heavy Metal Resistance of Pseudomonas putida X4.

    Science.gov (United States)

    Liu, Pulin; Chen, Xi; Huang, Qiaoyun; Chen, Wenli

    2015-07-27

    The role of different czcRS genes in metal resistance and the cross-link between czcRS and czcCBA in Pseudomonas putida X4 were studied to advance understanding of the mechanisms by which P. putida copes with metal stress. Similar to P. putida KT2440, two complete czcRS1 and czcRS2 two-component systems, as well as a czcR3 without the corresponding sensing component were amplified in P. putida X4. The histidine kinase genes czcS1 and czcS2 were inactivated and fused to lacZ by homologous recombination. The lacZ fusion assay revealed that Cd2+ and Zn2+ caused a decrease in the transcription of czcRS1, whereas Cd2+ treatment enhanced the transcription of czcRS2. The mutation of different czcRSs showed that all czcRSs are necessary to facilitate full metal resistance in P. putida X4. A putative gene just downstream of czcR3 is related to metal ion resistance, and its transcription was activated by Zn2+. Data from quantitative real-time polymerase chain reaction (qRT-PCR) strongly suggested that czcRSs regulate the expression of czcCBA, and a cross-link exists between different czcRSs.

  7. Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida.

    Science.gov (United States)

    Nogales, Juan; Canales, Angeles; Jiménez-Barbero, Jesús; Serra, Beatriz; Pingarrón, José Manuel; García, José Luis; Díaz, Eduardo

    2011-01-01

    Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor. © 2010 Blackwell Publishing Ltd.

  8. ISOLATION AND IDENTIFICATION OF A THERMOTOLERANT PLANT GROWTH PROMOTING PSEUDOMONAS PUTIDA PRODUCING TREHALOSE SYNTHASE

    Directory of Open Access Journals (Sweden)

    Ali Sk.Z.

    2013-08-01

    Full Text Available A thermotolerant plant growth promoting Pseudomonas isolate growing at 40oC producing trehalose synthase (TreS was isolated from rhizosphere soil under semi arid conditions of India. Trehalose synthase was extracted; purified and enzymatic activity was examined at various temperatures and pH. The optimum temperature and pH was 38oC and pH 7.5 and the activity declined at above or below the optimum pH and temperature. The enzyme was active on maltose and trehalose among saccharides tested. The enzyme had a higher catalytic activity for maltose with a trehalose yield of 72% than for trehalose where 30% yield of maltose was achieved, indicating maltose as preferred substrate. The isolate showed multiple plant growth promoting traits (indole acetic acid (IAA, phosphate solubilization, siderophore and ammonia both at ambient (28oC and high temperature (40oC. Based on phenotypic and 16SrRNA analysis the isolate was identified as Pseudomonas putida (Accession No. GU396283.

  9. Integrated foam fractionation for heterologous rhamnolipid production with recombinant Pseudomonas putida in a bioreactor.

    Science.gov (United States)

    Beuker, Janina; Steier, Anke; Wittgens, Andreas; Rosenau, Frank; Henkel, Marius; Hausmann, Rudolf

    2016-03-01

    Heterologeous production of rhamnolipids in Pseudomonas putida is characterized by advantages of a non-pathogenic host and avoidance of the native quorum sensing regulation in Pseudomonas aeruginosa. Yet, downstream processing is a major problem in rhamnolipid production and increases in complexity at low rhamnolipid titers and when using chemical foam control. This leaves the necessity of a simple concentrating and purification method. Foam fractionation is an elegant method for in situ product removal when producing microbial surfactants. However, up to now in situ foam fractionation is nearly exclusively reported for the production of surfactin with Bacillus subtilis. So far no cultivation integrated foam fractionation process for rhamnolipid production has been reported. This is probably due to excessive bacterial foam enrichment in that system. In this article a simple integrated foam fractionation process is reported for heterologous rhamnolipid production in a bioreactor with easily manageable bacterial foam enrichments. Rhamnolipids were highly concentrated in the foam during the cultivation process with enrichment factors up to 200. The described process was evaluated at different pH, media compositions and temperatures. Foam fractionation processes were characterized by calculating procedural parameter including rhamnolipid and bacterial enrichment, rhamnolipid recovery, YX/S, YP/X, and specific as well as volumetric productivities. Comparing foam fractionation parameters of the rhamnolipid process with the surfactin process a high effectiveness of the integrated foam fractionation for rhamnolipid production was demonstrated.

  10. Biological synthesis of the analgesic hydromorphone, an intermediate in the metabolism of morphine, by Pseudomonas putida M10.

    OpenAIRE

    Hailes, A M; Bruce, N C

    1993-01-01

    The morphine alkaloid hydromorphone (dihydromorphinone) was identified as an intermediary metabolite in the degradation of morphine by Pseudomonas putida M10. A constitutive NADH-dependent morphinone reductase capable of catalyzing the reduction of the 7,8-unsaturated bond of morphinone and codeinone, yielding hydromorphone and hydrocodone, respectively, was shown to be present in cell extracts. The structures have been identified by 1H nuclear magnetic resonance and mass spectrometry. Morphi...

  11. Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204.

    OpenAIRE

    Eaton, R W; Timmis, K N

    1986-01-01

    The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.

  12. Isolation of a strain of Pseudomonas putida capable of metabolizing anionic detergent sodium dodecyl sulfate (SDS).

    Science.gov (United States)

    Chaturvedi, V; Kumar, A

    2011-03-01

    Sodium Dodecyl Sulfate (SDS) is one of the most widely used anionic detergents. The present study deals with isolation and identification of SDS-degrading bacteria from a detergent contaminated pond situated in Varanasi city, India. Employing enrichment technique in minimal medium (PBM), SDS-degrading bacteria were isolated from pond water sample. Rate of degradation of SDS was studied in liquid PBM and also degradation of different concentrations of SDS was also studied to find out maximum concentration of SDS degraded by the potent isolates. Alkyl sulfatase activity (key enzyme in SDS degradation) was estimated in crude cell extracts and multiplicity of alkyl sulfatase was studied by Native PAGE Zymography. The potent isolate was identified by 16S rRNA sequence analysis. Using enrichment technique in minimal medium containing SDS as a sole carbon source, initially three SDS degrading isolates were recovered. However, only one isolate, SP3, was found to be an efficient degrader of SDS. It was observed that this strain could completely metabolize 0.1% SDS in 16 h, 0.2% SDS in 20 h and 0.3% SDS in 24 h of incubation. Specific activity of alkyl sulfatase was 0.087±0.004 µmol SDS/mg protein/min and Native PAGE Zymography showed presence of alkyl sulfatase of Rf value of 0.21. This isolate was identified as Pseudomonas putida strain SP3. This is the report of isolation of SDS-degrading strain of P. putida, which shows high rate of SDS degradation and can degrade up to 0.3% SDS. It appears that this isolate can be exploited for bioremediation of this detergent from water systems.

  13. Metabolic and Regulatory Rearrangements Underlying Efficient d-Xylose Utilization in Engineered Pseudomonas putida S12*

    Science.gov (United States)

    Meijnen, Jean-Paul; de Winde, Johannes H.; Ruijssenaars, Harald J.

    2012-01-01

    Previously, an efficient d-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the d-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor d-xylose utilization was the redistribution of 6-phospho-d-gluconate (6-PG) between the Entner-Doudoroff pathway and the oxidative pentose phosphate (PP) pathway. This redistribution increased the availability of 6-PG for oxidative decarboxylation to d-ribose-5-phosphate, which is essential for the utilization of d-xylose via the nonoxidative PP pathway. The metabolic redistribution of 6-PG was procured by modified HexR regulation, which in addition appeared to control periplasmic sugar oxidation. Because the absence of periplasmic d-xylonate formation was previously demonstrated to be essential for achieving a high biomass yield on d-xylose, the aberrant HexR control appeared to underlie both the improved growth rate and biomass yield of the evolved d-xylose utilizing P. putida strain. The increased oxidative PP pathway activity furthermore resulted in an elevated NADH/NAD+ ratio that caused the metabolic flux to be redirected from the TCA cycle to the glyoxylate shunt, which was also activated transcriptionally. Clearly, these findings may serve as an important case in point to engineer and improve the utilization of non-natural carbon sources in a wide range of industrial microorganisms. PMID:22416130

  14. The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4.

    Science.gov (United States)

    Chen, Qiongzhen; Tu, Hui; Luo, Xue; Zhang, Biying; Huang, Fei; Li, Zhoukun; Wang, Jue; Shen, Wenjing; Wu, Jiale; Cui, Zhongli

    2016-01-01

    Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2), para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR). It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

  15. The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4.

    Directory of Open Access Journals (Sweden)

    Qiongzhen Chen

    Full Text Available Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2, para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR. It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

  16. Proteomic Analysis of Pseudomonas putida Reveals an Organic Solvent Tolerance-Related Gene mmsB

    Science.gov (United States)

    Ni, Ye; Song, Liang; Qian, Xiaohong; Sun, Zhihao

    2013-01-01

    Organic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD660, whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications. PMID:23409067

  17. Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge

    Directory of Open Access Journals (Sweden)

    McCarthy Andrea T

    2010-05-01

    Full Text Available Abstract Background Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI [Cr(VI], the proteome of aerobically grown, Cr(VI-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB media and minimal (M9L media containing lactate as the sole carbon source. Results Growth studies demonstrated that F1 sensitivity to Cr(VI was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI conditions. Conclusions Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion

  18. Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge.

    Science.gov (United States)

    Thompson, Dorothea K; Chourey, Karuna; Wickham, Gene S; Thieman, Stephanie B; VerBerkmoes, Nathan C; Zhang, Bing; McCarthy, Andrea T; Rudisill, Matt A; Shah, Manesh; Hettich, Robert L

    2010-05-19

    Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source. Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose > acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions. Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport

  19. Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440

    Science.gov (United States)

    2013-01-01

    Background Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations. Results Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a PrpoS → rpoS‘-’lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria. Conclusions While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and

  20. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Science.gov (United States)

    2012-01-01

    Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l) with relative decolorization values of 91.2% (3 h) and 97.1% (18 h), as well as high activity to AR18 (1 g/l) by 80.5% (3 h) and 89.0% (18 h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h

  1. Effects of Cobalt on Manganese Oxidation by Pseudomonas putida MnB1

    Science.gov (United States)

    Pena, J.; Bargar, J.; Sposito, G.

    2005-12-01

    The oxidation of Mn(II) in the environment is thought to occur predominantly through biologically mediated pathways. During the stationary phase of growth, the well-characterized freshwater and soil bacterium Pseudomonas putida MnB1 oxidizes soluble Mn(II) to a poorly crystalline layer type Mn(IV) oxide. These Mn oxide particles (2 - 5 nm thickness) are deposited in a matrix of extracellular polymeric substances (EPS) surrounding the cell, creating a multi-component system distinct from commonly studied synthetic Mn oxides. Accurate characterization of the reactivity of these biomineral assemblages is essential to understanding trace metal biogeochemistry in natural waters and sediments. Moreover, these biogenic oxides may potentially be used for the remediation of surface and ground waters impacted by mining, industrial pollution, and other anthropogenic activities. In this study, we consider the interactions between Co, P. putida MnB1, and its biogenic Mn oxide. Cobalt is a redox-active transition metal which exists in the environment as Co(II) and Co(III). While Co is not generally found in the environment at toxic concentrations, it may be released as a byproduct of mining activities (e.g. levels of up to 20 μM are found in Pinal Creek, AZ, a stream affected by copper mining). In addition, the radionuclide 60Co, formed by neutron activation in nuclear reactors, is of concern at Department of Energy sites, such as that at Hanford, and has several industrial applications, including radiotherapy. We address the following questions: Do high levels of Co inhibit enzymatic processes such as Mn(II) oxidation? Can the multicopper oxidase enzyme involved in Mn(II) oxidation facilitate Co(II) oxidation? Lastly, does the organic matter surrounding the oxides affect Co or Mn oxide reactivity? These issues were approached via wet chemical analysis, synchrotron radiation X-ray diffraction (SR-XRD), and extended X-ray absorption fine structure (EXAFS) spectroscopy. In the

  2. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  3. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

    Science.gov (United States)

    Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

    2017-04-01

    Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one

  4. Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440.

    Science.gov (United States)

    Nikel, Pablo I; Chavarría, Max; Martínez-García, Esteban; Taylor, Anne C; de Lorenzo, Víctor

    2013-05-20

    Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations. Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd(2+) and Cu(2+)). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a PrpoS → rpoS'-'lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria. While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and biochemical networks that

  5. Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach.

    Science.gov (United States)

    Cao, Bin; Loh, Kai-Chee

    2008-12-15

    The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.

  6. Antioxidative enzyme profiling and biosorption ability of Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 under cadmium stress.

    Science.gov (United States)

    Shamim, Saba; Rehman, Abdul

    2015-03-01

    Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd)-resistant and -sensitive bacteria, respectively, to study their biosorption ability and their antioxidative enzymes. The minimal inhibitory concentration of C. metallidurans CH34 for Cd was found to be 30 mM, and for P. putida mt2 it was 1.25 mM. The tube dilution method revealed the heavy-metal resistance pattern of C. metallidurans CH34 as Ni(2+) (10 mM)>Zn(2+) (4 mM)>Cu(2+) (2 mM)>Hg(2+) (1 mM)>Cr(2+) (1 mM)>Pb(2+) (0 mM), whereas P. putida mt2 was only resistant to Zn(2+) (1 mM). Under Cd stress, the induction of GSH was higher in C. metallidurans CH34 (0.359 ± 0.010 mM g(-1)  FW) than in P. putida mt2 (0.286 ± 0.005 mM g(-1)  FW). Glutathione reductase was more highly expressed in the mt2 strain, in contrast to non-protein thiols and peroxidase. Unlike dead bacterial cells, live cells of both bacteria showed significant Cd biosorption, i.e. more than 80% at 48 h. C. metallidurans CH34 used only catalase, whereas P. putida mt2 used superoxide dismutase and ascorbate peroxidase to combat Cd stress. This study investigated the Cd biosorption ability and enzymes involved in the Cd detoxification mechanisms of C. metallidurans CH34 and P. putida mt2. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Synthesis and Physical Properties of Polyhydroxyalkanoate Polymers with Different Monomer Compositions by Recombinant Pseudomonas putida LS46 Expressing a Novel PHA SYNTHASE (PhaC116 Enzyme

    Directory of Open Access Journals (Sweden)

    Parveen K. Sharma

    2017-03-01

    Full Text Available A recombinant of Pseudomonas putida LS461 (deletion of the phaC1phaZphaC2 genes was constructed by introducing cosmid JC123 carrying a novel phaC116 gene from a metagenomic clone. The resulting strain, P. putida LS46123, was able to synthesize polyhydroxyalkanoate (PHA polymers with novel monomer compositions when cultured on glucose or free fatty acids, and accumulated PHAs from 9.24% to 27.09% of cell dry weight. The PHAs synthesized by P. putida LS46123 contained up to 50 mol % short chain length subunits (3-hydroxybutyrate and 3-hydroxyvalerate, with the remaining monomers consisting of various medium chain length subunits. The PhaC116 protein expressed by P. putida LS46123 had an amino acid sequence similarity of 45% with the PhaC1 protein of the parent strain, P. putida LS46. Predicted 3D structures of the PhaC116 proteins from P. putida LS46123 and P. putida LS46 revealed several differences in the numbers and locations of protein secondary structures. The physical and thermal properties of the novel polymers synthesized by P. putida LS46123 cultured with glucose or free fatty acids differed significantly from those produced by P. putida LS46 grown on the same substrates. PHA polymers with different subunit compositions, and hence different physical and thermal properties, can be tailor-made using novel PHA synthase for specific applications.

  8. Draft Genome Sequence Analysis of a Pseudomonas putida W15Oct28 Strain with Antagonistic Activity to Gram-Positive and Pseudomonas sp. Pathogens

    Science.gov (United States)

    Ye, Lumeng; Hildebrand, Falk; Dingemans, Jozef; Ballet, Steven; Laus, George; Matthijs, Sandra; Berendsen, Roeland; Cornelis, Pierre

    2014-01-01

    Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors. PMID:25369289

  9. Screening, identification and culture optimization of a newly isolated aromatic nitrilase-producing bacterium--Pseudomonas putida CGMCC3830.

    Science.gov (United States)

    Zhu, Xiaoyan; Gong, Jinsong; Li, Heng; Lu, Zhenming; Zhou, Zhemin; Shi, Jinsong; Xu, Zhenghong

    2014-03-01

    Microbial nitrilases have attracted increasing attention in nitrile hydrolysis for carboxylic acid production in recent years. A bacterium with nitrilase activity was isolated and identified as Pseudomonas putida CGMCC3830 based on its morphology, physiological and biochemical characteristics, as well as 16S rRNA gene sequence. The nitrilase production was optimized by varying culture conditions using the one-factor-at-a-time method and response surface methodology. Glycerol 13.54 g/L, tryptone 11.59 g/L, yeast extract 5.21 g/L, KH2PO4 1 g/L, NaCl 1 g/L, urea 1 g/L, initial pH 6.0 and culture temperature 30 degrees C were proved to be the optimal culture conditions. It resulted in the maximal nitrilase production of 36.12 U/mL from 2.02 U/mL. Investigations on substrate specificity demonstrate P. putida nitrilase preferentially hydrolyze aromatic nitriles. When applied in nicotinic acid synthesis, 2 mg/mL P. putida cells completely hydrolyzed 20.8 g/L 3-cyanopyridine into nicotinic acid in 90 min. The results indicated P. putida CGMCC3830 displayed potential for industrial production of nicotinic acid.

  10. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production

    Science.gov (United States)

    Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2014-01-01

    As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

  11. Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions.

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    Anatoly S Glukhov

    Full Text Available The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure--a blunt right end and a 4-nucleotide 3'-protruding left end--was observed. Secondly, 14 single-chain interruptions (nicks were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5'-TACT/RTGMC-3'. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

  12. Evaluation of Zosteric Acid for Mitigating Biofilm Formation of Pseudomonas putida Isolated from a Membrane Bioreactor System

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    Andrea Polo

    2014-05-01

    Full Text Available This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (−97% and thickness (−50%, and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

  13. A role for the regulator PsrA in the polyhydroxyalkanoate metabolism of Pseudomonas putida KT2440.

    Science.gov (United States)

    Fonseca, Pilar; de la Peña, Fernando; Prieto, María Auxiliadora

    2014-11-01

    Pseudomonas putida KT2440 is a Gram-negative bacterium capable of producing medium-chain-length-polyhydroxyalkanoates (mcl-PHA). When fatty acids are used as growth and polymer precursors, the biosynthesis is linked to fatty acid metabolism via ß-oxidation route. In the close-related Pseudomonas aeruginosa, the transcriptional repressor PsrA regulates the ß-oxidation, but little is known about the regulatory system in P. putida. To analyze the effect of the absence of psrA gene on the growth and PHA production in P. putida, a set of different carbon sources were assayed in the wild type strain and in a generated psrA deficient strain (KT40P). The growth rates were in all cases, lower for the mutant. The amount of PHA produced by the mutant strain is lower than the wild type. Moreover, the monomeric composition seems to be different among the strains, as there is enrichment in monomers with shorter carbon length in the mutant strain. To understand the role of the psrA gene on the metabolism of fatty acids, we have determined the expression profile of several genes related to fatty acid metabolism in the wild type and in the mutant strain. The results indicated that PsrA mostly negatively regulate genes related to fatty acid metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Enhanced biodegradation of mixed phenol and sodium salicylate by Pseudomonas putida in membrane contactors.

    Science.gov (United States)

    Juang, Ruey-Shin; Tsai, Shang-Yuan

    2006-11-01

    A polypropylene (PP) hollow fiber membrane contactor was used as a reactor to enhance the biodegradation of equimolar phenol and sodium salicylate (SA) by Pseudomonas putida CCRC 14365 at 30 degrees C and pH 7. Experiments were performed at a fixed initial cell density of 0.025 g/L and in the total substrate level range 5.32-63.8 mM. The degradation experiments by free cells were also studied for comparison. With pristine hydrophobic fibers, the degradation of SA was started only after phenol was completely consumed. Substrate inhibitory effect was avoided due to sufficiently low substrate levels in the cell medium; however, the biodegradation was time consuming. With ethanol-wetted fibers, both substrates were completely degraded much faster than the use of pristine fibers. Although the wetted fibers were unable to prevent movement of substrates through the pores, biofilm formed on the outer surfaces of the fibers could enhance the tolerance limit of substrate toxicity. This greatly extended the treatment range to high-level substrate mixtures, as long as the water was nearly neutral and free of concentrated inorganic salts.

  15. Dependence of toxicity of silver nanoparticles on Pseudomonas putida biofilm structure.

    Science.gov (United States)

    Thuptimdang, Pumis; Limpiyakorn, Tawan; Khan, Eakalak

    2017-12-01

    Susceptibility of biofilms with different physical structures to silver nanoparticles (AgNPs) was studied. Biofilms of Pseudomonas putida KT2440 were formed in batch conditions under different carbon sources (glucose, glutamic acid, and citrate), glucose concentrations (5 and 50 mM), and incubation temperatures (25 and 30 °C). The biofilms were observed using confocal laser scanning microscopy for their physical characteristics (biomass amount, thickness, biomass volume, surface to volume ratio, and roughness coefficient). The biofilms forming under different growth conditions exhibited different physical structures. The biofilm thickness and the roughness coefficient were found negatively and positively correlated with the biofilm susceptibility to AgNPs, respectively. The effect of AgNPs on biofilms was low (1-log reduction of cell number) when the biofilms had high biomass amount, high thickness, high biomass volume, low surface to volume ratio, and low roughness coefficient. Furthermore, the extracellular polymeric substance (EPS) stripping process was applied to confirm the dependence of susceptibility to AgNPs on the structure of biofilm. After the EPS stripping process, the biofilms forming under different conditions showed reduction in thickness and biomass volume, and increases in surface to volume ratio and roughness coefficient, which led to more biofilm susceptibility to AgNPs. The results of this study suggest that controlling the growth conditions to alter the biofilm physical structure is a possible approach to reduce the impact of AgNPs on biofilms in engineered and natural systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Electricity Generation and Wastewater Treatment of Oil Refinery in Microbial Fuel Cells Using Pseudomonas putida

    Science.gov (United States)

    Majumder, Dip; Maity, Jyoti Prakash; Tseng, Min-Jen; Nimje, Vanita Roshan; Chen, Hau-Ren; Chen, Chien-Cheng; Chang, Young-Fo; Yang, Tsui-Chu; Chen, Chen-Yen

    2014-01-01

    Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm2 in the third cycle with a maximum current density of 0.015 mA/cm2 in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10−2% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation. PMID:25247576

  17. Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

    Science.gov (United States)

    Isken, Sonja; Derks, Antoine; Wolffs, Petra F. G.; de Bont, Jan A. M.

    1999-01-01

    Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells. PMID:10347053

  18. Electricity Generation and Wastewater Treatment of Oil Refinery in Microbial Fuel Cells Using Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Dip Majumder

    2014-09-01

    Full Text Available Microbial fuel cells (MFCs represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059, a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm chemical oxygen demand (COD could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm2 in the third cycle with a maximum current density of 0.015 mA/cm2 in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10−2% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation.

  19. Daya Tahan Hidup Pseudomonas putida Strain Pf-20 dalam Beberapa Macam Inokulum

    Directory of Open Access Journals (Sweden)

    Yenny Wuryandari

    2004-07-01

    Full Text Available For any crop-protection agent, an efficient formulation is a necessity to translate laboratory activity into adequate field performance. There are particular challenges to be faced in formulation of biological control agents, because the active ingredient is a living organism that must be kept relatively immobile and inactive while in storage, but quickly resume its  normal metabolic processes once applied to the target site. The objective of the research was  to study survival of Pseudomonas putida strain Pf-20 in various formulations at the storage  time and germination. The twelve formulations include carriers, additives and concentration  of Pf-20. The efficacy of various formulation in maintaining the population of Pf-20 in storage was assessed. The research result showed that population of Pf 20  in the formulation  number seven was the highest, with the combination peat+talc, CMC+arginin and  concentration of Pf[20 10^10 CFU/ml. In peat+talc, CMC+arginin, Pf-20 10^10  CFU/ml based formulation the bacteria survived even up to 84 days of storage although the population declined. In all formulations, population of Pf-20 increased at the time of seed germination. At the time of seed germination, formulation number seven was the highest too.

  20. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity

    Energy Technology Data Exchange (ETDEWEB)

    Thuptimdang, Pumis, E-mail: pumis.th@gmail.com [International Program in Hazardous Substance and Environmental Management, Graduate School, Chulalongkorn University, Bangkok 10330 (Thailand); Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Limpiyakorn, Tawan, E-mail: tawan.l@chula.ac.th [Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Department of Environmental Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Research Unit Control of Emerging Micropollutants in Environment, Chulalongkorn University, Bangkok 10330 (Thailand); McEvoy, John, E-mail: john.mcevoy@ndsu.edu [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Prüß, Birgit M., E-mail: birgit.pruess@ndsu.edu [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Khan, Eakalak, E-mail: eakalak.khan@ndsu.edu [Department of Civil and Environmental Engineering, North Dakota State University, Fargo, ND 58108 (United States)

    2015-06-15

    Highlights: • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs. - Abstract: This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

  1. In vivo role of sulfite in photocontrol of urocanase from Pseudomonas putida

    International Nuclear Information System (INIS)

    Venema, R.C.; Hunter, J.K.; Hug, D.H.

    1985-01-01

    Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and is activated by UV radiation. Sulfite addition to the bound nicotinamide adenine dinucleotide coenzyme has previously been shown to inactivate the enzyme in vitro. The enzyme released sulfite upon photoactivation. Whether sulfite addition and dissociation is involved in the in vivo photoregulation of urocanase was examined. The dark reversion (inactivation) in cultures was markedly enhanced by growth at 32 0 C rather than at 24 0 C; cells grown at 32 0 C and resting cells were used to obtain in vivo-inactivated urocanase. The in vivo-inactivated enzyme was purified and the amount of sulfite released through photodissociation quantitated. One mole of sulfite was released per mole of urocanase. This is based on a molecular weight of 110 000 confirmed by gel electrophoresis and a protein estimation method. A previous report of sulfide inactivation of urocanase in vitro is now shown to have been mistaken; the inactivation resulted from the oxidation of sulfide to sulfite, which occurred in solution. (author)

  2. An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440.

    Science.gov (United States)

    Wang, Yuanyuan; Zhang, Chi; Gong, Ting; Zuo, Zhenqiang; Zhao, Fengjie; Fan, Xu; Yang, Chao; Song, Cunjiang

    2015-06-01

    A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA(MCL))-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P. mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA(MCL) depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P. mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA(MCL) polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P. putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp-based counterselection system could be further adapted for P. mendocina NK-01 and P. putida KT2440 and used for genome reduction and metabolic pathway engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The novel Pseudomonas putida plasmid p12969-2 harbors an In127-carrying multidrug-resistant region.

    Science.gov (United States)

    Xu, Yang; Niu, Yong; Sun, Fengjun; Yang, Ying; Luo, Wenbo; Wang, Zhe

    2017-06-01

    This study aims to characterize a multidrug-resistant (MDR) plasmid p12969-2 coexistent with the previously reported one p12969-DIM in clinical Pseudomonas putida. The complete sequence of p12969-2 was determined using next-generation sequencing technology. p12969-2 contains a 29.2 kb MDR region, which carries In127 harboring three resistance genes aadA2, qacED1 and sul1. The MDR region is derived from the connection of Tn5041D and Tn5045, which is facilitated by two copies of miniature inverted-repeat transposable element. Conclusion & future perspective: p12969-2 represents a novel lineage with the highest but limited nucleotide sequence similarity with the plasmid pGRT1 that does not carry any of the resistance genes. This is the first report of coexistence of two MDR plasmids in P. putida.

  4. The contribution of proteomics to the unveiling of the survival strategies used by Pseudomonas putida in changing and hostile environments.

    Science.gov (United States)

    Moreno, Renata; Rojo, Fernando

    2013-10-01

    Pseudomonas putida is a ubiquitous, metabolically very versatile, Gram-negative bacterium adapted to habitats as diverse as soil, water and the rhizosphere. Most strains are nonpathogenic, many are used as experimental models, and many others have biotechnological applications in the areas of agriculture, bioremediation, biocatalysis, and the production of bioplastics. This review summarizes the contribution of proteomic technologies to our understanding of how P. putida responds to different carbon sources, how it adapts to living at suboptimal temperatures or attached to surfaces, and how it responds to the presence of toxic compounds such as aromatic molecules and heavy metals. The examples described illustrate the value of proteomics in furthering our knowledge of the physiology and behavior of bacteria, knowledge that is important for understanding how they behave in their natural habitats and for optimizing their behavior in biotechnological applications. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital.

    Science.gov (United States)

    Lee, Kyungwon; Lim, Jong Back; Yum, Jong Hwa; Yong, Dongeun; Chong, Yunsop; Kim, June Myung; Livermore, David M

    2002-04-01

    We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4. bla(VIM) also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.

  6. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440.

    Science.gov (United States)

    Elmore, Joshua R; Furches, Anna; Wolff, Gara N; Gorday, Kent; Guss, Adam M

    2017-12-01

    Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli , the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Further, most heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. To improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, -35 sequences, and -10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. An additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.

  7. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440

    Energy Technology Data Exchange (ETDEWEB)

    Elmore, Joshua R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Furches, Anna [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Wolff, Gara N. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Gorday, Kent [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division

    2017-04-15

    Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Furthermore, heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. In order to improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, -35 sequences, and -10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. One additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.

  8. Enhanced cadmium resistance and accumulation in Pseudomonas putida KT2440 expressing the phytochelatin synthase gene of Schizosaccharomyces pombe.

    Science.gov (United States)

    Yong, X; Chen, Y; Liu, W; Xu, L; Zhou, J; Wang, S; Chen, P; Ouyang, P; Zheng, T

    2014-03-01

    Phytochelatins (PCs) are cysteine-rich peptides with high binding affinity for toxic metals. Expressing the PC synthase gene (PCS) in plant growth-promoting bacteria may enhance its metal resistance and accumulation, consequently increasing phytoremediation efficiency in heavy metal pollution. In this study, PCS from Schizosaccharomyces pombe was cloned and expressed in Pseudomonas putida KT2440, which was confirmed by real-time RT-PCR through an increase in SpPCS mRNA expression level when induced by 20 μmol of CdCl2 in the transformed Ps. putida cells. The recombined strain KT2440-SpPCS exhibited enhanced Cd, Ag and Hg resistance. Compared with the original strain, KT2440-SpPCS also displayed a threefold to fivefold increase in Cd accumulation (14·32 μmol g(-1) to 17·38 μmol g(-1) ; dry weight) when grown in 30 and 50 μmol CdCl2 , along with an increase in nonprotein thiols. Further experiments showed significantly enhanced germination rates and growth of wheat seeds in 0·1 mmol to 1·0 mmol Cd when inoculated with KT2440-SpPCS. This study shows potential use of Ps. putida KT2440-SpPCS in plants to construct a symbiotic system for an enhanced phytoremediation of heavy metal-contaminated environments. The symbiotic system of using plant growth-promoting bacteria Pseudomonas putida to express phytochelatin synthase gene of Schizosaccharomyces pombe together in plants resulted in high heavy metal resistance and high accumulation capacity, suggesting potential enhancement in phytoremediation of heavy metal-contaminated environments. © 2013 The Society for Applied Microbiology.

  9. Effects of Pseudomonas putida and Glomusintraradices Inoculations on Morphological and Biochemical Traitsin Trigonellafoenum-graecum L.

    Directory of Open Access Journals (Sweden)

    simin irankhah

    2017-02-01

    Full Text Available Introduction: Fenugreek (Trigonellafoenum-graecum L. is a traditional medicinal plant belonging to the legume family Fabaceae. Diverse groups of microorganisms are symbiotic with Fenugreek roots system. This integration leads to significant increases in the development and production by increasing nitrogen fixation, phytohormones production, siderophores and phosphate solubilization. Plant growth-promoting bacteria increase plant growth byimproving nutrientuptake and phytohormones production. In addition, the beneficial effect of these bacteria could be due totheirinteractionwithArbuscularMycorrhizal fungi(VAM. Drought is one of the major limiting factors for crop production in many parts of the world including Iran. Symbiotic microorganisms can enhance plant tolerance to drought. This experiment was carried out to investigate the effect of Vesicular ArbuscularMycorrhiza (VAM and Plant Growth Promoting Rhizobacteria (PGPR on morphological and biochemical characteristics of Fenugreek in drought stress conditions. Materials and Methods: The experiment was carried out in completely random design with 3 replications.There were four treatments including inoculation with Pseudomonas putida, inoculation with Glomusintraradices, combined association of Pseudomonas putida and Glomusintraradices and untreated as a check under drought stress (40% of field capacity and non-stress conditions (80% of field capacity. In this experiment fiveseeds were sowninplastic pots. Before sowing, seeds were inoculated with microorganisms. In order to inoculation ofseed with Mycorrhizal fungi, for each kilogram of soil, 100 grams of powder containing 10 to 15 thousand spores of fungal soil (produced by the biotech company Toos was added to three centimeters of soil in the pot. For seed inoculation with Plant Growth Promoting Rhizobacteria, the growth curve of the bacteria was drawn at first and then the best time for the growth of bacteria was determined. The bacteria at

  10. Global regulation of food supply by Pseudomonas putida DOT-T1E.

    Science.gov (United States)

    Daniels, Craig; Godoy, Patricia; Duque, Estrella; Molina-Henares, M Antonia; de la Torre, Jesús; Del Arco, José María; Herrera, Carmen; Segura, Ana; Guazzaroni, M Eugenia; Ferrer, Manuel; Ramos, Juan Luis

    2010-04-01

    Pseudomonas putida DOT-T1E was used as a model to develop a "phenomics" platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Deltacrc, Deltacrp, DeltacyoB, and DeltaptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine

  11. Characterization of a novel blaIMP gene, blaIMP-58, using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark

    DEFF Research Database (Denmark)

    Holmgaard, Dennis Back; Hansen, Frank; Hasman, Henrik

    2017-01-01

    A multidrug-resistant strain of Pseudomonas putida was isolated from the urine of a 65-year-old women hospitalized for serious clinical conditions. Using whole genome sequencing a novel blaIMP gene, blaIMP-58 was discovered and characterized.......A multidrug-resistant strain of Pseudomonas putida was isolated from the urine of a 65-year-old women hospitalized for serious clinical conditions. Using whole genome sequencing a novel blaIMP gene, blaIMP-58 was discovered and characterized....

  12. Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants.

    Science.gov (United States)

    Planchamp, Chantal; Glauser, Gaetan; Mauch-Mani, Brigitte

    2014-01-01

    Pseudomonas putida KT2440 (KT2440) rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots) and systemic (leaves) early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots 3 days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR) against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal growth in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as resistance inducers.

  13. Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants

    Directory of Open Access Journals (Sweden)

    Chantal ePlanchamp

    2015-01-01

    Full Text Available Pseudomonas putida KT2440 (KT2440 rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots and systemic (leaves early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots three days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal development in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as

  14. The Identification and Validation of Novel Small Proteins in Pseudomonas Putida KT-2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Long, Katherine

    2014-01-01

    and activities and may lead to the discovery of novel antimicrobial agents. Our project focuses on the identification, validation and characterization of novel s-­‐proteins in the bacterium Pseudomonas putida KT-­2440. As there is virtually no information on s-­‐proteins in pseudomonads, the first step......, total protein samples are prepared, fractionated, and analyzed with mass spectrometry (MS/MS). The MS/MS data are compared to a custom database containing >80000 putative sORF sequences to identify candidates for validation. A total of 56 and 22 putative sORFs were obtained from MS/MS data...

  15. Pseudomonas putida Strain FStm2 Isolated from Shark Skin: A Potential Source of Bacteriocin.

    Science.gov (United States)

    Ahmad, Asmat; Hamid, Rahimi; Dada, Ayokunle Christopher; Usup, Gires

    2013-09-01

    Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO4 and MnSO4 were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30-70 °C. Bacteriocin activity also was not affected within a wide pH range of 3-9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.

  16. Conversion and assimilation of furfural and 5-(hydroxymethylfurfural by Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Michael T. Guarnieri

    2017-06-01

    Full Text Available The sugar dehydration products, furfural and 5-(hydroxymethylfurfural (HMF, are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethylfurfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

  17. Identification and characterization of the PhhR regulon in Pseudomonas putida.

    Science.gov (United States)

    Herrera, M Carmen; Duque, Estrella; Rodríguez-Herva, José J; Fernández-Escamilla, Ana M; Ramos, Juan L

    2010-06-01

    Pseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p-hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl-CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR-proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl-CoA. A number of mutants in the paa genes encoding phenylacetyl-CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad-substrate-specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine-dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.

  18. Two distinct pathways for metabolism of theophylline and caffeine are coexpressed in Pseudomonas putida CBB5.

    Science.gov (United States)

    Yu, Chi Li; Louie, Tai Man; Summers, Ryan; Kale, Yogesh; Gopishetty, Sridhar; Subramanian, Mani

    2009-07-01

    Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.

  19. From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida.

    Science.gov (United States)

    Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2014-07-01

    Although DNA is the ultimate repository of biological information, deployment of its instructions is constrained by the metabolic and physiological status of the cell. To this end, bacteria have evolved intricate devices that connect exogenous signals (e.g. nutrients, physicochemical conditions) with endogenous conditions (metabolic fluxes, biochemical networks) that coordinately influence expression or performance of a large number of cellular functions. The phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) is a bacterial multi-protein phosphorylation chain which computes extracellular (e.g. sugars) and intracellular (e.g. phosphoenolpyruvate, nitrogen) signals and translates them into post-translational regulation of target activities through protein-protein interactions. The PTS of Pseudomonas putida KT2440 encompasses one complete sugar (fructose)-related system and the three enzymes that form the so-called nitrogen-related PTS (PTS(N) (tr) ), which lacks connection to transport of substrates. These two PTS branches cross-talk to each other, as the product of the fruB gene (a polyprotein EI-HPr-EIIA) can phosphorylate PtsN (EIIA(N) (tr) ) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIA(N) (tr) ) which, in turn, checks the activity of key metabolic and regulatory proteins. Such a control of bacterial physiology highlights the prominence of biochemical homeostasis over genetic ruling -and not vice versa. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  20. Metabolic Value Chemoattractants Are Preferentially Recognized at Broad Ligand Range Chemoreceptor of Pseudomonas putida KT2440

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    Matilde Fernández

    2017-05-01

    Full Text Available Bacteria have evolved a wide range of chemoreceptors with different ligand specificities. Typically, chemoreceptors bind ligands with elevated specificity and ligands serve as growth substrates. However, there is a chemoreceptor family that has a broad ligand specificity including many compounds that are not of metabolic value. To advance the understanding of this family, we have used the PcaY_PP (PP2643 chemoreceptor of Pseudomonas putida KT2440 as a model. Using Isothermal Titration Calorimetry we showed here that the recombinant ligand binding domain (LBD of PcaY_PP recognizes 17 different C6-ring containing carboxylic acids with KD values between 3.7 and 138 μM and chemoeffector affinity correlated with the magnitude of the chemotactic response. Mutation of the pcaY_PP gene abolished chemotaxis to these compounds; phenotype that was restored following gene complementation. Growth experiments using PcaY_PP ligands as sole C-sources revealed functional relationships between their metabolic potential and affinity for the chemoreceptor. Thus, only 7 PcaY_PP ligands supported growth and their KD values correlated with the length of the bacterial lag phase. Furthermore, PcaY_PP ligands that did not support growth had significantly higher KD values than those that did. The receptor has thus binds preferentially compounds that serve as C-sources and amongst them those that rapidly promote growth. Tightest binding compounds were quinate, shikimate, 3-dehydroshikimate and protocatechuate, which are at the interception of the biosynthetic shikimate and catabolic quinate pathways. Analytical ultracentrifugation studies showed that ligand free PcaY_PP-LBD is present in a monomer-dimer equilibrium (KD = 57.5 μM. Ligand binding caused a complete shift to the dimeric state, which appears to be a general feature of four-helix bundle LBDs. This study indicates that the metabolic potential of compounds is an important parameter in the molecular recognition

  1. Phenol biodegradation by immobilized Pseudomonas putida FNCC-0071 cells in alginate beads

    Science.gov (United States)

    Hakim, Lukman Nul; Rochmadi, Sutijan

    2017-06-01

    Phenol is one of industrial liquid waste which is harmful to the environment, so it must be degraded. It can be degraded by immobilized Pseudomonas putida FNCC-0071 cells. It needs the kinetics and mass transfer data to design this process which can be estimated by the proposed dynamic model in this study. This model involves simultaneous diffusion and reaction in the alginate bead and liquid bulk. The preliminary stage of phenol biodegradation process was acclimatization cells. This is the stage where cells were acclimated to phenol as carbon source (substrate). Then the acclimated cells were immobilized in alginate beads by extrusion method. The variation of the initial phenol concentration in the solution is 350 to 850 ppm where 60 g alginate bead contained by cells loaded into its solution in reactor batch, so then biodegradation occurs. In this study, the average radius of alginate bead was 0.152 cm. The occurred kinetic reaction process can be explained by Blanch kinetic model with the decreasing of parameter μmax' while the increasing values of initial phenol concentration in the same time, but the parameters KM, KM', and kt were increasing by the rising values of initial phenol concentration. The value of the parameter β is almost zero. Effective diffusivity of phenol and cells are 1.11 × 10-5±4.5% cm2 s-1 and 1.39 × 10-7± 0.04% cm2 s-1. The partition coefficient of phenol and cells are 0.39 ± 15% and 2.22 ± 18%.

  2. Metabolite profiling reveals abiotic stress tolerance in Tn5 mutant of Pseudomonas putida.

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    Vasvi Chaudhry

    Full Text Available Pseudomonas is an efficient plant growth-promoting rhizobacteria (PGPR; however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5 mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR spectroscopy and gas chromatography-mass spectrometry (GC-MS. Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic

  3. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

    Science.gov (United States)

    Gong, Ting; Xu, Xiaoqing; Che, You; Liu, Ruihua; Gao, Weixia; Zhao, Fengjie; Yu, Huilei; Liang, Jingnan; Xu, Ping; Song, Cunjiang; Yang, Chao

    2017-08-01

    An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP + ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

  4. Bioconversion of styrene to poly(hydroxyalkanoate) (PHA) by the new bacterial strain Pseudomonas putida NBUS12.

    Science.gov (United States)

    Tan, Giin-Yu Amy; Chen, Chia-Lung; Ge, Liya; Li, Ling; Tan, Swee Ngin; Wang, Jing-Yuan

    2015-01-01

    Styrene is a toxic pollutant commonly found in waste effluents from plastic processing industries. We herein identified and characterized microorganisms for bioconversion of the organic eco-pollutant styrene into a valuable biopolymer medium-chain-length poly(hydroxyalkanoate) (mcl-PHA). Twelve newly-isolated styrene-degrading Pseudomonads were obtained and partial phaC genes were detected by PCR in these isolates. These isolates assimilated styrene to produce mcl-PHA, forming PHA contents between 0.05±0.00 and 23.10±3.25% cell dry mass (% CDM). The best-performing isolate was identified as Pseudomonas putida NBUS12. A genetic analysis of 16S rDNA and phaZ genes revealed P. putida NBUS12 as a genetically-distinct strain from existing phenotypically-similar bacterial strains. This bacterium achieved a final biomass of 1.28±0.10 g L(-1) and PHA content of 32.49±2.40% CDM. The extracted polymer was mainly comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanoate (C8 ), 3-hydroxydecanoate (C10 ), 3-hydroxydodecanoate (C12 ), and 3-hydroxytetradecanoate (C14 ) monomers at a ratio of 2:42:1257:17:1. These results collectively suggested that P. putida NBUS12 is a promising candidate for the biotechnological conversion of styrene into mcl-PHA.

  5. Protein as chemical cue: non-nutritional growth enhancement by exogenous protein in Pseudomonas putida KT2440.

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    Hiren Joshi

    Full Text Available Research pertaining to microbe-microbe and microbe-plant interactions has been largely limited to small molecules like quorum sensing chemicals. However, a few recent reports have indicated the role of complex molecules like proteins and polysaccharides in microbial communication. Here we demonstrate that exogenous proteins present in culture media can considerably accelerate the growth of Pseudomonas putida KT2440, even when such proteins are not internalized by the cells. The growth enhancement is observed when the exogenous protein is not used as a source of carbon or nitrogen. The data show non-specific nature of the protein inducing growth; growth enhancement was observed irrespective of the protein type. It is shown that growth enhancement is mediated via increased siderophore secretion in response to the exogenous protein, leading to better iron uptake. We highlight the ecological significance of the observation and hypothesize that exogenous proteins serve as chemical cues in the case of P.putida and are perceived as indicator of the presence of competitors in the environment. It is argued that enhanced siderophore secretion in response to exogenous protein helps P.putida establish numerical superiority over competitors by way of enhanced iron assimilation and quicker utilization of aromatic substrates.

  6. Lead Sorption from Aqueous Solutions by Pseudomonas putida (p168 and its Composites with Palygorskite and Sepiolite Clays

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    Marzieh tavanaei

    2017-02-01

    Full Text Available Introduction: Heavy metals contamination due to natural and anthropogenic sources is a global environmental concern. Lead (Pb is one of the very toxic heavy metals. Industrial production processes and their emissions, mining operation, smelting, combustion sources and solid waste incinerators are the primary sources of lead. This heavy metal has aberrant effects on the environment and living organisms. Hence, proper treatment of lead from soil and industrial wastewaters is very important. In order to remove toxic heavy metals from contaminated water systems, conventional methods such as chemical precipitation, coagulation, ion exchange, solvent extraction and filtration, evaporation and membrane methods are being used. These conventional methods generally have high costs and technical problems. Therefore, biosorption processes, in which microorganisms are used as sorbents, have been considered as economical and environmentally friendly options for removal of heavy metals from aqueous solution. Clay minerals are another group of sorbents used in removal of heavy metals from polluted environments. Furthermore, bacterial cells can be attached on clay mineral surfaces and form bacteria-mineral composites. These composites adsorb heavy metals and convert them into forms with low mobility and bioavailability. Pseudomonas putida is a unique microorganism with a high tendency to sorb and/or degrade certain environmental pollutants. Palygorskite and sepiolite are the fibrous clay minerals of arid and semiarid regions; their structures consist of ribbons and channels. These fibrous minerals have various applications in industry and the environment because of its large surface area and high adsorption capacity. The present study was conducted in order to determine the ability of Pseudomonas putida (P168, and its composites with palygorskite and sepiolite in lead sorption. Materials and Methods: The bacterial strain used in the present study was Pseudomonas

  7. Genetic Dissection of the Regulatory Network Associated with High C-di-GMP Levels in Pseudomonas putida KT2440

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    María Isabel Ramos-González

    2016-07-01

    Full Text Available Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony. A total of nineteen different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two

  8. Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

    Science.gov (United States)

    Wigneswaran, Vinoth; Nielsen, Kristian Fog; Sternberg, Claus; Jensen, Peter Ruhdal; Folkesson, Anders; Jelsbak, Lars

    2016-10-24

    Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C 10 fatty acids. This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without

  9. The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42

    DEFF Research Database (Denmark)

    Aparicio, Tomás; Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2016-01-01

    of reference strain KT2440) is still a time-consuming endeavor. In this work we have investigated the in vivo activity of the Ssr protein encoded by the open reading frame T1E_1405 from Pseudomonas putida DOT-T1E, a plausible functional homologue of the β protein of the Red recombination system of λ phage...... of Escherichia coli. A test based on the phenotypes of pyrF mutants of P. putida (the yeast’s URA3 ortholog) was developed for quantifying the ability of Ssr to promote invasion of the genomic DNA replication fork by synthetic oligonucleotides. The efficiency of the process was measured by monitoring...

  10. Colony morphology and transcriptome profiling of Pseudomonas putida KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials.

    Science.gov (United States)

    Gulez, Gamze; Altıntaş, Ali; Fazli, Mustafa; Dechesne, Arnaud; Workman, Christopher T; Tolker-Nielsen, Tim; Smets, Barth F

    2014-08-01

    Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS components under mild water limitation. To create environmentally realistic water limited conditions as observed in soil, we used the Pressurized Porous Surface Model. Our main hypothesis was that under water limitation and in the absence of alginate other exopolysaccharides would be more active to maintain homeostasis. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 wild type and its mutants deficient in synthesis of either alginate or all known EPS. Overall our results support that alginate is an important exopolysaccharide under water limitation and in the absence of alginate other tolerance mechanisms are activated. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Production of Medium Chain Length Polyhydroxyalkanoates From Oleic Acid Using Pseudomonas putida PGA1 by Fed Batch Culture

    Directory of Open Access Journals (Sweden)

    Sidik Marsudi

    2010-10-01

    Full Text Available Bacterial polyhydroxyalkanoates (PHAs are a class of p0lymers currently receiving much attention because of their potential as renewable and biodegradable plastics. A wide variety of bacteria has been reported to produce PHAs including Pseudomonas strains. These strains are known as versatile medium chain length PHAs (PHAs-mcl producers using fatty acids as carbon source. Oleic acid was used to produce PHAs-mcl using Pseudomonas putida PGA 1 by continuous feeding of both nitrogen and carbon source, in a fed batch culture. During cell growth, PHAs also accumulated, indicating that PHA production in this organism is growth associated. Residual cell increased until the nitrogen source was depleted. At the end of fermentation, final cell concentration, PHA content, and roductivity were 30.2 g/L, 44.8 % of cell dry weight, and 0.188 g/l/h, respectively.

  12. Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

    Science.gov (United States)

    Wang, Weiwei; Xu, Ping; Tang, Hongzhi

    2015-11-17

    Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

  13. Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84.

    Science.gov (United States)

    Omidinia, Eskandar; Mahdizadehdehosta, Rahman; Mohammadi, Hamid Shahbaz

    2013-09-01

    The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

  14. Taxis of Pseudomonas putida F1 toward Phenylacetic Acid Is Mediated by the Energy Taxis Receptor Aer2

    Science.gov (United States)

    Luu, Rita A.; Schneider, Benjamin J.; Ho, Christie C.; Nesteryuk, Vasyl; Ngwesse, Stacy E.; Liu, Xianxian; Parales, Juanito V.; Ditty, Jayna L.

    2013-01-01

    The phenylacetic acid (PAA) degradation pathway is a widely distributed funneling pathway for the catabolism of aromatic compounds, including the environmental pollutants styrene and ethylbenzene. However, bacterial chemotaxis to PAA has not been studied. The chemotactic strain Pseudomonas putida F1 has the ability to utilize PAA as a sole carbon and energy source. We identified a putative PAA degradation gene cluster (paa) in P. putida F1 and demonstrated that PAA serves as a chemoattractant. The chemotactic response was induced during growth with PAA and was dependent on PAA metabolism. A functional cheA gene was required for the response, indicating that PAA is sensed through the conserved chemotaxis signal transduction system. A P. putida F1 mutant lacking the energy taxis receptor Aer2 was deficient in PAA taxis, indicating that Aer2 is responsible for mediating the response to PAA. The requirement for metabolism and the role of Aer2 in the response indicate that P. putida F1 uses energy taxis to detect PAA. We also revealed that PAA is an attractant for Escherichia coli; however, a mutant lacking a functional Aer energy receptor had a wild-type response to PAA in swim plate assays, suggesting that PAA is detected through a different mechanism in E. coli. The role of Aer2 as an energy taxis receptor provides the potential to sense a broad range of aromatic growth substrates as chemoattractants. Since chemotaxis has been shown to enhance the biodegradation of toxic pollutants, the ability to sense PAA gradients may have implications for the bioremediation of aromatic hydrocarbons that are degraded via the PAA pathway. PMID:23377939

  15. Nitrogen contribution proportion from soil, fertilizer and pseudomonas putida like in sorghum plantation on South Sumatra's inceptisols

    International Nuclear Information System (INIS)

    Kesumadewi, A.A.I.; Anas, Iswandi; Santosa, D.A.; Sisworo, Elsje L.

    2000-01-01

    The proportion of N which absorbed either from natural source (soil), N-fertilizer or N sub.2-fixing microorganism on sorghum plantation in marginal alang-alang lands that group to inceptisol should be studied in securing the soil fertility. In this experiment, N contribution of those N sources was determined during vegetative stage of sorghum on three subgroup of south sumatera's inceptisols. A greenhouse experiment that arranged in randomized complete block design using 2 factorial split plot was carried out in IPB Bogor from May-December 1998. The observation was focused on partition of N contribution from the soil, N-fertilizer and Pseudomonas putida like(N sub.2-fixing microorganism) on total plant-content 4 and 8 weeks after plantations (WAP). N-partition from those N-sources was done based o A-value method. Sorghum was absorb larger proportion of N from soil(63,36%-48,83% on 4 WAP and 64,58 % on WAP) than from fertilizer and P.putida like on all of the soil subgroups. Soil-N absorption was highest on oxic dystropept. Pseudomonas putida like was not able to subtite soil-N and fertilizer-N in sufficient amount to vigorous plant growth particularly on typica humitropept and typic dystropept. The microorganism was only provide a small amount of N to sorghum on oxic dystropept (23,05-23,95 % on 4 WAP and 15,91-34,44 % on 8WAP). The increment of plant root surface area could be enhance in greater extent of plant-N absorption than N sub.2-fixation. Thus, plantation of marginal tolerant sorghum still need addition of N-fertilizer

  16. Liquid chromatography time of flight mass spectrometry based environmental metabolomics for the analysis of Pseudomonas putida Bacteria in potable water.

    Science.gov (United States)

    Kouremenos, Konstantinos A; Beale, David J; Antti, Henrik; Palombo, Enzo A

    2014-09-01

    Water supply biofilms have the potential to harbour waterborne diseases, accelerate corrosion, and contribute to the formation of tuberculation in metallic pipes. One particular species of bacteria known to be found in the water supply networks is Pseudomonas sp., with the presence of Pseudomonas putida being isolated to iron pipe tubercles. Current methods for detecting and analysis pipe biofilms are time consuming and expensive. The application of metabolomics techniques could provide an alternative method for assessing biofilm risk more efficiently based on bacterial activity. As such, this paper investigates the application of metabolomic techniques and provides a proof-of-concept application using liquid chromatography coupled with time-of-flight mass spectrometry (LC-ToF-MS) to three biologically independent P. putida samples, across five different growth conditions exposed to solid and soluble iron (Fe). Analysis of the samples in +ESI and -ESI mode yielded 887 and 1789 metabolite features, respectively. Chemometric analysis of the +ESI and -ESI data identified 34 and 39 significant metabolite features, respectively, where features were considered significant if the fold change was greater than 2 and obtained a p-value less than 0.05. Metabolite features were subsequently identified according to the Metabolomics Standard Initiative (MSI) Chemical Analysis Workgroup using analytical standards and standard online LC-MS databases. Possible markers for P. putida growth, with and without being exposed to solid and soluble Fe, were identified from a diverse range of different chemical classes of metabolites including nucleobases, nucleosides, dipeptides, tripeptides, amino acids, fatty acids, sugars, and phospholipids. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid.

    Science.gov (United States)

    Graf, Nadja; Altenbuchner, Josef

    2014-01-01

    Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficient to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86% within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

  18. Degradation of chloroaromatics : Purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31

    NARCIS (Netherlands)

    Kaschabek, Stefan R.; Kasberg, Thomas; Müller, Dagmar; Mars, Astrid E.; Janssen, Dick B.; Reineke, Walter

    A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31

  19. Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31

    NARCIS (Netherlands)

    Mars, Astrid E.; Kingma, Jaap; Kaschabek, Stefan R.; Reineke, Walter; Janssen, Dick B.

    Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth, A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol

  20. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: Verification and complementation of hypotheses derived from transcriptomics

    NARCIS (Netherlands)

    Wierckx, N.; Ruijssenaars, H.J.; Winde, J.H.de; Schmid, A.; Blank, L.M.

    2009-01-01

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase

  1. Multivariate analysis of microarray data by principal component discriminant analysis: Prioritizing relevant transcripts linked to the degradation of different carbohydrates in Pseudomonas putida S12

    NARCIS (Netherlands)

    Werf, M.J. van der; Pieterse, B.; Luijk, N. van; Schuren, F.; Werff van der - Vat, B. van der; Overkamp, K.; Jellema, R.H.

    2006-01-01

    The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four

  2. Whole-Genome Sequence of Pseudomonas putida Strain UASWS0946, a Highly Ammonia-Tolerant Nitrifying Bacterium Isolated from Sewage Sludge Aerobic Granules.

    Science.gov (United States)

    Crovadore, Julien; Calmin, Gautier; Cochard, Bastien; Chablais, Romain; Grizard, Damien; Berthon, Jean-Yves; Lefort, François

    2015-10-08

    We report here the genome of Pseudomonas putida strain UASWS0946, a highly ammonia-tolerant nitrifying strain isolated from sewage sludge aerobic granules, which displays adequate genetic equipment for soil depollution, sludge treatment, and biological fertilization in agriculture. Copyright © 2015 Crovadore et al.

  3. Draft Genome Sequence of Pseudomonas putida CBF10-2, a Soil Isolate with Bioremediation Potential in Agricultural and Industrial Environmental Settings

    OpenAIRE

    Iyer, Rupa; Damania, Ashish

    2016-01-01

    Pseudomonas putida CBF10-2 is a microorganism isolated from farmland soil in Fairchild, TX, found to degrade high-impact xenobiotics, including organophosphate insecticides, petroleum hydrocarbons, and both monocyclic and polycyclic aromatics. The versatility of CBF10-2 makes it useful for multipurpose bioremediation of contaminated sites in agricultural and industrial environments.

  4. Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

    NARCIS (Netherlands)

    Kieboom, J; Bruinenberg, R; Keizer-Gunnink, [No Value; de Bont, JAM

    2001-01-01

    Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a

  5. Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose

    NARCIS (Netherlands)

    Nijkamp, K.; Westerhof, R.G.M.; Ballerstedt, H.; Bont, J.A.M.de; Wery, J.

    2007-01-01

    A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular L-tyrosine availability, and formation of the by-product cinnamate via L-phenylalanine. The

  6. Characterization of a novel blaIMPgene, blaIMP-58, using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark.

    Science.gov (United States)

    Holmgaard, Dennis Back; Hansen, Frank; Hasman, Henrik; Justesen, Ulrik S; Hammerum, Anette M

    2017-01-01

    A multidrug-resistant strain of Pseudomonas putida was isolated from the urine of a 65-year-old women hospitalized for serious clinical conditions. Using whole genome sequencing a novel blaIMP gene, blaIMP-58 was discovered and characterized. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Study on the efficiency of the two phase partitioning stirred tank bioreactor on the toluene filtration from the airstream by Pseudomonas putida via

    Directory of Open Access Journals (Sweden)

    2013-02-01

    Full Text Available Introduction: There are different methods for controlling gaseous pollutants formed from air pollution sources that one of the most economical and efficient of them, is bio-filtration. The purpose of this study is Toluene removal from airstream by using the pure Pseudomonas putida bacteria as a fluidized bed in a two phase partitioning stirred tank bioreactor.Toluene ( Metyle benzene is one of the aromatic compounds which uses as a chemical solvent.low to moderate concentration of Toluene causes fatigue, dizziness, weakness,unbalance behaviour, memory loss, insomnia, loss of appetite, loss of vision and hearing. .Material and Method: In this experimental study at first, pure Pseudomonas putida in an aqueous phase containing nutrients and trace elements solution was duplicated and accustomed with Toluene. then solution contained microorganisms with 10% silicon oil was entered to bioreactor. The amount of CO2 and pollutant concentrations in the entrance and exhaust of bioreactor containing Pseudomonas putida was studied during 17 days for each variable. .Result: Experimental findings showed that in the 0.06 m3/h and 0.12 m3/h flow rate, the efficiency of bioreactor containing Pseudomonas putida in the concentration ranges of 283 Mg/m3 to 4710 Mg/m3 was at least 97% and 25% respectively. Statistical analysis (ANOVA showed that in two flow rates of 0.06 m3/h and 0.12 m3/h removal efficiency and mineralization percentage had significant differences .(Pvalue =0.01. .Conclusion: Achieving high efficiencies in pollutants removal was because of the prepared optimum conditions for Pseudomonas putida in the two phase partitioning stirred tank bioreactor with 10% organic phase.

  8. Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.

    Science.gov (United States)

    Beckers, Veronique; Poblete-Castro, Ignacio; Tomasch, Jürgen; Wittmann, Christoph

    2016-05-03

    Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network. Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol. This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This

  9. NER enzymes maintain genome integrity and suppress homologous recombination in the absence of exogenously induced DNA damage in Pseudomonas putida.

    Science.gov (United States)

    Sidorenko, Julia; Ukkivi, Kärt; Kivisaar, Maia

    2015-01-01

    In addition to its prominence in producing genetic diversity in bacterial species, homologous recombination (HR) plays a key role in DNA repair and damage tolerance. The frequency of HR depends on several factors, including the efficiency of DNA repair systems as HR is involved in recovery of replication forks perturbed by DNA damage. Nucleotide excision repair (NER) is one of the major DNA repair pathways involved in repair of a broad range of DNA lesions generally induced by exogenous chemicals or UV-irradiation and its functions in the cells not exposed to DNA-damaging agents have attracted less attention. In this study we have developed an assay that enables to investigate HR between chromosomal loci of the soil bacterium Pseudomonas putida both in growing and stationary-phase cells. The present assay detects HR events between two non-functional alleles of phenol degrading genes that produce a functional allele and allow the growth of bacteria on phenol as a sole carbon source. Our results indicate that HR between chromosomal loci takes place mainly in the growing cells and the frequency of HR is reduced during the following starvation in NER-proficient P. putida but not in the case when bacteria lack UvrA or UvrB enzymes. The absence of UvrA or UvrB resulted in a hyper-recombination phenotype in P. putida, the cells were filamented and their growth was impaired even in the absence of exogenous DNA damage. However, NER-deficient derivatives that overcame growth defects emerged rapidly. Such adaptation resulted in the decline of the frequency of HR. Although HR in actively replicating P. putida was still elevated in the adapted variants of the UvrA- and UvrB-deficient strains, the dynamics of emergence of the recombinants in these strains turned similar to NER-proficient bacteria. Additionally, we observed that HR was enhanced in the absence of the transcription repair coupling factor Mfd in growing cells but not during starvation. The frequency of HR was not

  10. Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

    Science.gov (United States)

    Mohan, Karishma

    2017-01-01

    ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the

  11. Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86.

    Science.gov (United States)

    Mohan, Karishma; Phale, Prashant S

    2017-04-15

    Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz , veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer , ver , and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O -demethylases, viz , VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O -demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic

  12. Contrasting colonization and plant growth promoting capacity between wild type and gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

    Energy Technology Data Exchange (ETDEWEB)

    Weyens N.; van der Lelie D.; Boulet, J.; Adriaensen, D.; Timmermans, J.-P.; Prinsen, E.; Van Oevelen, S.; D" Haen, J.; Smeets, K.; Taghavi, S.; Vangronsveld, J.

    2011-06-09

    This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

  13. Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Weiwei; Chen, Lingxin; Liu, Dongyan [Chinese Academy of Sciences, Yantai, SD (China). Yantai Inst. of Coastal Zone Research (YICCAS); Chinese Academy of Sciences, Yantai, SD (China). Shandong Provincial Key Lab. of Coastal Zone Environmental Processes

    2012-02-15

    The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 {mu}M HgCl{sub 2}. SP1 was also highly resistant to other metals, including CdCl{sub 2}, CoCl{sub 2}, CrCl{sub 3}, CuCl{sub 2}, PbCl{sub 2}, and ZnSO{sub 4}, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl{sub 2} and the removal of HgCl{sub 2} by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg{sup 2+} to volatile and relatively inert monoatomic Hg{sup 0} vapor, was around 5.0. LD50 of P. putida SP1 to flounder and turbot was 1.5 x 10{sup 9} CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl{sub 2} contamination over a broad range of pH. (orig.)

  14. Differential infectivity of two Pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera).

    Science.gov (United States)

    Schneider, M; Dorn, A

    2001-10-01

    Pseudomonas aeruginosa and Pseudomonas putida show a profound differential infectivity after inoculation in Oncopeltus fasciatus. Whereas P. putida has no significant impact on nymphs, P. aeruginosa kills all experimental animals within 48 h. Both Pseudomonas species, however, induce the same four hemolymph peptides in O. fasciatus. Also injection of saline solution and injury induced these peptides. In general peptide induction was stronger in nymphs than in adult males. A significantly higher number of nymphs survived a challenge with P. aeruginosa when an immunization with P. putida preceded. The antibacterial properties of the hemolymph were demonstrated in inhibition experiments with P. putida. Two of the four inducible peptides (peptides 1 and 4) could be partially sequenced after Edman degradation and were compared with known antibacterial peptides. Peptide 1, of 15 kDa, showed 47.1% identity with the glycine-rich hemiptericin of Pyrrhocoris apterus. Peptide 4, of 2 kDa, had a 77.8% identity with the proline-rich pyrrhocoricin of P. apterus and a 76.9% identity with metalnikowin 1 of Palomena prasina. Peptides 2 and 3 are also small, with molecular weights of 8 and 5 kDa.

  15. Antimicrobial effect of Al2O3, Ag and Al2O3/Ag thin films on Escherichia coli and Pseudomonas putida

    International Nuclear Information System (INIS)

    Angelov, O; Stoyanova, D; Ivanova, I; Todorova, S

    2016-01-01

    The influence of Al 2 O 3 , Ag and Al 2 O 3 /Ag thin films on bacterial growth of Gramnegative bacteria Pseudomonas putida and Escherichia coli is studied. The nanostructured thin films are deposited on glass substrates without intentional heating through r.f. magnetron sputtering in Ar atmosphere of Al 2 O 3 and Ag targets or through sequential sputtering of Al 2 O 3 and Ag targets, respectively. The individual Ag thin films (thickness 8 nm) have a weak bacteriostatic effect on Escherichia coli expressed as an extended adaptive phase of the bacteria up to 5 hours from the beginning of the experiment, but the final effect is only 10 times lower bacterial density than in the control. The individual Al 2 O 3 film (20 nm) has no antibacterial effect against two strains E. coli - industrial and pathogenic. The Al 2 O 3 /Ag bilayer films (Al 2 O 3 20 nm/Ag 8 nm) have strong bactericidal effect on Pseudomonas putida and demonstrate an effective time of disinfection for 2 hours. The individual films Al2O3 and Ag have not pronounced antibacterial effect on Pseudomonas putida . A synergistic effect of Al2O3/Ag bilayer films in formation of oxidative species on the surface in contact with the bacterial suspension could be a reason for their antimicrobial effect on E. coli and P. putida . (paper)

  16. Antimicrobial effect of Al2O3, Ag and Al2O3/Ag thin films on Escherichia coli and Pseudomonas putida

    Science.gov (United States)

    Angelov, O.; Stoyanova, D.; Ivanova, I.; Todorova, S.

    2016-10-01

    The influence of Al2O3, Ag and Al2O3/Ag thin films on bacterial growth of Gramnegative bacteria Pseudomonas putida and Escherichia coli is studied. The nanostructured thin films are deposited on glass substrates without intentional heating through r.f. magnetron sputtering in Ar atmosphere of Al2O3 and Ag targets or through sequential sputtering of Al2O3 and Ag targets, respectively. The individual Ag thin films (thickness 8 nm) have a weak bacteriostatic effect on Escherichia coli expressed as an extended adaptive phase of the bacteria up to 5 hours from the beginning of the experiment, but the final effect is only 10 times lower bacterial density than in the control. The individual Al2O3 film (20 nm) has no antibacterial effect against two strains E. coli - industrial and pathogenic. The Al2O3/Ag bilayer films (Al2O3 20 nm/Ag 8 nm) have strong bactericidal effect on Pseudomonas putida and demonstrate an effective time of disinfection for 2 hours. The individual films Al2O3 and Ag have not pronounced antibacterial effect on Pseudomonas putida. A synergistic effect of Al2O3/Ag bilayer films in formation of oxidative species on the surface in contact with the bacterial suspension could be a reason for their antimicrobial effect on E. coli and P. putida.

  17. Multiple Modes of Nematode Control by Volatiles of Pseudomonas putida 1A00316 from Antarctic Soil against Meloidogyne incognita

    Directory of Open Access Journals (Sweden)

    Yile Zhai

    2018-02-01

    Full Text Available Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs, and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24–48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography–mass spectrometry (SPME-GC/MS included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.

  18. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.

    Science.gov (United States)

    Puchałka, Jacek; Oberhardt, Matthew A; Godinho, Miguel; Bielecka, Agata; Regenhardt, Daniela; Timmis, Kenneth N; Papin, Jason A; Martins dos Santos, Vítor A P

    2008-10-01

    A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to

  19. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.

    Directory of Open Access Journals (Sweden)

    Jacek Puchałka

    2008-10-01

    Full Text Available A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile

  20. Interaction of Pseudomonas putida with kaolinite and montmorillonite: a combination study by equilibrium adsorption, ITC, SEM and FTIR.

    Science.gov (United States)

    Rong, Xingmin; Huang, Qiaoyun; He, Xiaomin; Chen, Hao; Cai, Peng; Liang, Wei

    2008-06-15

    Equilibrium adsorption along with isothermal titration calorimetry (ITC), Fourier transform infrared spectra (FTIR) and scanning electron microscopy (SEM) techniques were employed to investigate the adsorption of Pseudomonas putida on kaolinite and montmorillonite. A higher affinity as well as larger amounts of adsorption of P. putida was found on kaolinite. The majority of sorbed bacterial cells (88.7%) could be released by water from montmorillonite, while only a small proportion (9.3%) of bacteria desorbed from kaolinite surface. More bacterial cells were observed to form aggregates with kaolinite, while fewer cells were within the larger bacteria-montmorillonite particles. The sorption of bacteria on kaolinite was enthalpically more favorable than that on montmorillonite. Based on our findings, it is proposed that the non-electrostatic forces other than electrostatic force play a more important role in bacterial adsorption by kaolinite and montmorillonite. Adsorption of bacteria on clay minerals resulted in obvious shifts of infrared absorption bands of water molecules, showing the importance of hydrogen bonding in bacteria-clay mineral adsorption. The enthalpies of -4.1+/-2.1 x 10(-8) and -2.5+/-1.4 x 10(-8)mJ cell(-1) for the adsorption of bacteria on kaolinite and montmorillonite, respectively, at 25 degrees C and pH 7.0 were firstly reported in this paper. The enthalpy of bacteria-mineral adsorption was higher than that reported previously for bacteria-biomolecule interaction but lower than that of bacterial coaggregation. The bacteria-mineral adsorption enthalpies increased at higher temperature, suggesting that the enthalpy-entropy compensation mechanism could be involved in the adsorption of P. putida on clay minerals. Data obtained in this study would provide valuable information for a better understanding of the mechanisms of mineral-microorganism interactions in soil and associated environments.

  1. High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply.

    Science.gov (United States)

    Davis, Reeta; Duane, Gearoid; Kenny, Shane T; Cerrone, Federico; Guzik, Maciej W; Babu, Ramesh P; Casey, Eoin; O'Connor, Kevin E

    2015-04-01

    High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L(-1) h(-1) ) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L(-1) was achieved in 33 h with a biomass productivity of 3.1 g L(-1) h(-1) which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L(-1) h(-1) ) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA(-1) . © 2014 Wiley Periodicals, Inc.

  2. Genotypic and phenotypic diversity of polyhydroxybutyrate (PHB) producing Pseudomonas putida isolates of Chhattisgarh region and assessment of its phosphate solubilizing ability.

    Science.gov (United States)

    Agrawal, Toshy; Kotasthane, Anil S; Kushwah, Renu

    2015-02-01

    A diverse and versatile spectrum of metabolic activities among isolates of fluorescent Pseudomonas putida indicates their adaptability to various niches. These polyhydroxybutyrate producing and phosphate solubilizing isolates showed a high level of functional and genetic versatility among themselves. One of the potential P. putida isolate P132 can contribute as a candidate agent for both biocontrol and PGPR applications. Identified as one of the most efficient PHB producer and phosphate solubilizer, in vitro detection of P132 showed the presence of genes for phenazine, pyrrolnitrin, pyoluteorin and 2,4 diacetylphloroglucinol along with polyhydroxyalkanoate.

  3. Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols.

    Science.gov (United States)

    Rehdorf, Jessica; Behrens, Geoffrey A; Nguyen, Giang-Son; Kourist, Robert; Bornscheuer, Uwe T

    2012-02-01

    An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.

  4. Bioremoval of Basic Violet 3 and Acid Blue 93 by Pseudomonas putida and its adsorption isotherms and kinetics.

    Science.gov (United States)

    Arunarani, A; Chandran, Preethy; Ranganathan, B V; Vasanthi, N S; Sudheer Khan, S

    2013-02-01

    Basic Violet 3 and Acid Blue 93 are the most important group of synthetic colourants extensively used in textile industries for dyeing cotton, wool, silk and nylon. Release of these dye pollutants in to the environment adversely affects the human health and aquatic organisms. The present study we used Pseudomonas putida MTCC 4910 for the adsorptive removal of Basic Violet 3 and Acid Blue 93 from the aqueous solutions. The pH (4-9) and NaCl concentrations (1mM-1M) did not influence the adsorption process. The equilibrium adsorption process fitted well to Freundlich model than Langmuir model. The kinetics of adsorption fitted well by pseudo-second-order. Thus in the present study an attempt has been made to exploit the dye removal capability of P. putida MTCC 4910, and it was found to be an efficient microbe that could be used for bio removal of dyes from textile effluents. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Pathway-Consensus Approach to Metabolic Network Reconstruction for Pseudomonas putida KT2440 by Systematic Comparison of Published Models.

    Science.gov (United States)

    Yuan, Qianqian; Huang, Teng; Li, Peishun; Hao, Tong; Li, Feiran; Ma, Hongwu; Wang, Zhiwen; Zhao, Xueming; Chen, Tao; Goryanin, Igor

    2017-01-01

    Over 100 genome-scale metabolic networks (GSMNs) have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design.

  6. Toxicities effects of pharmaceutical, olive mill and textile wastewaters before and after degradation by Pseudomonas putida mt-2

    Directory of Open Access Journals (Sweden)

    Mansour Hedi

    2012-02-01

    Full Text Available Abstract Background Removal of numerous classes of chemical pollutants from the industrial wastewater such as textile, pharmaceutical and olive mill using conventional wastewater treatment, is incomplete and several studies suggested that improvement of this situation would require the application of biological treatment techniques. Dyes, polyphenols and drugs are an environmental pollutants extremely toxics to plants and other living organisms including humans. These effluents were previously treated by Pseudomonas putida. The main of this work was to evaluate the in vivo toxicity of the three wastewaters. Methods Writhes and convulsant effect of effluents were carried out and were compared to the treated effluents. Only pharmaceutical wastewater was exhibited a convulsant effect which observed in mice treated by effluent. On the other hand, all industrial wastewater induced significantly an algogenic effects particularly when mice were treated by the pharmaceutical wastewater (Number of writhes = 44. Conclusion Toxicity was totally removed when mice were treated by the bio remediated effluent. This indicates that P. putida was able to completely detoxify the toxic industrial effluent.

  7. N-acyl Homoserine Lactone-Producing Pseudomonas putida Strain T2-2 from Human Tongue Surface

    Directory of Open Access Journals (Sweden)

    Yeun Mun Choo

    2013-09-01

    Full Text Available Bacterial cell-to-cell communication (quorum sensing refers to the regulation of bacterial gene expression in response to changes in microbial population density. Quorum sensing bacteria produce, release and respond to chemical signal molecules called autoinducers. Bacteria use two types of autoinducers, namely autoinducer-1 (AI-1 and autoinducer-2 (AI-2 where the former are N-acylhomoserine lactones and the latter is a product of the luxS gene. Most of the reported literatures show that the majority of oral bacteria use AI-2 for quorum sensing but rarely the AI-1 system. Here we report the isolation of Pseudomonas putida strain T2-2 from the oral cavity. Using high resolution mass spectrometry, it is shown that this isolate produced N-octanoylhomoserine lactone (C8-HSL and N-dodecanoylhomoserine lactone (C12-HSL molecules. This is the first report of the finding of quorum sensing of P. putida strain T2-2 isolated from the human tongue surface and their quorum sensing molecules were identified.

  8. Pathway-Consensus Approach to Metabolic Network Reconstruction for Pseudomonas putida KT2440 by Systematic Comparison of Published Models.

    Directory of Open Access Journals (Sweden)

    Qianqian Yuan

    Full Text Available Over 100 genome-scale metabolic networks (GSMNs have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design.

  9. A Pseudomonas putida double mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform.

    Science.gov (United States)

    Cuenca, María Del Sol; Molina-Santiago, Carlos; Gómez-García, María R; Ramos, Juan L

    2016-03-01

    Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Antimicrobial effect of TiO2 doped with Ag and Cu on Escherichia coli and Pseudomonas putida

    Science.gov (United States)

    Angelov, O.; Stoyanova, D.; Ivanova, I.

    2016-10-01

    Antimicrobial effect of TiO2 doped with Ag and Cu on Gram-negative bacteria Escherichia coli and Pseudomonas putida is studied. The thin films are deposited on glass substrates without heating during the deposition by r.f. magnetron co-sputtering of TiO2 target and pieces of Ag and Cu. The studied films, thickness about 65 nm, were as deposited and annealed (5200C, 4h, N2+5%H2, 4Pa). The as deposited thin films TiO2:Ag:Cu have band gap energy of 3.56 eV little higher than the band gap of crystalline anatase TiO2 which can be explained with the quantum effect of the granular structure of r.f. magnetron sputtered films. The annealed samples have band gap of 2.52 eV due to formation of donor levels from Ag and Cu atoms near the bottom of the conduction band. The toxic effect was determined through the classical Koch's method and the optical density measurements at λ=610 nm. The as deposited TiO2:Ag:Cu thin films demonstrate stronger inhibition effect - bactericidal for P. putida and bacteriostatic for E. coli (up to the 6th hour) in comparison with the annealed samples. The both methods of study show the same trends of the bacterial growth independently of their different sensitivity which confirms the observed effect.

  11. Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF.

    Directory of Open Access Journals (Sweden)

    Andrio Lahesaare

    Full Text Available The biofilm matrix of the rhizospheric bacterium Pseudomonas putida consists mainly of a proteinaceous component. The two largest P. putida proteins, adhesins LapA and LapF, are involved in biofilm development but prevail in different developmental stages of the biofilm matrix. LapA is abundant in the initial stage of biofilm formation whereas LapF is found in the mature biofilm. Although the transcriptional regulation of the adhesins is not exhaustively studied, some factors that can be involved in their regulation have been described. For example, RpoS, the major stress response sigma factor, activates, and Fis represses LapF expression. This study focused on the LapF expression control by Fis. Indeed, using DNase I footprint analysis a Fis binding site Fis-F2 was located 150 bp upstream of the lapF gene coding sequence. The mapped 5' end of the lapF mRNA localized the promoter to the same region, overlapping with the Fis binding site Fis-F2. Monitoring the lapF promoter activity by a β-galactosidase assay revealed that Fis overexpression causes a 4-fold decrease in the transcriptional activity. Furthermore, mutations that diminished Fis binding to the Fis-F2 site abolished the repression of the lapF promoter. Thus, these data suggest that Fis is involved in the biofilm regulation via repression of LapF expression.

  12. DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

    Science.gov (United States)

    Jatsenko, Tatjana; Sidorenko, Julia; Saumaa, Signe; Kivisaar, Maia

    2017-01-01

    Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

  13. Biological production of hydroxylated aromatics : Optimization strategies for Pseudomonas putida S12

    NARCIS (Netherlands)

    Verhoef, A.

    2010-01-01

    To replace environmentally unfriendly petrochemical production processes, the demand for bio-based production of organic chemicals is increasing. This thesis focuses on the biological production of hydroxylated aromatics from renewable substrates by engineered P. putida S12 including several cases

  14. Tn5090-like class 1 integron carrying bla(VIM-2) in a Pseudomonas putida strain from Portugal.

    Science.gov (United States)

    Santos, C; Caetano, T; Ferreira, S; Mendo, S

    2010-10-01

    Three Pseudomonas putida strains containing bla(VIM-2) were isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro. A novel class 1 integron was found in strain Pp2 (aacA4/bla(VIM-2)/aac6'-IIc disrupted by an insertion sequence IS1382), and strain Pp1 was found to carry a class 1 integron (aacA7/bla(VIM-2)/aacC1/aacA4), which is described for the first time in this species. Strain PF1 carries a class 1 integron associated with a Tn5090-like transposon, constituting the first finding of this type of arrangement in a strain from Portugal. This association highlights further dissemination of bla(VIM-2) in environmental hospital isolates. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.

  15. Cow Dung Substrate for the Potential Production of Alkaline Proteases by Pseudomonas putida Strain AT in Solid-State Fermentation

    Directory of Open Access Journals (Sweden)

    Ponnuswamy Vijayaraghavan

    2014-01-01

    Full Text Available Cow dung and agroresidues were used as the substrates for the production of alkaline proteases by Pseudomonas putida strain AT in solid-state fermentation. Among the various substrates evaluated, cow dung supported maximum (1351±217 U/g protease production. The optimum conditions for the production of alkaline proteases were a fermentation period of 48 h, 120% (v/w moisture, pH 9, and the addition of 6% (v/w inoculum, 1.5% (w/w trehalose, and 2.0% (w/w yeast extract to the cow dung substrate. The enzyme was active over a range of temperatures (50–70°C and pHs (8–10, with maximum activity at 60°C and pH 9. These enzymes showed stability towards surfactants, detergents, and solvent and digested various natural proteins.

  16. Effects of LB broth, naphthalene concentration, and acetone on the naphthalene degradation activities by Pseudomonas putida G7.

    Science.gov (United States)

    Chang, Su-Yun; Liu, Xue-Gong; Ren, Bi-Qiong; Liu, Bo; Zhang, Kai; Zhang, Honggui; Wan, Yao

    2015-01-01

    Luria-Bertani broth and acetone were usually used in naphthalene degradation experiments as nutrient and solvent. However, their effect on the degradation was seldom mentioned. In this work, we investigated the effect of LB, naphthalene concentration, and acetone on the degradation of naphthalene by Pseudomonas putida G7, which is useful for the degradation of naphthalene on future field remediation. By adding LB, the naphthalene degradation efficiencies and naphthalene dioxygenase were both decreased by 98%, while the catechol dioxygenase was decreased by 90%. Degradation of naphthalene was also inhibited when naphthalene concentration was 56 ppm and higher, which was accompanied with the accumulation of orange-colored metabolism products. However, acetone can stimulate the degradation of naphthalene, and the stimulation was more obvious when naphthalene concentration was lower than 2000 ppm. By assaying the enzyme activities of naphthalene dioxygenase and catechol dioxygenase, it was thought that the degradation efficiency was depending on the more sensitive enzymes on the complicated conditions.

  17. Mechanisms of trace metal sorption in Pseudomonas putida-birnessite assemblages

    Science.gov (United States)

    Peña, J.; Kwon, K. D.; Bargar, J. R.; Sposito, G.

    2012-04-01

    Biogenic manganese oxides (MnO2) are ubiquitous nanoparticulate minerals that contribute strongly to the adsorption of nutrient and toxicant metals in aquatic and terrestrial environments. The formation of these minerals is catalyzed by a diverse and widely-distributed group of bacteria and fungi, often through the enzymatic oxidation of aqueous Mn(II) to Mn(IV). The biogenic Mn(IV) oxide found in field settings, as well as that produced by model bacteria in laboratory culture, is typically layer-type hexagonal birnessite containing abundant cation vacancy sites and enmeshed in an organic matrix of bacterial cells and extracellular polymeric substances. In this talk I summarize the results from laboratory-scale research designed to investigate the mechanisms of metal sorption by the bacterial biomass-birnessite assemblages formed by Pseudomonas putida GB-1 when grown in the presence of 1 mM Mn(II) at circumneutral pH values. The goals of this research were first, to identify the structure of the surface complexes formed by trace metals (e.g., Ni, Cu and Zn) on biogenic birnessite and second, to determine the conditions under which the bacterial cell surfaces and extracellular polymeric substances contribute to metal sorption. Macroscopic and spectroscopic experiments were performed at varying pH values (6 - 8) and over a wide-range of metal concentrations. Extended X-ray absorption fine structure (EXAFS) spectroscopy and first-principles calculations based on density functional theory showed that cation vacancy sites in birnessite drive mineral reactivity, but that surface speciation varies from metal to metal. For, Ni we identified two species, Ni bonded to three surface oxygen atoms vacancy sites as a triple-corner-sharing (TCS) complex and Ni incorporated at vacancy sites, with surface speciation varying with pH and surface loading. Zinc formed TCS complexes at vacancy sites, with the proportion of Zn in tetrahedral or octahedral coordination geometry influenced

  18. Toxicity evaluation of selected ammonium-based ionic liquid forms with MCPP and dicamba moieties on Pseudomonas putida.

    Science.gov (United States)

    Piotrowska, Aleksandra; Syguda, Anna; Wyrwas, Bogdan; Chrzanowski, Łukasz; Heipieper, Hermann J

    2017-01-01

    Combination of the hydrophilic herbicidal anion with hydrophobic, antimicrobial ammonium cation allows to obtain compounds in ionic liquid form with better properties then conventional herbicides. Both cation and anion can be modified by selection of herbicide and the length of alkyl chains in cation structure. However the knowledge of their potential toxic effects are still limited. Furthermore, the relation between hydrophobicity associated with the length of alkyl chains and toxicity for ionic liquids has not been thoroughly studied. Therefore we investigated toxic effects of herbicidal ionic liquid forms on growth inhibition, given as EC 50, of the common soil bacterium Pseudomonas putida. We thereby concentrated on quaternary ammonium salts. Analyzed compounds were composed of dicamba or MCPP moieties and cation with various alkyl chain lengths (n = 6,8,10) We compared them with commercial herbicides, and ammonium-based ionic liquids with neutral anion (Br - ). In addition, cis-trans isomerisation of unsaturated membrane fatty acids in Pseudomonas putida was applied as the proxy for toxicity and membrane activity. We showed that toxicity increased with the length of alkyl chains. However, this correlation is only valid for six and eight carbon atom in alkyl chains, where for n = 10 the EC 50 values rise by one order of magnitude. In our studies, the herbicidal ionic liquids [C 10 ,C 10 ,C 1 ,C 1 N][MCPP] and [C 10 ,C 10 ,C 1 ,C 1 N][dicamba] showed the lowest toxicity among analyzed quaternary ammonium salts and comparable toxicity with corresponding herbicides. No clear increase in toxicity could be followed by changing the anion moieties for ammonium-based ionic liquid forms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Identification and validation of novel small proteins in Pseudomonas putida

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Ingemann Jensen, Sheila; Wulff, Tune

    2016-01-01

    Small proteins of fifty amino acids or less have been understudied due to difficulties that impede their annotation and detection. In order to obtain information on small open reading frames (sORFs) in P. putida, bioinformatic and proteomic approaches were used to identify putative small open...... reading frames (sORFs) in the well-characterized strain KT2440. A plasmid-based system was established for sORF validation, enabling expression of C-terminal sequential peptide affinity (SPA) tagged variants and their detection via protein immunoblotting. Out of 22 tested putative sORFs, the expression...... and CysB involved in biofilm formation and cysteine biosynthesis, respectively. The work sheds light on the P. putida small proteome and small protein identification, a necessary first step towards gaining insights into their functions and possible evolutionary implications....

  20. Unusual poly(3-hydroxyalkanoate) (PHA) biosynthesis behavior of Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002 isolated from palm oil mill effluent.

    Science.gov (United States)

    Razaif-Mazinah, Mohd Rafais Mohd; Anis, Siti Nor Syairah; Harun, Hazwani Izzati; Rashid, Khairunnisa Abdul; Annuar, Mohamad Suffian Mohamad

    2017-03-01

    Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002, isolated from palm oil mill effluent, accumulated poly(3-hydroxyalkanoates) (PHAs) when grown on aliphatic fatty acids, sugars, and glycerol. The substrates were supplied at 20:1 C/N mole ratio. Among C-even n-alkanoic acids, myristic acid gave the highest PHA content 26 and 28 wt% in P. putida and D. tsuruhatensis, respectively. Among C-odd n-alkanoic acids, undecanoic gave the highest PHA content at 40 wt% in P. putida and 46 wt% in D. tsuruhatensis on pentadecanoic acid. Sugar and glycerol gave putida, the major hydroxyalkanoates unit was 3-hydroxyoctanoate and 3-hydroxydecanoate when grown on C-even acids. Conversely, 3-hydroxyheptanoate, 3-hydrxoynonanoate, and 3-hydroxyundecanoate were accumulated with C-odd acids. Weight-averaged molecular weight (M w ) was in the range of 53-81 kDa and 107-415 kDa for P. putida and D. tsuruhatensis, respectively. Calorimetric analyses indicated that both bacteria synthesized semicrystalline polymer with good thermal stability with degradation temperature (T d ) ranging from 178 to 282 °C. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  1. Combination of pseudomonas putida and EK method to reduce the amount of mercury on landfill soil

    Science.gov (United States)

    Nabila, A. T. A.; Azhar, A. T. S.; Nurshuhaila, M. S.; Azim, M. A. M.; Amirah, S. N.

    2017-11-01

    Landfills usually lack of environment measures especially on soil. There are no guarantee that the landfill soil is free from being contaminated. It may cause harm for humans, animals and plants at surrounding area. In order to solve this problem, advance remediation technique is essential such as the electrokinetic combined with microorganisms known as electrokinetic bioremediation technique. The aim of this study is to investigate the performance of P.putida with 15 volt electric current supply (Ek-bio) and without electric current (Bio) in removal of mercury in landfill soil. Both treatments were running throughout 14 days. The P.putida was placed at anode compartment meanwhile sterile distilled water poured at cathode compartment. According to the both results, Ek-bio was removed mercury up to 48 % but by using standard bioremediation treatment, the removal only 32 %. Besides that, the migration of P.putida react more aggressively during the present of electric current compared with bioremediation. As the results, it was proven that by using Ek-bio technique can increase the activity of bacteria beside and the removal of mercury. Therefore, Ek-bio method can be commercialized to the parties concerned to solve the contaminated soil by mercury.

  2. Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine.

    Science.gov (United States)

    Li, Aiwen; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Wang, Yuhong; Tong, Lu; Jiang, Jiandong; Chen, Jianmeng

    2017-05-31

    The presence of nicotine and nicotinic acid (NA) in the marine environment has caused great harm to human health and the natural environment. Therefore, there is an urgent need to use efficient and economical methods to remove such pollutants from the environment. In this study, a nicotine and NA-degrading bacterium-strain JQ581-was isolated from sediment from the East China Sea and identified as a member of Pseudomonas putida based on morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. The relationship between growth and nicotine/NA degradation suggested that strain JQ581 was a good candidate for applications in the bioaugmentation treatment of nicotine/NA contamination. The degradation intermediates of nicotine are pseudooxynicotine (PN) and 3-succinoyl-pyridine (SP) based on UV, high performance liquid chromatography, and liquid chromatography-mass spectrometry analyses. However, 6-hydroxy-3-succinoyl-pyridine (HSP) was not detected. NA degradation intermediates were identified as 6-hydroxynicotinic acid (6HNA). The whole genome of strain JQ581 was sequenced and analyzed. Genome sequence analysis revealed that strain JQ581 contained the gene clusters for nicotine and NA degradation. This is the first report where a marine-derived Pseudomonas strain had the ability to degrade nicotine and NA simultaneously.

  3. Effect of silver nanoparticles and silver ions on growth and adaptive response mechanisms of Pseudomonas putida mt-2.

    Science.gov (United States)

    Hachicho, Nancy; Hoffmann, Philipp; Ahlert, Kristin; Heipieper, Hermann J

    2014-06-01

    The distribution and use of nanoparticles increased rapidly during the last years, while the knowledge about mode of action, ecological tolerance and biodegradability of these chemicals is still insufficient. The effect of silver nanoparticles (AgNP) and free silver ions (Ag(+) , AgNO3 ) on Pseudomonas putida mt-2 as one of the best described bacterial strains for stress response were investigated. The effective concentration (EC50) causing 50% growth inhibition for AgNP was about 250 mg L(-1) , whereas this was only 0.175 mg L(-1) for AgNO3 . However, when calculating the amount of free silver ions released from AgNP both tested compounds showed very similar results. Therefore, the antibacterial activity of AgNP can be explained and reduced, respectively, to the amount of silver ions released from the nanoparticles. Both tested compounds showed a strong activation of the unique membrane adaptive response of Pseudomonas strains, the cis-trans isomerization of unsaturated fatty acids, whereas another important adaptive response of these bacteria, changes in cell surface hydrophobicity, measured as water contact angle, was not activated. These results are important informations for the estimation of environmental tolerance of newly developed, active ingredients like silver nanoparticles. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  4. Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440.

    Directory of Open Access Journals (Sweden)

    Jana Hoffmann

    Full Text Available A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.

  5. Pseudomonas putida growing at low temperature shows increased levels of CrcZ and CrcY sRNAs, leading to reduced Crc-dependent catabolite repression.

    Science.gov (United States)

    Fonseca, Pilar; Moreno, Renata; Rojo, Fernando

    2013-01-01

    The Crc protein of Pseudomonas inhibits the expression of genes involved in the transport and assimilation of a number of non-preferred carbon sources when preferred substrates are available, thus coordinating carbon metabolism. Crc acts by binding to target mRNAs, inhibiting their translation. In Pseudomonas putida, the amount of free Crc available is controlled by two sRNAs, CrcY and CrcZ, which bind to and sequester Crc. The levels of these sRNAs vary according to metabolic conditions. Pseudomonas putida grows optimally at 30°C, but can also thrive at 10°C. The present work shows that when cells grow exponentially at 10°C, the repressive effect of Crc on many genes is significantly reduced compared with that seen at 30°C. Total Crc levels were similar at both temperatures, but those of CrcZ and CrcY were significantly higher at 10°C. Therefore, Crc-mediated repression may, at least in part, be reduced at 10°C because the fraction of Crc protein sequestered by CrcZ and CrcY is larger, reducing the amount of free Crc available to bind its targets. This may help P. putida to face cold stress. The results reported might help understanding the behaviour of this bacterium in bioremediation or rhizoremediation strategies at low temperatures. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  6. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

    DEFF Research Database (Denmark)

    Dueholm, Morten Simonsen; Søndergaard, Mads; Nilsson, Martin

    2013-01-01

    . fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently......The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P...

  7. Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR regulator of Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Max Chavarría

    2014-01-01

    Full Text Available Fructose-1-phosphate (F1P is the preferred effector of the catabolite repressor/activator (Cra protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP was submitted to mobility shift assays with target DNA sequences (the PfruB promoter and candidate effectors fructose-1,6-bisphosphate (FBP, glucose 6-phosphate (G6P, and fructose-6-phosphate (F6P. 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between CraPP and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that CraPP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of CraPP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

  8. Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.

    Science.gov (United States)

    Chavarría, Max; Durante-Rodríguez, Gonzalo; Krell, Tino; Santiago, César; Brezovsky, Jan; Damborsky, Jiri; de Lorenzo, Víctor

    2014-01-01

    Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra(PP)) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra(PP) and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra(PP) represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra(PP) for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

  9. Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR

    International Nuclear Information System (INIS)

    Maenaka, Katsumi; Fukushi, Kouji; Aramaki, Hironori; Shirakihara, Yasuo

    2005-01-01

    The P. putida cytochrome P450cam operon repressor CamR has been expressed in E. coli and crystallized in space group P2 1 2 1 2. The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer d-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12–14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P i , 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl 2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2 1 2 1 2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 Å. Native and selenomethionyl derivative data sets were collected to 3 Å resolution at SPring-8 and the Photon Factory

  10. Integrated organic-aqueous biocatalysis and product recovery for quinaldine hydroxylation catalyzed by living recombinant Pseudomonas putida.

    Science.gov (United States)

    Ütkür, F Ozde; Thanh Tran, Tan; Collins, Jonathan; Brandenbusch, Christoph; Sadowski, Gabriele; Schmid, Andreas; Bühler, Bruno

    2012-07-01

    In an earlier study, biocatalytic carbon oxyfunctionalization with water serving as oxygen donor, e.g., the bioconversion of quinaldine to 4-hydroxyquinaldine, was successfully achieved using resting cells of recombinant Pseudomonas putida, containing the molybdenum-enzyme quinaldine 4-oxidase, in a two-liquid phase (2LP) system (Ütkür et al. J Ind Microbiol Biotechnol 38:1067-1077, 2011). In the study reported here, key parameters determining process performance were investigated and an efficient and easy method for product recovery was established. The performance of the whole-cell biocatalyst was shown not to be limited by the availability of the inducer benzoate (also serving as growth substrate) during the growth of recombinant P. putida cells. Furthermore, catalyst performance during 2LP biotransformations was not limited by the availability of glucose, the energy source to maintain metabolic activity in resting cells, and molecular oxygen, a possible final electron acceptor during quinaldine oxidation. The product and the organic solvent (1-dodecanol) were identified as the most critical factors affecting biocatalyst performance, to a large extent on the enzyme level (inhibition), whereas substrate effects were negligible. However, none of the 13 alternative solvents tested surpassed 1-dodecanol in terms of toxicity, substrate/product solubility, and partitioning. The use of supercritical carbon dioxide for phase separation and an easy and efficient liquid-liquid extraction step enabled 4-hydroxyquinaldine to be isolated at a purity of >99.9% with recoveries of 57 and 84%, respectively. This study constitutes the first proof of concept on an integrated process for the oxyfunctionalization of toxic substrates with a water-incorporating hydroxylase.

  11. Inoculation effects of Pseudomonas putida, Gluconacetobacter azotocaptans, and Azospirillum lipoferum on corn plant growth under greenhouse conditions.

    Science.gov (United States)

    Mehnaz, Samina; Lazarovits, George

    2006-04-01

    Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for

  12. Responses of Pseudomonas putida to Zinc Excess Determined at the Proteome Level: Pathways Dependent and Independent of ColRS.

    Science.gov (United States)

    Mumm, Karl; Ainsaar, Kadi; Kasvandik, Sergo; Tenson, Tanel; Hõrak, Rita

    2016-12-02

    Zinc is an important micronutrient for bacteria, but its excess is toxic. Recently, the ColRS two-component system was shown to detect and respond to zinc excess and to contribute to zinc tolerance of Pseudomonas putida. Here, we applied a label-free whole-cell proteome analysis to compare the zinc-induced responses of P. putida and colR knockout. We identified dozens of proteins that responded to zinc in a ColR-independent manner, among others, known metal efflux systems CzcCBA1, CzcCBA2, CadA2 and CzcD. Nine proteins were affected in a ColR-dependent manner, and besides known ColR targets, four new candidates for ColR regulon were identified. Despite the relatively modest ColR-dependent changes of wild-type, colR deficiency resulted in drastic proteome alterations, with 122 proteins up- and 62 down-regulated by zinc. This zinc-promoted response had remarkable overlap with the alternative sigma factor AlgU-controlled regulon in P. aeruginosa. The most prominent hallmark was a high induction of alginate biosynthesis proteins and regulators. This response likely alleviates the zinc stress, as the AlgU-regulated alginate regulator AmrZ was shown to contribute to zinc tolerance of colR knockout. Thus, the ColRS system is important for zinc homeostasis, and in its absence, alternative stress response pathways are activated to support the zinc tolerance.

  13. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

    Directory of Open Access Journals (Sweden)

    Libera Latino

    Full Text Available A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31 is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10 with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

  14. Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process.

    Science.gov (United States)

    Elbahloul, Yasser; Steinbüchel, Alexander

    2009-02-01

    The suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO) was investigated in this study. Three fed-batch cultivations of P. putida GPo1 at the 350- or 400-liter scale in a bioreactor with a capacity of 650 liters were done in mineral salts medium containing initially 20 mM sodium octanoate as the carbon source. The feeding solution included ammonium octanoate, which was fed at a relatively low concentration to promote PHO accumulation under nitrogen-limited conditions. During cultivation, the pH was regulated by addition of NaOH, NH(4)OH, or octanoic acid, which was used as an additional carbon source. Partial O(2) pressure (pO(2)) was adjusted to 20 to 40% by controlling the airflow and stirrer speed. Under the optimized conditions, P. putida GPo1 was able to grow to cell densities as high as 18, 37, and 53 g cells (dry mass) (CDM) per liter containing 49, 55, and 60% (wt/wt) of PHO, respectively. The resulting 40 kg CDM from these three cultivations was used directly for extraction of PHO. Three different methods of extraction of PHO were applied. From these, only acetone extraction showed better performance and resulted in 94% recovery of the PHO contents of cells. A novel mixture of precipitation solvents composed of 70% (vol/vol) methanol and 70% (vol/vol) ethanol was identified in this study. The ratio of PHO concentrate to the mixture was 0.2:1 (vol/vol) and allowed complete precipitation of PHO as white flakes. However, at a ratio of 1:1 (vol/vol) of the solvent mixture to PHO concentrate, a highly purified PHO was obtained. Precipitation yielded a dough-like polymeric material which was cast into thin layers and then shredded into small strips to allow evaporation of the remaining solvents. Gas chromatographic analysis revealed a purity of about 99% +/- 0.2% (wt/wt) of the polymer, which consisted mainly of 3-hydroxyoctanoic acid (96 mol%).

  15. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis

    DEFF Research Database (Denmark)

    Johansen, Helle Krogh; Gøtzsche, Peter C

    2013-01-01

    Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed.......Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed....

  16. Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol.

    Science.gov (United States)

    Simon, Oliver; Klebensberger, Janosch; Mükschel, Björn; Klaiber, Iris; Graf, Nadja; Altenbuchner, Josef; Huber, Armin; Hauer, Bernhard; Pfannstiel, Jens

    2015-06-03

    To increase the efficiency of biocatalysts a thorough understanding of the molecular response of the biocatalyst to precursors, products and environmental conditions applied in bioconversions is essential. Here we performed a comprehensive proteome and phospholipid analysis to characterize the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. Using complementary quantitative proteomics approaches we were able to identify and quantify 1467 proteins, corresponding to 28% of the total KT2440 proteome. 256 proteins were altered in abundance in response to n-butanol. The proteome response entailed an increased abundance of enzymes involved in n-butanol degradation including quinoprotein alcohol dehydrogenases, aldehyde dehydrogenases and enzymes of fatty acid beta oxidation. From these results we were able to construct a pathway for the metabolism of n-butanol in P. putida. The initial oxidation of n-butanol is catalyzed by at least two quinoprotein ethanol dehydrogenases (PedE and PedH). Growth of mutants lacking PedE and PedH on n-butanol was significantly impaired, but not completely inhibited, suggesting that additional alcohol dehydrogenases can at least partially complement their function in KT2440. Furthermore, phospholipid profiling revealed a significantly increased abundance of lyso-phospholipids in response to n-butanol, indicating a rearrangement of the lipid bilayer. n-butanol is an important bulk chemical and a promising alternative to gasoline as a transportation fuel. Due to environmental concerns as well as increasing energy prices there is a growing interest in sustainable and cost-effective biotechnological production processes for the production of bulk chemicals and transportation fuels from renewable resources. n-butanol fermentation is well established in Clostridiae, but the efficiency of n-butanol production is mainly limited by its toxicity. Therefore bacterial strains with

  17. Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

    NARCIS (Netherlands)

    Braun, P; Bitter, W; Tommassen, J

    2000-01-01

    The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes

  18. Medium-chain-length polyhydroxyalkanoates synthesis by Pseudomonas putida KT2440 relA/spoT mutant: bioprocess characterization and transcriptome analysis.

    Science.gov (United States)

    Mozejko-Ciesielska, Justyna; Dabrowska, Dorota; Szalewska-Palasz, Agnieszka; Ciesielski, Slawomir

    2017-12-01

    Pseudomonas putida KT2440 is a model bacteria used commonly for medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production using various substrates. However, despite many studies conducted on P. putida KT2440 strain, the molecular mechanisms of leading to mcl-PHAs synthesis in reaction to environmental stimuli are still not clear. The rearrangement of the metabolism in response to environmental stress could be controlled by stringent response that modulates the transcription of many genes in order to promote survival under nutritional deprivation conditions. Therefore, in this work we investigated the relation between mcl-PHAs synthesis and stringent response. For this study, a relA/spoT mutant of P. putida KT2440, unable to induce the stringent response, was used. Additionally, the transcriptome of this mutant was analyzed using RNA-seq in order to examine rearrangements of the metabolism during cultivation. The results show that the relA/spoT mutant of P. putida KT2440 is able to accumulate mcl-PHAs in both optimal and nitrogen limiting conditions. Nitrogen starvation did not change the efficiency of mcl-PHAs synthesis in this mutant. The transition from exponential growth to stationary phase caused significant upregulation of genes involved in transport system and nitrogen metabolism. Transcriptional regulators, including rpoS, rpoN and rpoD, did not show changes in transcript abundance when entering the stationary phase, suggesting their limited role in mcl-PHAs accumulation during stationary phase.

  19. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    International Nuclear Information System (INIS)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

    2011-01-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

  20. Potential of the TCE-degrading endophyte Pseudomonas putida W619-TCE to improve plant growth and reduce TCE phytotoxicity and evapotranspiration in poplar cuttings

    Energy Technology Data Exchange (ETDEWEB)

    Weyens, N.; van der Lelie, D.; Truyens, S.; Dupae, J.; Newman, L.; Taghavi, S.; Carleer, R.; Vangronsveld, J.

    2010-09-01

    The TCE-degrading poplar endophyte Pseudomonas putida W619-TCE was inoculated in poplar cuttings, exposed to 0, 200 and 400 mg l{sup -1} TCE, that were grown in two different experimental setups. During a short-term experiment, plants were grown hydroponically in half strength Hoagland nutrient solution and exposed to TCE for 3 days. Inoculation with P. putida W619-TCE promoted plant growth, reduced TCE phytotoxicity and reduced the amount of TCE present in the leaves. During a mid-term experiment, plants were grown in potting soil and exposed to TCE for 3 weeks. Here, inoculation with P. putida W619-TCE had a less pronounced positive effect on plant growth and TCE phytotoxicity, but resulted in strongly reduced amounts of TCE in leaves and roots of plants exposed to 400 mg l{sup -1} TCE, accompanied by a lowered evapotranspiration of TCE. Dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), which are known intermediates of TCE degradation, were not detected. The endophyte P. putida W619-TCE degrades TCE during its transport through the xylem, leading to reduced TCE concentrations in poplar, and decreased TCE evapotranspiration.

  1. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia); Kivisaar, Maia, E-mail: maiak@ebc.ee [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia)

    2011-09-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif{sup r} mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

  2. Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

    Science.gov (United States)

    Tozakidis, Iasson E P; Brossette, Tatjana; Lenz, Florian; Maas, Ruth M; Jose, Joachim

    2016-06-10

    The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps. The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h. We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.

  3. FleQ of Pseudomonas putida KT2440 is a multimeric cyclic diguanylate binding protein that differentially regulates expression of biofilm matrix components.

    Science.gov (United States)

    Molina-Henares, María Antonia; Ramos-González, María Isabel; Daddaoua, Abdelali; Fernández-Escamilla, Ana María; Espinosa-Urgel, Manuel

    2017-01-01

    The intracellular signal molecule cyclic di-GMP (c-di-GMP) is an important element in regulation of biofilm formation by bacteria. In Pseudomonas aeruginosa, FleQ functions as a c-di-GMP-dependent transcriptional regulator of expression of flagellar genes and the exopolysaccharide (EPS) Pel, a component of the biofilm extracellular matrix. In the plant-beneficial bacterium Pseudomonas putida KT2440, a mutation in fleQ reduces biofilm formation and colonization of plant surfaces. Using isothermal titration calorimetry and electrophoretic mobility shift assays, we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes. Data obtained by analytical gel filtration and ultracentrifugation indicate that FleQ is in multiple oligomeric states in solution (dimers, tetramers and hexamers), which do not show altered equilibrium in the presence of c-di-GMP. DNA binding is independent of c-diGMP, although it is favored by the second messenger in the case of the promoter of the operon responsible for synthesis of the species-specific EPS Pea. Analysis of expression using transcriptional fusions showed an influence of FleQ upon two of the four EPS operons under regular growth conditions. Finally, a consensus sequence for promoter recognition by FleQ in P. putida is also proposed. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. A lung segmental model of chronic Pseudomonas infection in sheep.

    Directory of Open Access Journals (Sweden)

    David Collie

    Full Text Available Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep.Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>10(4 cfu/g.The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.

  5. A lung segmental model of chronic Pseudomonas infection in sheep.

    Science.gov (United States)

    Collie, David; Govan, John; Wright, Steven; Thornton, Elisabeth; Tennant, Peter; Smith, Sionagh; Doherty, Catherine; McLachlan, Gerry

    2013-01-01

    Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep. Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>10(4) cfu/g). The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.

  6. Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Kremling Andreas

    2012-12-01

    Full Text Available Abstract Background Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru and a second branch (PTSNtr, which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. Results A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. Conclusion Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.

  7. Significantly enhanced substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma and cell immobilization.

    Science.gov (United States)

    Dong, Ting-Ting; Gong, Jin-Song; Gu, Bing-Chen; Zhang, Qiang; Li, Heng; Lu, Zhen-Ming; Lu, Mao-Lin; Shi, Jin-Song; Xu, Zheng-Hong

    2017-11-01

    The objective of the study was to enhance the substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma (ARTP) and cell immobilization. The mutant library was constructed by ARTP and rapidly screened by an OPA-TCA microscale reaction. A mutant strain of mut-D3 was obtained and its optimum substrate concentration was improved to 150mM from 100mM. It could accumulate 189g/L nicotinic acid (NA) from 3-cyanopyridine (3-CP), which was increased by 42% compared with that of wild type (WT). Additionally, composite immobilization of mut-D3 was performed and SA-PVA immobilized cells could catalyze 250mM 3-CP each batch with finally accumulating 346g/L NA, while free cells accumulated 175g/L NA. These results indicated that the free or immobilized catalysts of mut-D3 could serve as a good choice for NA production. This is the first report on mutation breeding of nitrilase-producing microorganisms by ARTP. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.

    Science.gov (United States)

    Blaschke, Lisa; Wagner, Wenke; Werkmeister, Christina; Wild, Marion; Gihring, Adrian; Rupp, Steffen; Zibek, Susanne

    2017-01-10

    Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Preparation and characterization of monodisperse microcapsules with alginate and bentonite via external gelation technique encapsulating Pseudomonas putida Rs-198.

    Science.gov (United States)

    Li, Xuan; Wu, Zhansheng; He, Yanhui; Ye, Bang-Ce; Wang, Jun

    2017-10-01

    This paper evaluated the external gelation technique for preparing microcapsules. The microcapsules were consisted of Pseudomonas putida Rs-198 (Rs-198) core and sodium alginate (NaAlg)-bentonite (Bent) shell. Different emulsification rotation speeds and core/shell ratios were used to prepare the microcapsules of each formulation. The near-spherical microcapsules were monodisperse with a mean diameter of 25-100 μm and wrinkled surfaces. Fourier transform infrared spectrophotometry (FTIR) and thermogravimetric analysis (TGA) revealed the physical mixture of the wall material and the superior thermal stability of the microcapsules. Percentage yield, water content, and encapsulation efficiency were evaluated and correlated with the changes in emulsification rotation speed and core/shell ratio. In vitro release experiments demonstrated that 60% of the bacteria were released from the NaAlg-Bent microcapsules within three days. Considerably better survival was observed for encapsulated cells compared to free cells, especially in pH 4.0 and 10.0. In summary, the desired properties of microcapsules can be obtained by external gelation technique and the microcapsules on the bacteria had a good protective effect.

  10. Growth stimulation of Bacillus cereus and Pseudomonas putida using nanostructured ZnO thin film as transducer element

    Energy Technology Data Exchange (ETDEWEB)

    Loukanov, Alexandre, E-mail: loukanov@mail.saitama-u.ac.jp [Saitama University, Department of Chemistry, Faculty of Science (Japan); Filipov, Chavdar [University of Forestry, Department of Infectious pathology, hygiene, technology and control of food stuffs of animal origin, Faculty of Veterinary Medicine (Bulgaria); Valcheva, Violeta [Bulgarian Academy of Science, Department of Infectious Diseases, Institute of microbiology (Bulgaria); Lecheva, Marta [University of Mining and Geology “St. Ivan Rilski”, Laboratory of Engineering NanoBiotechnology, Department of Engineering Geoecology (Bulgaria); Emin, Saim [University of Nova Gorica, Materials Research Laboratory (Slovenia)

    2015-04-15

    The semiconductor zinc oxide nanomaterial (ZnO or ZnO:H) is widely used in advanced biosensor technology for the design of highly-sensitive detector elements for various applications. In the attempt to evaluate its effect on common microorganisms, two types of nanostructured transducer films have been used (average diameter 600–1000 nm). They have been prepared by using both wet sol–gel method and magnetron sputtering. Their polycrystalline structure and specific surface features have been analyzed by X-ray diffraction (XRD), scanning electron microscope, and atomic force microscope. The assessment of growth stimulation of bacteria was determined using epifluorescent microscope by cell staining with Live/Dead BacLight kit. In our experiments, the growth stimulation of Gram-positive and Gram-negative bacteria on nanostructured ZnO film is demonstrated by Bacillus cereus and Pseudomonas putida. These two bacterial species have been selected, because they are well known and studied in biosensor technologies, with structural difference of their cell walls. These pathogens are easy for with common source in the liquid food or some commercial products. Our data has revealed that the method of transducer film preparation influences strongly bacterial inhibition and division. These results present the transforming signal precisely, when ZnO is used in biosensor applications.

  11. Crystallization and Preliminary X-ray Diffraction Analysis of Recombinant Chlorocatechol 1 2-dioxygenase from Pseudomonas Putida

    Energy Technology Data Exchange (ETDEWEB)

    J Rustiguel; M Pinheiro; A Araujo; M Nonato

    2011-12-31

    Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapor-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6{sub 1}22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 {angstrom} resolution.

  12. Growth stimulation of Bacillus cereus and Pseudomonas putida using nanostructured ZnO thin film as transducer element

    Science.gov (United States)

    Loukanov, Alexandre; Filipov, Chavdar; Valcheva, Violeta; Lecheva, Marta; Emin, Saim

    2015-04-01

    The semiconductor zinc oxide nanomaterial (ZnO or ZnO:H) is widely used in advanced biosensor technology for the design of highly-sensitive detector elements for various applications. In the attempt to evaluate its effect on common microorganisms, two types of nanostructured transducer films have been used (average diameter 600-1000 nm). They have been prepared by using both wet sol-gel method and magnetron sputtering. Their polycrystalline structure and specific surface features have been analyzed by X-ray diffraction (XRD), scanning electron microscope, and atomic force microscope. The assessment of growth stimulation of bacteria was determined using epifluorescent microscope by cell staining with Live/Dead BacLight kit. In our experiments, the growth stimulation of Gram-positive and Gram-negative bacteria on nanostructured ZnO film is demonstrated by Bacillus cereus and Pseudomonas putida. These two bacterial species have been selected, because they are well known and studied in biosensor technologies, with structural difference of their cell walls. These pathogens are easy for with common source in the liquid food or some commercial products. Our data has revealed that the method of transducer film preparation influences strongly bacterial inhibition and division. These results present the transforming signal precisely, when ZnO is used in biosensor applications.

  13. Broad host range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    Calero Valdayo, Patricia; Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2016-01-01

    of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine......Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set...... and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest...

  14. A newly isolated Pseudomonas putida S-1 strain for batch-mode-propanethiol degradation and continuous treatment of propanethiol-containing waste gas

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Dong-Zhi, E-mail: cdz@zjut.edu.cn [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Sun, Yi-Ming; Han, Li-Mei [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Chen, Jing [College of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316004 (China); Ye, Jie-Xu; Chen, Jian-Meng [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China)

    2016-01-25

    Highlights: • A novel strain capable of effectively degrading 1-propanethiol (PT) was isolated. • Cells could be feasibly cultured in nutrition-rich media for PT degradation. • A possible pathway for PT degradation was proposed. • Pseudomonas putida S-1 could degrade mixed pollutants with diauxic growth. • Continuous removal of gaseous PT with or without isopropanol was demonstrated. - Abstract: Pseudomonas putida S-1 was isolated from activated sludge. This novel strain was capable of degrading malodorous 1-propanethiol (PT). PT degradation commenced with no lag phase by cells pre-grown in nutrition-rich media, such as Luria–Bertani (LB), and PT-contained mineral medium at specific growth rates of 0.10–0.19 h{sup −1}; this phenomenon indicated the operability of a large-scale cell culture. A possible PT degradation pathway was proposed on the basis of the detected metabolites, including dipropyl disulfide, 3-hexanone, 2-hexanone, 3-hexanol, 2-hexanol, S{sup 0}, SO{sub 4}{sup 2−}, and CO{sub 2}. P. putida S-1 could degrade mixed pollutants containing PT, diethyl disulfide, isopropyl alcohol, and acetaldehyde, and LB-pre-cultured cells underwent diauxic growth. Waste gas contaminated with 200–400 mg/m{sup 3} PT was continuously treated by P. putida S-1 pre-cultured in LB medium in a completely stirred tank reactor. The removal efficiencies exceeded 88% when PT stream was mixed with 200 mg/m{sup 3} isopropanol; by contrast, the removal efficiencies decreased to 60% as the empty bed residence time was shortened from 40 s to 20 s.

  15. Influence of growth stage on activities of polyhydroxyalkanoate (PHA polymerase and PHA depolymerase in Pseudomonas putida U

    Directory of Open Access Journals (Sweden)

    Zinn Manfred

    2010-10-01

    Full Text Available Abstract Background Medium chain length (mcl- polyhydroxyalkanoates (PHA are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC and depolymerase (PhaZ. Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary. Results Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI from the granules PhaC activity decreased further. Conclusion Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of

  16. Pseudomonas putida as a microbial cell factory

    DEFF Research Database (Denmark)

    Wigneswaran, Vinoth

    The extensive use of fossil fuels has a severe influence on the environment. In order to reduce the dependency on these limited resources and to protect the environment substantial effort is being made to implement renewable resources. One part of this transition is to develop methods for sustain......The extensive use of fossil fuels has a severe influence on the environment. In order to reduce the dependency on these limited resources and to protect the environment substantial effort is being made to implement renewable resources. One part of this transition is to develop methods....... The applicability of glycerol as a substrate was also investigated. Since glycerol is a poor substrate adaptive evolution was made in order to improve the capabilities of P. putida to proliferate on glycerol. The evolved lineages all had significantly increased growth rate, enhanced cell density and reduced lag...... phase. The genomic alterations were identified by genome sequencing and revealed parallel evolution. Glycerol was also shown to be able to support biofilm growth and as a result of this it can be used as an alternative substrate for producing biochemicals in conventional and biofilm reactors. The use...

  17. Isolation of a gene responsible for the oxidation of trans-anethole to para-anisaldehyde by Pseudomonas putida JYR-1 and its expression in Escherichia coli.

    Science.gov (United States)

    Han, Dongfei; Ryu, Ji-Young; Kanaly, Robert A; Hur, Hor-Gil

    2012-08-01

    A plasmid, pTA163, in Escherichia coli contained an approximately 34-kb gene fragment from Pseudomonas putida JYR-1 that included the genes responsible for the metabolism of trans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyze trans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter in E. coli catalyzed oxidative cleavage of a propenyl group of trans-anethole to an aldehyde group, resulting in the production of para-anisaldehyde, and this gene was designated tao (trans-anethole oxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein of Agrobacterium vitis S4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water into p-anisaldehyde using (18)O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase from Pseudomonas putida IE27 and Pseudomonas nitroreducens Jin1, TAO from P. putida JYR-1 catalyzed isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts of E. coli (pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

  18. Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain

    OpenAIRE

    Galvão, Teca Calcagno; de Lorenzo, Víctor

    2005-01-01

    A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin ...

  19. Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442

    DEFF Research Database (Denmark)

    Tripathi, Lakshmi; Wu, Lin-Ping; Chen, Jinchun

    2012-01-01

    BACKGROUND: Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB...... structural related mechanical properties.......) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. RESULTS: The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR...

  20. Expression of the diguanylate cyclase GcbA is regulated by FleQ in response to cyclic di-GMP in Pseudomonas putida KT2440.

    Science.gov (United States)

    Xiao, Yujie; Nie, Hailing; Liu, Huizhong; Chen, Wenli; Huang, Qiaoyun

    2016-12-01

    Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA is a well conserved DGC among Pseudomonas species, and has been reported to influence biofilm formation and flagellar motility in Pseudomonas fluorescens and Pseudomonas aeruginosa. Here we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Directory of Open Access Journals (Sweden)

    Ahmad Mohammed Gumel

    Full Text Available The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW basis were observed when fatty acids ranging from octanoic acid (C(8:0 to oleic acid (C(18:1 were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d of 264.6 to 318.8 (± 0.2 (oC, melting temperature (T(m of 43. (± 0.2 (oC, glass transition temperature (T(g of -1.0 (± 0.2 (oC and apparent melting enthalpy of fusion (ΔH(f of 100.9 (± 0.1 J g(-1.

  2. Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5▿ †

    Science.gov (United States)

    Yu, Chi Li; Louie, Tai Man; Summers, Ryan; Kale, Yogesh; Gopishetty, Sridhar; Subramanian, Mani

    2009-01-01

    Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria. PMID:19447909

  3. Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily.

    Science.gov (United States)

    Zahniser, Megan P D; Prasad, Shreenath; Kneen, Malea M; Kreinbring, Cheryl A; Petsko, Gregory A; Ringe, Dagmar; McLeish, Michael J

    2017-03-01

    Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Genetic analysis of plant endophytic Pseudomonas putida BP25 and chemo-profiling of its antimicrobial volatile organic compounds.

    Science.gov (United States)

    Sheoran, Neelam; Valiya Nadakkakath, Agisha; Munjal, Vibhuti; Kundu, Aditi; Subaharan, Kesavan; Venugopal, Vibina; Rajamma, Suseelabhai; Eapen, Santhosh J; Kumar, Aundy

    2015-04-01

    Black pepper associated bacterium BP25 was isolated from root endosphere of apparently healthy cultivar Panniyur-5 that protected black pepper against Phytophthora capsici and Radopholus similis - the major production constraints. The bacterium was characterized and mechanisms of its antagonistic action against major pathogens are elucidated. The polyphasic phenotypic analysis revealed its identity as Pseudomonas putida. Multi locus sequence typing revealed that the bacterium shared gene sequences with several other isolates representing diverse habitats. Tissue localization assays exploiting green fluorescence protein expression clearly indicated that PpBP25 endophytically colonized not only its host plant - black pepper, but also other distantly related plants such as ginger and arabidopsis. PpBP25 colonies could be enumerated from internal tissues of plants four weeks post inoculation indicated its stable establishment and persistence in the plant system. The bacterium inhibited broad range of pathogens such as Phytophthora capsici, Pythium myriotylum, Giberella moniliformis, Rhizoctonia solani, Athelia rolfsii, Colletotrichum gloeosporioides and plant parasitic nematode, Radopholus similis by its volatile substances. GC/MS based chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1-3-Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25 metabolite. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence of aromatic compounds such as 1-Undecene;Disulfide dimethyl; Pyrazine, methyl-Pyrazine, 2,5-dimethyl-; Isoamyl alcohol; Pyrazine, methyl-; Dimethyl trisulfide, etc. The work paved way for profiling of broad spectrum antimicrobial VOCs in endophytic PpBP25 for crop protection. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. Inhibited transport of graphene oxide nanoparticles in granular quartz sand coated with Bacillus subtilis and Pseudomonas putida biofilms.

    Science.gov (United States)

    He, Jian-Zhou; Wang, Deng-Jun; Fang, Huan; Fu, Qing-Long; Zhou, Dong-Mei

    2017-02-01

    Increasing production and use of graphene oxide nanoparticles (GONPs) boost their wide dissemination in the subsurface environments where biofilms occur ubiquitously, representative of the physical and chemical heterogeneities. This study aimed at investigating the influence of Gram-positive Bacillus subtilis (BS) and Gram-negative Pseudomonas putida (PP) biofilms on the transport of GONPs under different ionic strengths (0.1, 0.5, and 1.0 mM CaCl 2 ) at neutral pH 7.2 in water-saturated porous media. Particularly, the X-ray micro-computed tomography was used to quantitatively characterize the pore structures of sand columns in the presence and absence of biofilms. Our results indicated that the presence of biofilms reduced the porosity and narrowed down the pore sizes of packed columns. Transport experiments in biofilm-coated sand showed that biofilms, irrespective of bacterial species, significantly inhibited the mobility of GONPs compared to that in cleaned sand. This could be due to the Ca 2+ complexation, increased surface roughness and charge heterogeneities of collectors, and particularly enhanced physical straining caused by biofilms. The two-site kinetic retention model-fitted value of maximum solid-phase concentration (S max2 ) for GONPs was higher for biofilm-coated sand than for cleaned sand, demonstrating that biofilms act as favorable sites for GONPs retention. Our findings presented herein are important to deepen our current understanding on the nature of particle-collector interactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids.

    Science.gov (United States)

    Guzik, Maciej W; Narancic, Tanja; Ilic-Tomic, Tatjana; Vojnovic, Sandra; Kenny, Shane T; Casey, William T; Duane, Gearoid F; Casey, Eoin; Woods, Trevor; Babu, Ramesh Padamati; Nikodinovic-Runic, Jasmina; O'Connor, Kevin E

    2014-08-01

    Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (β-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7. © 2014 The Authors.

  7. Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-07-01

    Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

  8. Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis.

    Science.gov (United States)

    Xia, Zhenyuan; Zhang, Wei; Lei, Liping; Liu, Xingzhong; Wei, Hai-Lei

    2015-08-01

    Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.

  9. A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

    Directory of Open Access Journals (Sweden)

    Zinn Manfred

    2011-04-01

    Full Text Available Abstract Background The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. Results To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate, PHA-unrelated (gluconate and poor PHA substrate (citrate. The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. Conclusions The study illustrates that the recruitment of a pleiotropic mutation, whose effects might

  10. Quantitative 'Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-01-01

    Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the

  11. Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V.; Zhang, Xiangli; Fristensky, Brian; Cicek, Nazim; Sparling, Richard; Levin, David. B.

    2015-01-01

    Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. ‘Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the ‘Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining

  12. Quantitative 'Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids.

    Directory of Open Access Journals (Sweden)

    Jilagamazhi Fu

    Full Text Available Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA. The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1 glycerol transportation; 2 enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA. Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in

  13. A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

    Science.gov (United States)

    2011-01-01

    Background The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. Results To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. Conclusions The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into

  14. Metabolomics Analysis Reveals the Participation of Efflux Pumps and Ornithine in the Response of Pseudomonas putida DOT-T1E Cells to Challenge with Propranolol.

    Science.gov (United States)

    Sayqal, Ali; Xu, Yun; Trivedi, Drupad K; AlMasoud, Najla; Ellis, David I; Rattray, Nicholas J W; Goodacre, Royston

    2016-01-01

    Efflux pumps are critically important membrane components that play a crucial role in strain tolerance in Pseudomonas putida to antibiotics and aromatic hydrocarbons that result in these toxicants being expelled from the bacteria. Here, the effect of propranolol on P. putida was examined by sudden addition of 0.2, 0.4 and 0.6 mg mL-1 of this β-blocker to several strains of P. putida, including the wild type DOT-T1E and the efflux pump knockout mutants DOT-T1E-PS28 and DOT-T1E-18. Bacterial viability measurements reveal that the efflux pump TtgABC plays a more important role than the TtgGHI pump in strain tolerance to propranolol. Mid-infrared (MIR) spectroscopy was then used as a rapid, high-throughput screening tool to investigate any phenotypic changes resulting from exposure to varying levels of propranolol. Multivariate statistical analysis of these MIR data revealed gradient trends in resultant ordination scores plots, which were related to the concentration of propranolol. MIR illustrated phenotypic changes associated with the presence of this drug within the cell that could be assigned to significant changes that occurred within the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during the growth of bacteria following toxic perturbation with the same concentration levels of propranolol. Metabolic profiling revealed that ornithine, which was only produced by P. putida cells in the presence of propranolol, presents itself as a major metabolic feature that has important functions in propranolol stress tolerance mechanisms within this highly significant and environmentally relevant species of bacteria.

  15. Toxicity of fungal-generated silver nanoparticles to soil-inhabiting Pseudomonas putida KT2440, a rhizospheric bacterium responsible for plant protection and bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Indarchand R. [Nanobiotechnology Laboratory, Department of Biotechnology, S.G.B. Amravati University, Amravati 444602, Maharashtra (India); Department of Biotechnology, Institute of Science, Nipat Niranjan Nagar, Caves Road, Aurangabad 431004, Maharashtra (India); Anderson, Anne J. [Department of Biology, Utah State University, Logan, Utah 84321 (United States); Rai, Mahendra, E-mail: mahendrarai@sgbau.ac.in [Nanobiotechnology Laboratory, Department of Biotechnology, S.G.B. Amravati University, Amravati 444602, Maharashtra (India); Laboratório de Química Biológica, Instituto de Química, UNICAMP, Cidade Universitária “Zefferino Vaz” Barão Geraldo, CEP 13083-970, Caixa Postal 6150, Campinas, SP (Brazil)

    2015-04-09

    Highlights: • This study incorporates the mycosynthesis of AgNPs and their characterisation by various methods. • A first attempt demonstrating the toxicity assessment of AgNPs on beneficial soil microbe. • Use of biosensor in Pseudomonas putida KT2440, gave accurate antimicrobial results. - Abstract: Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV–vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 – a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4 μg/ml, which warrants further detailed investigations concerning toxicity.

  16. ABILITY OF BACTERIAL CONSORTIUM: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp. and Pseudomonas putida IN BIOREMEDIATION OF WASTE WATER IN CISIRUNG WASTE WATER TREATMENT PLANT

    Directory of Open Access Journals (Sweden)

    Ratu SAFITRI

    2015-10-01

    Full Text Available This study was conducted in order to determine the ability of bacterial consortium: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp., and Pseudomonas putida in bioremediation of wastewater origin Cisirung WWTP. This study uses an experimental method completely randomized design (CRD, which consists of two treatment factors (8x8 factorial design. The first factor is a consortium of bacteria (K, consisting of 8 level factors (k1, k2, k3, k4, k5, k6, k7, and k8. The second factor is the time (T, consisting of a 7 level factors (t0, t1, t2, t3, t4, t5, t6, and t7. Test parameters consist of BOD (Biochemical Oxygen Demand, COD (Chemical Oxygen Demand, TSS (Total Suspended Solid, Ammonia and Population of Microbes during bioremediation. Data were analyzed by ANOVA, followed by Duncan test. The results of this study showed that the consortium of Bacillus pumilus, Bacillus subtilis, Bacillus coagulans, Nitrosomonas sp., and Pseudomonas putida with inoculum concentration of 5% (k6 is a consortium of the most effective in reducing BOD 71.93%, 64.30% COD, TSS 94.85%, and 88.58% of ammonia.

  17. Crystallization and preliminary X-ray diffraction analysis of cyclic imide hydrolase (CIH) from Pseudomonas putida YZ-26

    OpenAIRE

    Fan, Zheng; Qi, Jianxun; Shi, Yawei; Liu, Yiwei

    2011-01-01

    A recombinant cyclic imide hydrolase from P. putida YZ-26 has been crystallized by the hanging-drop method. This will be helpful in understanding the role of CIH in pyrimidine metabolism and organic acid bioconversion.

  18. Evidence for the Involvement of Lipid Rafts and Plasma Membrane Sphingolipid Hydrolases in Pseudomonas aeruginosa Infection of Cystic Fibrosis Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Domitilla Schiumarini

    2017-01-01

    Full Text Available Cystic fibrosis (CF is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF.

  19. Effects of exogenous pyoverdines on Fe availability and their impacts on Mn(II) oxidation by Pseudomonas putida GB-1

    Science.gov (United States)

    Lee, Sung-Woo; Parker, Dorothy L.; Geszvain, Kati; Tebo, Bradley M.

    2014-01-01

    Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental

  20. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

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    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  1. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Science.gov (United States)

    Jung, Heung-Chae; Kwon, Seok-Joon; Pan, Jae-Gu

    2006-01-01

    Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA) from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP) anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC). The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents. PMID:16620394

  2. Transcriptional Modulation of Transport- and Metabolism-Associated Gene Clusters Leading to Utilization of Benzoate in Preference to Glucose in Pseudomonas putida CSV86.

    Science.gov (United States)

    Choudhary, Alpa; Modak, Arnab; Apte, Shree K; Phale, Prashant S

    2017-10-01

    The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Soil isolate Pseudomonas putida CSV86 displays a unique ability to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at the glc and ben loci, respectively, as two distinct operons. When grown on glucose plus benzoate, CSV86 displayed significantly higher expression of the ben locus in the first log phase and of the glc locus in the second log phase. Kinetics of substrate uptake and metabolism matched the transcription profiles. The inability of succinate to suppress benzoate transport and metabolism resulted in coutilization of succinate and benzoate. When challenged with succinate or benzoate, glucose-grown cells showed rapid reduction in glc locus transcription, glucose transport, and metabolic activity, with succinate being more effective at the functional level. Benzoate and succinate failed to interact with or inhibit the activities of glucose transport components or metabolic enzymes. The data suggest that succinate and benzoate suppress glucose transport and metabolism at the transcription level, enabling P. putida CSV86 to preferentially metabolize benzoate. This strain thus has the potential to be an ideal host to engineer diverse metabolic pathways for efficient bioremediation. IMPORTANCE Pseudomonas strains play an important role in carbon cycling in the environment and display a hierarchy in carbon utilization: organic acids first, followed by glucose, and aromatic substrates last. This limits their exploitation for bioremediation. This study demonstrates the substrate-dependent modulation of ben and glc operons in Pseudomonas putida CSV86, wherein benzoate suppresses glucose transport and metabolism at the transcription level, leading to preferential

  3. Occurrence of pseudomonas aeruginosa in post-operative wound infection

    International Nuclear Information System (INIS)

    Oguntibeju, O.O.; Nwobu, R.A.U.

    2004-01-01

    Objective: To determine the prevalence of Pseudomonas aeruginosa in post-operative wound infection. Results: Out of the 60 bacterial isolates found in post-operative wound infection, 20 (33.3%) were Pseudomonas aeruginosa, followed by Staphylococcus aureus 13(21.7%), Klebsiella species 10(16.7%), Escherichia coli 7(11.7%), Atypical coliform 4(6.7%), Proteus species 4(6.7%), Streptococcus pyogenes 1(1.7%) and Enterococcus faecalis 1(1.7%) in the order. Pseudomonas aeruginosa infections was higher in female than male, ratio 3:2 and was found more among young and elderly debilitated patients. The in vitro sensitivity pattern of 20 isolates of Pseudomonas aeruginosa showed colistin (100%), gentamicin (75%), streptomycin (30%), and tetracycline (10%). Conclusion: The role of Pseudomonas aeruginosa as an agent of nosocomial infection is re-emphasised. (author)

  4. Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Le Meur Sylvaine

    2012-08-01

    Full Text Available Abstract Background Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs. To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. Results The genes encoding xylose isomerase (XylA and xylulokinase (XylB from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. Conclusions Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

  5. Surface display of monkey metallothionein {alpha} tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    He, Xiaochuan; Chen, Wenli; Huang, Qiaoyun [Huazhong Agricultural Univ., Wuhan (China). State Key Lab. of Agricultural Microbiology

    2012-09-15

    Monkey metallothionein {alpha} domain tandem repeats (4mMT{alpha}), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC - 10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMT{alpha}-EGFP fusion, was constructed and used to target 4mMT{alpha} and EGFP on the surface of Pseudomonas putida X4 (CCTCC - 209319). The surface location of the INAXN-4mMT{alpha}-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMT{alpha} on the engineered strains was four times higher than that of the wild-type X4. The Cd{sup 2+} accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu{sup 2+} and Zn{sup 2+}. Moreover, the surface-engineered strains could effectively bind Cd{sup 2+} under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMT{alpha}-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMT{alpha}-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants. (orig.)

  6. High performance and prospective application of xanthate-modified thiourea chitosan sponge-combined Pseudomonas putida and Talaromyces amestolkiae biomass for Pb(II) removal from wastewater.

    Science.gov (United States)

    Wang, Nana; Xu, Xingjian; Li, Haiyan; Wang, Quanying; Yuan, Lizhu; Yu, Hongwen

    2017-06-01

    Biosorption using microbes has been proved to be an efficient technology to remove heavy metals from wastewater, whereas the imperfections in mechanical property and separation limit their practical application. In this study, Pseudomonas putida I3 and Talaromyces amestolkiae Pb respectively combined with xanthate-modified thiourea chitosan sponge (PXTCS and TXTCS) were synthesized to investigate the Pb(II) removal ability from solutions. The prepared biosorbents possessed a three-dimensional macroporous structure convenient for separation. Experimental data indicated their biosorption behaviors well followed the pseudo-second-order kinetics and Langmuir isotherm model. The maximum biosorption capacities of PXTCS and TXTCS were 232.03 and 241.61mgg -1 with 40% P. putida I3 and 15% T. amestolkiae Pb, respectively. For the effects of co-existing metal ions on Pb(II) biosorption, the promoting degree followed the sequence: Zn(II)>Na(I)≈K(I)>Ca(II)>Mg(II)≈Al(III)≫Cd(II)>Fe(III). Both prepared biosorbents were effective in removing heavy metals from simulated industrial effluents containing various trace-level heavy metals or high concentration Pb(II). Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. iTRAQ-based quantitative proteomic analysis of the global response to 17β-estradiol in estrogen-degradation strain Pseudomonas putida SJTE-1

    Science.gov (United States)

    Xu, Jing; Zhang, Lei; Hou, Jingli; Wang, Xiuli; Liu, Huan; Zheng, Daning; Liang, Rubing

    2017-01-01

    Microorganism degradation is efficient to remove the steroid hormones like 17β-estradiol (E2); but their degradation mechanism and metabolic network to these chemicals are still not very clear. Here the global responses of the estrogen-degradation strain Pseudomonas putida SJTE-1 to 17β-estradiol and glucose were analyzed and compared using the iTRAQ (isobaric tags for relative and absolute quantization) strategy combined with LC-MS/MS (liquid chromatography-tandem mass spectrometry). 78 proteins were identified with significant changes in expression; 45 proteins and 33 proteins were up-regulated and down-regulated, respectively. These proteins were mainly involved in the processes of stress response, energy metabolism, transportation, chemotaxis and cell motility, and carbon metabolism, considered probably responding to 17β-estradiol and playing a role in its metabolism. The up-regulated proteins in electron transfer, energy generation and transport systems were thought crucial for efficient uptake, translocation and transformation of 17β-estradiol. The over-expression of carbon metabolism proteins indicated cells may activate related pathway members to utilize 17β-estradiol. Meanwhile, proteins functioning in glucose capture and metabolism were mostly down-regulated. These findings provide important clues to reveal the 17β-estradiol degradation mechanism in P. putida and promote its bioremediation applications. PMID:28155874

  8. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: verification and complementation of hypotheses derived from transcriptomics.

    Science.gov (United States)

    Wierckx, Nick; Ruijssenaars, Harald J; de Winde, Johannes H; Schmid, Andreas; Blank, Lars M

    2009-08-20

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism.

  9. Stochasticity of TOL plasmid catabolic promoters sets a bimodal expression regime in Pseudomonas putida mt-2 exposed to m-xylene.

    Science.gov (United States)

    Silva-Rocha, Rafael; de Lorenzo, Víctor

    2012-10-01

    The expression dynamics of the catabolic promoters of the TOL plasmid pWW0 has been examined in single cells of Pseudomonas putida mt-2 exposed to m-xylene. To this end, we employed an á la carte bi-cistronic gfp-lacZ reporter system for generating monocopy transcriptional fusions to each of the four promoters (Pr, Ps, Pu and Pm) of the regulatory network. Whereas expression of xylR (the master regulatory gene of the TOL system) behaved in a unimodal fashion, the activation of Pu and Pm displayed a high degree of stochasticity. This event resulted in a time-dependent shift in the bacterial population between two induction states without intermediate expression levels. Bimodality of the Pm promoter for the lower TOL pathway was largely due to the propagation of the Ps output throughout the network, which caused stochastic overproduction of the second regulator of the system, XylS. Surprisingly, activation of Pm by the form of XylS that was bound to the pathway intermediate 3-methylbenzoate was relatively minor. These stochastic phenomena in response to m-xylene disappeared in the stationary phase. Taken together, the data suggests that genetically homogeneous populations of P. putida mt-2 employ a strategy of phenotypic variation (metabolic bet-hedging) when confronted with mixtures of nutrients. © 2012 Blackwell Publishing Ltd.

  10. The Crc/CrcZ-CrcY global regulatory system helps the integration of gluconeogenic and glycolytic metabolism in Pseudomonas putida.

    Science.gov (United States)

    La Rosa, Ruggero; Nogales, Juan; Rojo, Fernando

    2015-09-01

    In metabolically versatile bacteria, carbon catabolite repression (CCR) facilitates the preferential assimilation of the most efficient carbon sources, improving growth rates and fitness. In Pseudomonas putida, the Crc and Hfq proteins and the CrcZ and CrcY small RNAs, which are believed to antagonize Crc/Hfq, are key players in CCR. Unlike that seen in other bacterial species, succinate and glucose elicit weak CCR in this bacterium. In the present work, metabolic, transcriptomic and constraint-based metabolic flux analyses were combined to clarify whether P. putida prefers succinate or glucose, and to identify the role of the Crc protein in the metabolism of these compounds. When provided simultaneously, succinate was consumed faster than glucose, although both compounds were metabolized. CrcZ and CrcY levels were lower when both substrates were present than when only one was provided, suggesting a role for Crc in coordinating metabolism of these compounds. Flux distribution analysis suggested that, when both substrates are present, Crc works to organize a metabolism in which carbon compounds flow in opposite directions: from glucose to pyruvate, and from succinate to pyruvate. Thus, our results support that Crc not only favours the assimilation of preferred compounds, but balances carbon fluxes, optimizing metabolism and growth. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. The pWW0 plasmid imposes a stochastic expression regime to the chromosomal ortho pathway for benzoate metabolism in Pseudomonas putida.

    Science.gov (United States)

    Silva-Rocha, Rafael; de Lorenzo, Victor

    2014-07-01

    Environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. The pWW0 plasmid born by Pseudomonas putida mt-2 encodes the TOL pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. These can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemical conflict. In this context, we have investigated how the introduction of the pWW0 plasmid into P. putida affects behaviour of the promoter of the ben pathway (Pb) in single cells. Using a series of standardized transcriptional fusions to green fluorescent protein, we found that acquisition of the TOL pathway switches the activation course of the Pb promoter from unimodal/graded to bimodal/stochastic when cells were exposed to benzoate. This behaviour was propagated downstream into the Pc promoter of the cat gene cluster, which responds to the benzoate-degradation intermediate cis,cis-muconate. The TOL plasmid thus imposes expression of the chromosomal Pb with a stochastic behaviour likely to result in biochemical heterogeneity of the otherwise genetically clonal population when exposed to benzoate as a growth substrate.

  12. Complex pathways for regulation of pyrimidine metabolism by carbon catabolite repression and quorum sensing in Pseudomonas putida RU-KM3S.

    Science.gov (United States)

    Matcher, Gwynneth Felicity; Jiwaji, Meesbah; de la Mare, Jo-Anne; Dorrington, Rosemary Ann

    2013-07-01

    Pseudomonads are metabolically versatile microbes that employ complex regulatory networks to control gene expression, particularly with respect to carbon and nitrogen metabolism. The aim of this study was to characterise the regulatory networks that control pyrimidine metabolism (hydantoin-hydrolysing activity) in Pseudomonas putida strain RU-KM3S, focussing on transcriptional activation of dihydropyrimidinase (Dhp) and β-ureidopropionase (Bup), encoding dhp and bup, respectively. The two genes are arranged divergently on the chromosome and are separated by ORF1, encoding a putative transporter, which lies upstream of and in the same orientation as bup. The results from this study reveal that pyrimidine metabolism, as a function of Bup and Dhp activity in P. putida RU-KM3S, is controlled by a complex regulatory network including several global pathways in addition to induction by the substrate. Three major control pathways act at the level of transcriptional and include: (1) induction of transcriptional activation in the presence of hydantoin, (2) carbon catabolite repression mediated via a pathway independent of Crc and (3) quorum sensing that does not require a putative lux box located upstream of the dhp transcriptional start. Finally, the data suggest a minor role for the global regulators Anr, Vfr and Crc, likely through regulation of the activity of transcription factors interacting directly with the bup/ORF1-dhp promoter.

  13. In-vitro antibacterial activities of the essential oils of aromatic plants against Erwinia herbicola (Lohnis and pseudomonas putida (Kris Hamilton

    Directory of Open Access Journals (Sweden)

    Pandey Abhay K.

    2012-01-01

    Full Text Available This study was designed to examine in vitro antibacterial activities of essential oils extracted from 53 aromatic plants of Gorakhpur Division (UP, INDIA for the control of two phytopathogenic bacteria namely Erwinia herbicola and Pseudomonas putida causing several post-harvest diseases in fruits and vegetables. Out of 53 oils screened, 8 oils such as Chenopodium ambrosioides, Citrus aurantium, Clausena pentaphylla, Hyptis suaveolens, Lippia alba, Mentha arvensis, Ocimum sanctum and Vitex negundo completely inhibited the growth of test bacteria. Furthermore MIC & MBC values of C. ambrosioides oil were least for Erw. herbicola (0.25 & 2.0 μl/ml and Ps. putida (0.12 & 1.0 μl/ml respectively than other 7 oils as well as Agromycin and Streptomycin drugs used in current study. GC and GC-MS analysis of Chenopodium oil revealed presence of 125 major and minor compounds, out of them, 14 compounds were recognized. The findings concluded that Chenopodium oil may be regarded as safe antibacterial agent for the management of post-harvest diseases of fruits and vegetables.

  14. Co-synthesis of medium-chain-length polyhydroxyalkanoates and CdS quantum dots nanoparticles in Pseudomonas putida KT2440.

    Science.gov (United States)

    Oliva-Arancibia, Barbara; Órdenes-Aenishanslins, Nicolás; Bruna, Nicolas; Ibarra, Paula S; Zacconi, Flavia C; Pérez-Donoso, José M; Poblete-Castro, Ignacio

    2017-12-20

    Microbial polymers and nanomaterials production is a promising alternative for sustainable bioeconomics. To this end, we used Pseudomonas putida KT2440 as a cell factory in batch cultures to coproduce two important nanotechnology materials- medium-chain-length (MCL)-polyhydroxyalkanoates (PHAs) and CdS fluorescent nanoparticles (i.e. quantum dots [QDots]). Due to high cadmium resistance, biomass and PHA yields were almost unaffected by coproduction conditions. Fluorescent nanocrystal biosynthesis was possible only in presence of cysteine. Furthermore, this process took place exclusively in the cell, displaying the classical emission spectra of CdS QDots under UV-light exposure. Cell fluorescence, zeta potential values, and particles size of QDots depended on cadmium concentration and exposure time. Using standard PHA-extraction procedures, the biosynthesized QDots remained associated with the biomass, and the resulting PHAs presented no traces of CdS QDots. Transmission electron microscopy located the synthesized PHAs in the cell cytoplasm, whereas CdS nanocrystals were most likely located within the periplasmic space, exhibiting no apparent interaction. This is the first report presenting the microbial coproduction of MCL-PHAs and CdS QDots in P. putida KT2440, thus constituting a foundation for further bioprocess developments and strain engineering towards the efficient synthesis of these highly relevant bioproducts for nanotechnology. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. [Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

    Science.gov (United States)

    Shaburova, O V; Krylov, S V; Veĭko, V P; Pleteneva, E A; Burkal'tseva, M V; Miroshnokov, K A; Kornelissen, A; Lavogne, R; Sykilinda, N N; Kadykov, V A; Mesianzhinov, V V; Volckaert, G; Krylov, V N

    2009-02-01

    Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.

  16. In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA

    International Nuclear Information System (INIS)

    Pineda-Molina, E.; Daddaoua, A.; Krell, T.; Ramos, J. L.; García-Ruiz, J. M.; Gavira, J. A.

    2012-01-01

    The crystallization of both native P. putida transcriptional regulator PtxS and its complex with its DNA recognition sequence using the counter-diffusion method are reported. Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space group P3, with unit-cell parameters a = b = 213.71, c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution

  17. Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

    Science.gov (United States)

    Zhang, Rong; Xu, Xingjian; Chen, Wenli; Huang, Qiaoyun

    2016-02-01

    A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

  18. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

    Science.gov (United States)

    Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-10-23

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440.

    Science.gov (United States)

    Calero, Patricia; Jensen, Sheila I; Nielsen, Alex T

    2016-07-15

    Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as useful tools for optimizing gene expression in a variety of bacteria.

  20. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways*

    Science.gov (United States)

    Nikel, Pablo I.; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-01-01

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using 13C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. PMID:26350459

  1. Medium chain length polyhydroxyalkanoates biosynthesis in Pseudomonas putida mt-2 is enhanced by co-metabolism of glycerol/octanoate or fatty acids mixtures.

    Science.gov (United States)

    Fontaine, Paul; Mosrati, Ridha; Corroler, David

    2017-05-01

    The synthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas putida mt-2 was investigated under nitrogen-rich then deficient conditions with glycerol/octanoate or long-chain fatty acids (LCFAs) as carbon sources. When mixed, glycerol and octanoate were co-assimilated regardless of nitrogen availability but provided that glycerol uptake has been already triggered under non-limiting nutrient conditions. This concomitant consumption allowed to enhance mcl-PHAs accumulation (up to 57% of cell dry weight (CDW)) under both non-limiting and nitrogen deficient conditions. Octanoate then mostly drove anabolism of the polyester with 3-hydroxyoctanoate (3HO) synthesized as the main monomer (83%). If the preferred PHA precursor octanoate was supplied, glycerol was mainly involved in cell growth and/or maintenance but very little in PHA production even under nitrogen starvation. P. putida cells accumulated higher amounts of mcl-PHAs when grown on mixtures of LCFAs compared to LCFAs supplied as single substrate (25% and 9% of CDW, respectively). However, only a weak enrichment of the polyester was observed after transfer of cells in a fresh nitrogen-free medium containing the same combination of LCFAs. Some typical units within the polyester were related to the LCFAs ratio supplied in the medium indicating that tailor-made monomers could be synthesized. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. The LapG protein plays a role in Pseudomonas aeruginosa biofilm formation by controlling the presence of the CdrA adhesin on the cell surface

    DEFF Research Database (Denmark)

    Rybtke, Morten; Berthelsen, Jens; Yang, Liang

    2015-01-01

    formation and biofilm dispersal. The P. aeruginosa LapG protein is shown to be a functional homolog of the Pseudomonas putida LapG protein which has previously been shown to function as a periplasmic protease that targets the surface adhesin LapA. Transposon mutagenesis and characterization of defined......Pseudomonas aeruginosa is a clinically relevant species involved in biofilm-based chronic infections. We provide evidence that the P. aeruginosa LapG protein functions as a periplasmic protease that can cleave the protein adhesin CdrA off the cell surface, and thereby plays a role in biofilm...

  3. Production of natural fragrance aromatic acids by coexpression of trans-anethole oxygenase and p-anisaldehyde dehydrogenase genes of Pseudomonas putida JYR-1 in Escherichia coli.

    Science.gov (United States)

    Han, Dongfei; Kurusarttra, Somwang; Ryu, Ji-Young; Kanaly, Robert A; Hur, Hor-Gil

    2012-12-05

    A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli , the engineered strain has potential to be applied in the fragrance industry.

  4. Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4.

    Science.gov (United States)

    Cheng, Yongmei; Ma, Li; Deng, Chao; Xu, Zhenghong; Chen, Jinghua

    2014-08-01

    Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4 was studied. Through using PEG as porogen, the environmental tolerance as well as the biocatalytic reaction efficiency of immobilized cells was greatly improved, i.e., Ca-alginate-PEG immobilized cells got better temperature and substrate concentration tolerance than Ca-alginate immobilized cells and showed similar efficiency with free cells, suggesting that the intrinsic mass transfer resistance of immobilization obviously decreased. It was also observed that the pore diameter and porosity of immobilization beads were related with the molecular weight of PEG. PEG400 was found to be a relatively suitable porogen for Ca-alginate-PEG immobilized cells catalyzed hydrolysis of glycinonitrile. It was noteworthy that the Ca-alginate-PEG immobilized cells could be reused more than 18 times with little loss of enzyme activity which had shown good operation ability and great application potential.

  5. Identification and characterization of an N-acylhomoserine lactone-dependent quorum-sensing system in Pseudomonas putida strain IsoF

    DEFF Research Database (Denmark)

    Steidle, A.; Allesen-Holm, M.; Riedel, K.

    2002-01-01

    cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly...... similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity...

  6. Application of Biosurfactants Produced by Pseudomonas putida using Crude Palm Oil (CPO) as Substrate for Crude Oil Recovery using Batch Method

    Science.gov (United States)

    Suryanti, V.; Handayani, D. S.; Masykur, A.; Septyaningsih, I.

    2018-03-01

    The application of biosurfactants which have been produced by Pseudomonas putida in nutrient broth medium supplemented with NaCl and crude palm oil (CPO) for oil recovery has been evaluated. The crude and purified biosurfactants have been examined for oil recovery from a laboratory oil-contaminated sand in agitated flask (batch method). Two synthetic surfactants and water as control was also performed for oil recovery as comparisons. Using batch method, the results showed that removing ability of crude oil from the oil-contaminated sand by purified and crude biosurfactants were 79.40±3.10 and 46.84±2.23 %, respectively. On other hand, the recoveries obtained with the SDS, Triton X-100 and water were 94.33±0.47, 74.84±7.39 and 34.42±1.21%respectively.

  7. Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442.

    Science.gov (United States)

    Tripathi, Lakshmi; Wu, Lin-Ping; Chen, Jinchun; Chen, Guo-Qiang

    2012-04-05

    Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and -16.4°C, one melting temperature (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.

  8. Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases inPseudomonas putidaKT2440.

    Science.gov (United States)

    Wehrmann, Matthias; Billard, Patrick; Martin-Meriadec, Audrey; Zegeye, Asfaw; Klebensberger, Janosch

    2017-06-27

    The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca 2+ -dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La 3+ , Ce 3+ , Pr 3+ , Sm 3+ , or Nd 3+ Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH , most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides. IMPORTANCE Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also

  9. Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1.

    Science.gov (United States)

    De Vrind, Johannes; De Groot, Arjan; Brouwers, Geert Jan; Tommassen, Jan; De Vrind-De Jong, Elisabeth

    2003-02-01

    The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described 'regular'xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.

  10. Carbapenem stewardship: does ertapenem affect Pseudomonas susceptibility to other carbapenems? A review of the evidence.

    Science.gov (United States)

    Nicolau, David P; Carmeli, Yehuda; Crank, Christopher W; Goff, Debra A; Graber, Christopher J; Lima, Ana Lucia L; Goldstein, Ellie J C

    2012-01-01

    The group 2 carbapenems (imipenem, meropenem and, more recently, doripenem) have been a mainstay of treatment for patients with serious hospital infections caused by Pseudomonas aeruginosa, Enterobacteriaceae and other difficult-to-treat Gram-negative pathogens as well as mixed aerobic/anaerobic infections. When ertapenem, a group 1 carbapenem, was introduced, questions were raised about the potential for ertapenem to select for imipenem- and meropenem-resistant Pseudomonas. Results from ten clinical studies evaluating the effect of ertapenem use on the susceptibility of Pseudomonas to carbapenems have uniformly shown that ertapenem use does not result in decreased Pseudomonas susceptibility to these antipseudomonal carbapenems. Here we review these studies evaluating the evidence of how ertapenem use affects P. aeruginosa as well as provide considerations for ertapenem use in the context of institutional stewardship initiatives. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  11. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis

    DEFF Research Database (Denmark)

    Johansen, Helle Krogh; Gøtzsche, Peter C

    2015-01-01

    BACKGROUND: Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed....... This is an update of a previously published review. OBJECTIVES: To assess the effectiveness of vaccination against Pseudomonas aeruginosa in cystic fibrosis. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register using the terms vaccines AND pseudomonas (last search 30...... March 2015). We previously searched PubMed using the terms vaccin* AND cystic fibrosis (last search 30 May 2013). SELECTION CRITERIA: Randomised trials (published or unpublished) comparing Pseudomonas aeruginosa vaccines (oral, parenteral or intranasal) with control vaccines or no intervention in cystic...

  12. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis

    DEFF Research Database (Denmark)

    Johansen, H.K.; Gøtzsche, Peter C.; Johansen, Helle Krogh

    2008-01-01

    BACKGROUND: Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed. OBJECTIVES......: To assess the effectiveness of vaccination against Pseudomonas aeruginosa in cystic fibrosis. SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register using the terms vaccines AND pseudomonas (last search May 2008) and PubMed using the terms vaccin* AND cystic...... fibrosis (last search May 2008). SELECTION CRITERIA: Randomised trials (published or unpublished) comparing Pseudomonas aeruginosa vaccines (oral, parenteral or intranasal) with control vaccines or no intervention in cystic fibrosis. DATA COLLECTION AND ANALYSIS: The authors independently selected trials...

  13. APPLICATION OF PSEUDOMONAS PUTIDA AND RHODOCOCCUS SP. BY BIODEGRADATION OF PAH(S, PCB(S AND NEL SOIL SAMPLES FROM THE HAZARDOUS WASTE DUMP IN POZĎÁTKY (CZECH REPUBLIC

    Directory of Open Access Journals (Sweden)

    Radmila Kucerova

    2006-12-01

    Full Text Available The objective of the project was a laboratory check of biodegradation of soil samples contaminated by PAH(s, PCB(s and NEL from the hazardous waste dump in the Pozďátky locality. For the laboratory check, pure bacterial cultures of Rhodococcus sp. and Pseudomonas putida have been used. It is apparent from the laboratory experiments results that after one-month bacterial leaching, applying the bacterium of Rhodococcus sp. there is a 83 % removal of NEL, a 79 % removal of PAH(s and a 14 % removal of PCB(s. Applying a pure culture of Pseudomonas putida there is a 87 % removal of NEL, a 81 % removal of PAH(s and a 14 % removal of PCB(s.

  14. Pseudomonas aeruginosa burn wound infection in a dedicated ...

    African Journals Online (AJOL)

    Background. Pseudomonas aeruginosa infection is a major cause of morbidity in burns patients. There is a paucity of publications dealing with this infection in the paediatric population. We describe the incidence, microbiology and impact of P. aeruginosa infection in a dedicated paediatric burns unit. Methods.

  15. Decolorization of irgalite dye by immobilized Pseuodomonas putida ...

    African Journals Online (AJOL)

    ... using Pseuodomonas putida (MTCC 1194) with activated carbon prepared from mustard straw showed removal of 83.3% higher SAB, with respect to adsorption (70%). For biodegradation of irgalite by P. putida at shake flask level Haldane's growth model fitted the best. Key words: activated carbon, Pseudomonas putida, ...

  16. Optimización de un medio de cultivo para la producción de biomasa de la cepa Pseudomonas putida UA 44 aislada del suelo bananero de Uraba –Antioquia

    OpenAIRE

    Becerra Mejía, Camilo Andrés

    2007-01-01

    En el presente trabajo se diseñó y optimizó un medio de cultivo químicamente definido para la bacteria Pseudomonas putida UA44 con potencial PGPR aislada del suelo de la zona bananera de Uraba por Ramirez (2004). Los resultados obtenidos en el medio químicamente definido (MD) se compararon con los hallados en un medio de cultivo semicomplejo (TSB: Triptic Soy Broth por sus siglas en Ingles).

  17. Evaluation of nutrients removal (NO3-N, NH3-N and PO4-P) with Chlorella vulgaris, Pseudomonas putida, Bacillus cereus and a consortium of these microorganisms in the treatment of wastewater effluents.

    Science.gov (United States)

    Gómez-Guzmán, Abril; Jiménez-Magaña, Sergio; Guerra-Rentería, A Suggey; Gómez-Hermosillo, César; Parra-Rodríguez, F Javier; Velázquez, Sergio; Aguilar-Uscanga, Blanca Rosa; Solis-Pacheco, Josue; González-Reynoso, Orfil

    2017-07-01

    In this research removal of NH 3 -N, NO 3 -N and PO 4 -P nutrients from municipal wastewater was studied, using Chlorella vulgaris, Pseudomonas putida, Bacillus cereus and an artificial consortium of them. The objective is to analyze the performance of these microorganisms and their consortium, which has not been previously studied for nutrient removal in municipal wastewater. A model wastewater was prepared simulating the physicochemical characteristics found at the wastewater plant in Chapala, Mexico. Experiments were carried out without adding an external carbon source. Results indicate that nutrient removal with Chlorella vulgaris was the most efficient with a removal of 24.03% of NO 3 -N, 80.62% of NH 3 -N and 4.30% of PO 4 -P. With Bacillus cereus the results were 8.40% of NO 3 -N, 28.80% of NH 3 -N and 3.80% of PO 4 -P. The removals with Pseudomonas putida were 2.50% of NO 3 -N, 41.80 of NH 3 -N and 4.30% of PO 4 -P. The consortium of Chlorella vulgaris-Bacillus cereus-Pseudomonas putida removed 29.40% of NO 3 -N, 4.2% of NH 3 -N and 8.4% of PO 4 -P. The highest biomass production was with Bacillus cereus (450 mg/l) followed by Pseudomonas putida (444 mg/l), the consortium (205 mg/l) and Chlorella vulgaris (88.9 mg/l). This study highlights the utility of these microorganisms for nutrient removal in wastewater treatments.

  18. Forage Quantity and Quality of Berseem Clover (Trifolium ‎alexandrinum L. as Affected by Uses of Pseudomonas putida ‎Strains and Phophorus Fertilizer in the Second Crop

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Ansari

    2017-05-01

    Full Text Available Effects of phosphate fertilizer and pseudomonas putida strains on the quantity and quality of forage of berseem clover as a second crop was studied in a factorial field experiment using randomized complete block design with three replications at Fooman, Guilan province, Iran. Treatments consisted of phosphate fertilizer with three levels (0, 75 and 150 kg/ha as triple super phosphate and Pseudomonas putida strains with four levels (M21, M5, M168 and control. The results showed that use of phosphate fertilizers increased the soil pH during growing season while bacterial inoculation adjusted soil pH. The bacterial inoculation increased amount of crude protein, digestible protein, acidic and alkaline phosphatase activity compared to non-inoculated treatment, but it decreased crude fiber of the forage. Clover forage yield, protein yield and phosphorus content of foliage also were influenced by the interaction of bacterial strains and phosphate fertilizer. The highest forage and protein yield were obtained by using strain M5+150 kg P ha-1. Significant increases in forage and protein yield were found to be 16.49% and 8.01%, respectively, as compared with non-inoculated treatment. Based on the result of this experiment, application of 150 kg P ha-1 and Pseudomonas putida strain M5 inoculation can be used to obtain highest forage yield and quality of berseem clover as second crop in the experimental site.

  19. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    International Nuclear Information System (INIS)

    Schmidberger, Jason W.; Wilce, Jackie A.; Weightman, Andrew J.; Wilce, Matthew C. J.

    2008-01-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2 1 ), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary

  20. Molecular level biodegradation of phenol and its derivatives through dmp operon of Pseudomonas putida: A bio-molecular modeling and docking analysis.

    Science.gov (United States)

    Ray, Sujay; Banerjee, Arundhati

    2015-10-01

    Participation of Pseudomonas putida-derived methyl phenol (dmp) operon and DmpR protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has DmpR protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon, phenol hydroxylase encoded by dmpN gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from DmpR protein and of the DNA sequences from the two Upstream Activation Sequences (UAS) present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation. Copyright © 2015. Published by Elsevier B.V.

  1. Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4.

    Science.gov (United States)

    Chen, Qiongzhen; Tu, Hui; Huang, Fei; Wang, Yicheng; Dong, Weiliang; Wang, Wenhui; Li, Zhoukun; Wang, Fei; Cui, Zhongli

    2016-06-01

    LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para-nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL-ΔpnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL-ΔpnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, γ-aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL-ΔpnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

    Science.gov (United States)

    Nikel, Pablo I.; Romero-Campero, Francisco J.; Zeidman, Joshua A.; Goñi-Moreno, Ángel

    2015-01-01

    ABSTRACT The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states. PMID:25827416

  3. Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo, In Vitro, and In Silico Studies

    Science.gov (United States)

    Mielecki, Damian; Saumaa, Signe; Wrzesiński, Michał; Maciejewska, Agnieszka M.; Żuchniewicz, Karolina; Sikora, Anna; Piwowarski, Jan; Nieminuszczy, Jadwiga; Kivisaar, Maia; Grzesiuk, Elżbieta

    2013-01-01

    Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against

  4. Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.

    Directory of Open Access Journals (Sweden)

    Damian Mielecki

    Full Text Available Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB, characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB, PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems

  5. Role of extracellular polymeric substances (EPS) from Pseudomonas putida strain MnB1 in dissolution of natural rhodochrosite

    Science.gov (United States)

    Wang, H.; Pan, X.

    2014-05-01

    Microbially mediated oxidation of Mn(II) to Mn oxides have been demonstrated in previous studies, however, the mechanisms of bacteria how to dissolve and oxidize using a solid Mn(II) origin are poorly understood. In this study, we examined the role of extracellular polymeric substances (EPS) from P. putida strain MnB1 in enhancing dissolution of natural rhodochrosite. The results showed that P. putida strain MnB1 cell can effectively dissolve and oxidize natural rhodochrosite to generate Mn oxides, and EPS were found to play an important role in increasing dissolution of natural rhodochrosite. Compared with EPS-free treatment, dissolution rate of natural rhodochrosite in the presence of bacterial EPS was significantly increased with decreasing initial pH and increasing EPS concentration, ionic strength and rhodochrosite dosage (p < 0.05). The fourier-transform infrared spectroscopy (FTIR) analysis implies that the functional groups like N-H, C=O and C-H in EPS contributed to the dissolution of natural rhodochrosite. This study is helpful for understanding the mechanisms of the formation of biogenic Mn oxides using a solid Mn(II) origin.

  6. Multiple Roles for Two Efflux Pumps in the Polycyclic Aromatic Hydrocarbon-Degrading Pseudomonas putida Strain B6-2 (DSM 28064).

    Science.gov (United States)

    Yao, Xuemei; Tao, Fei; Zhang, Kunzhi; Tang, Hongzhi; Xu, Ping

    2017-12-15

    Microbial bioremediation is a promising approach for the removal of polycyclic aromatic hydrocarbon (PAH) contaminants. Many degraders of PAHs possess efflux pump genes in their genomes; however, their specific roles in the degradation of PAHs have not been clearly elucidated. In this study, two efflux pumps, TtgABC and SrpABC, were systematically investigated to determine their functions in a PAH-degrading Pseudomonas putida strain B6-2 (DSM 28064). The disruption of genes ttgABC or srpABC resulted in a defect in organic solvent tolerance. TtgABC was found to contribute to antibiotic resistance; SrpABC only contributed to antibiotic resistance under an artificial overproduced condition. Moreover, a mutant strain without srpABC did not maintain its activity in long-term biphenyl (BP) degradation, which correlated with the loss of cell viability. The expression of SrpABC was significantly upregulated in the course of BP degradation. BP, 2-hydroxybiphenyl, 3-hydroxybiphenyl, and 2,3-dihydroxybiphenyl (2,3-DHBP) were revealed to be the inducers of srpABC 2,3-DHBP was verified to be a substrate of pump SrpABC; SrpABC can enhance the tolerance to 2,3-DHBP by pumping it out. The mutant strain B6-2Δ srpS prolonged BP degradation with the increase of srpABC expression. These results suggest that the pump SrpABC of strain B6-2 plays a positive role in BP biodegradation by pumping out metabolized toxic substances such as 2,3-DHBP. This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs. IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are notorious for their recalcitrance to degradation in the environment. A high frequency of the occurrence of the efflux pump genes was observed in the genomes of effective PAH degraders; however, their specific roles in the degradation of PAHs are still obscure. The significance of our study is in the identification of the function and mechanism of the

  7. Chronic Pseudomonas aeruginosa lung infection in normal and athymic rats

    DEFF Research Database (Denmark)

    Johansen, H K; Espersen, F; Pedersen, S S

    1993-01-01

    We have compared a chronic lung infection with Pseudomonas aeruginosa embedded in alginate beads in normal and athymic rats with an acute infection with free live P. aeruginosa bacteria. The following parameters were observed and described: mortality, macroscopic and microscopic pathologic changes...

  8. Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates from Saponified Palm Kernel Oil by Pseudomonas putida: Kinetics of Batch and Fed-Batch Fermentations

    Directory of Open Access Journals (Sweden)

    Annuar, M. S. M.

    2006-01-01

    Full Text Available The kinetics of medium-chain-length poly(3-hydroxyalkanoates, PHAMCL production by Pseudomonas putida PGA1 in batch and fed-batch fermentations were studied. With saponified palm kernel oil (SPKO supplying the free fatty acids mixture as the sole carbon and energy source, PHAMCL accumulation is encouraged under ammonium-limited condition, which is a nitrogen stress environment. The amount of PHAMCL accumulated and its specific production rate, qPHA were influenced by the residual ammonium concentration level in the culture medium. It was observed that in both fermentation modes, when the residual ammonium was exhausted (< 0.05 gL-1, the PHAMCL accumulation (11.9% and qPHA (0.0062 h-1 were significantly reduced. However, this effect can be reversed by feeding low amount of ammonium to the culture, resulting in significantly improved PHAMCL yield (71.4% and specific productivity (0.6 h-1. It is concluded that the feeding of low ammonium concentration to the culture medium during the PHAMCL accumulation has a positive effect on sustaining the PHAMCL biosynthetic capability of the organism. It was also found that increasing SPKO concentration in the medium significantly reduced (up to 50% the volumetric oxygen transfer coefficient (KLa of the fermentation system.

  9. Protein SgpR of Pseudomonas putida strain AK5 is a LysR-type regulator of salicylate degradation through gentisate.

    Science.gov (United States)

    Filatova, Irina Yu; Kazakov, Alexei S; Muzafarov, Evgeny N; Zakharova, Marina V

    2017-07-03

    Pseudomonas putida strain AK5 was the first characterized natural strain containing the 'classical' nah1 operon and nahR gene along with genes whose products are responsible for the less explored pathway of salicylate degradation through gentisate (the sgp operon). The sgp operon was found to be preceded by the divergently directed sgpR gene. The amino acid sequence of the sgpR product qualifies it as a LysR-type transcriptional regulator (LTTR) and suggests its potential function as an sgp operon transcriptional regulator. This study focused on verification of SgpR's involvement in regulation of transcription of the operon genes and characterization of its interaction with the sgp promoter. We determined the transcription start for sgpAIKGHB and identified the SgpR binding site. The equilibrium dissociation constant (KD) of the SgpR-DNA complex determined in the presence and absence of the inducer salicylate appeared to be, on the whole, at the lower end of the range for KD values reported for LTTRs. RT-qPCR showed that in the presence of salicylate, efficiency of transcription of the sgpAIKGHB operon increased by three orders of magnitude and reached the highest values so far observed for LTTR-controlled operons, thus holding much promise for further studies of the mechanism of transcriptional regulation that involves SgpR. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates produced by Pseudomonas putida Bet001.

    Science.gov (United States)

    Anis, Siti Nor Syairah; Mohamad Annuar, Mohamad Suffian; Simarani, Khanom

    2017-09-14

    In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.

  11. A new family of intrinsically disordered proteins: structural characterization of the major phasin PhaF from Pseudomonas putida KT2440.

    Directory of Open Access Journals (Sweden)

    Beatriz Maestro

    Full Text Available Phasins are intracellular polyhydroxyalkanoat4e (PHA-associated proteins involved in the stabilization of these bacterial carbon storage granules. Despite its importance in PHA metabolism and regulation, only few reports have focused so far on the structure of these proteins. In this work we have investigated the structure and stability of the PhaF phasin from Pseudomonas putida KT2440, a protein that is involved in PHA granule stabilization and distribution to daughter cells upon cell division. A structural, three-dimensional model of the protein was built from homology modeling procedures and consensus secondary structure predictions. The model predicts that PhaF is an elongated protein, with a long, amphipathic N-terminal helix with PHA binding capacity, followed by a short leucine zipper involved in protein oligomerization and a superhelical C-terminal domain wrapped around the chromosomal DNA. Hydrodynamic, spectroscopical and thermodynamic experiments validated the model and confirmed both that free PhaF is a tetramer in solution and that most part of the protein is intrinsically disordered in the absence of its ligands. The results lay a molecular basis for the explanation of the biological role of PhaF and, along with an exhaustive analysis of phasin sequence databases, suggest that intrinsic disorder and oligomerization through coiled-coils may be a widespread mechanism among these proteins.

  12. Alkanols and chlorophenols cause different physiological adaptive responses on the level of cell surface properties and membrane vesicle formation in Pseudomonas putida DOT-T1E.

    Science.gov (United States)

    Baumgarten, Thomas; Vazquez, José; Bastisch, Christian; Veron, Wilfried; Feuilloley, Marc G J; Nietzsche, Sandor; Wick, Lukas Y; Heipieper, Hermann J

    2012-01-01

    In order to cope with the toxicity imposed by the exposure to environmental hydrocarbons, many bacteria have developed specific adaptive responses such as modifications in the cell envelope. Here we compared the influence of n-alkanols and chlorophenols on the surface properties of the solvent-tolerant bacterium Pseudomonas putida DOT-T1E. In the presence of toxic concentrations of n-alkanols, this strain significantly increased its cell surface charge and hydrophobicity with changes depending on the chain length of the added n-alkanols. The adaptive response occurred within 10 min after the addition of the solvent and was demonstrated to be of physiological nature. Contrary to that, chlorophenols of similar hydrophobicity and potential toxicity as the corresponding alkanols caused only minor effects in the surface properties. To our knowledge, this is the first observation of differences in the cellular adaptive response of bacteria to compound classes of quasi equal hydrophobicity and toxicity. The observed adaptation of the physico-chemical surface properties of strain DOT-T1E to the presence of alkanols was reversible and correlated with changes in the composition of the lipopolysaccharide content of the cells. The reaction is explained by previously described reactions allowing the release of membrane vesicles that was demonstrated for cells affected by 1-octanol and heat shock, whereas no membrane vesicles were released after the addition of chlorophenols.

  13. 3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

    Science.gov (United States)

    Konti, Aikaterini; Mamma, Diomi; Hatzinikolaou, Dimitios G; Kekos, Dimitris

    2016-10-01

    3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω ≪ 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

  14. Crystallization and crystallographic analysis of the ligand-binding domain of the Pseudomonas putida chemoreceptor McpS in complex with malate and succinate

    International Nuclear Information System (INIS)

    Gavira, J. A.; Lacal, J.; Ramos, J. L.; García-Ruiz, J. M.; Krell, T.; Pineda-Molina, E.

    2012-01-01

    The crystallization of the ligand-binding domain of the methyl-accepting chemotaxis protein chemoreceptor McpS (McpS-LBD) is reported. Methyl-accepting chemotaxis proteins (MCPs) are transmembrane proteins that sense changes in environmental signals, generating a chemotactic response and regulating other cellular processes. MCPs are composed of two main domains: a ligand-binding domain (LBD) and a cytosolic signalling domain (CSD). Here, the crystallization of the LBD of the chemoreceptor McpS (McpS-LBD) is reported. McpS-LBD is responsible for sensing most of the TCA-cycle intermediates in the soil bacterium Pseudomonas putida KT2440. McpS-LBD was expressed, purified and crystallized in complex with two of its natural ligands (malate and succinate). Crystals were obtained by both the counter-diffusion and the hanging-drop vapour-diffusion techniques after pre-incubation of McpS-LBD with the ligands. The crystals were isomorphous and belonged to space group C2, with two molecules per asymmetric unit. Diffraction data were collected at the ESRF synchrotron X-ray source to resolutions of 1.8 and 1.9 Å for the malate and succinate complexes, respectively

  15. Biocatalytic Production of Perillyl Alcohol from Limonene by Using a Novel Mycobacterium sp. Cytochrome P450 Alkane Hydroxylase Expressed in Pseudomonas putida

    Science.gov (United States)

    van Beilen, Jan B.; Holtackers, René; Lüscher, Daniel; Bauer, Ulrich; Witholt, Bernard; Duetz, Wouter A.

    2005-01-01

    A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (−)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (−)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams. PMID:15811996

  16. Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

    2015-05-01

    We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

  17. Effects of even and odd number fatty acids cofeeding on PHA production and composition in Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Science.gov (United States)

    Mohd Razaif-Mazinah, Mohd Rafais; Mohamad Annuar, Mohamad Suffian; Sharifuddin, Yusrizam

    2016-01-01

    The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  18. Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator.

    Science.gov (United States)

    Talavera, Ariel; Tamman, Hedvig; Ainelo, Andres; Hadǽi, San; Garcia-Pino, Abel; Hõrak, Rita; Konijnenberg, Albert; Loris, Remy

    2017-08-01

    The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P2 1 , with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P4 1 or P4 3 , with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P3 1 or P3 2 , with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution.

  19. The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F. [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom); Lau, Peter C. [National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2 (Canada); Hasegawa, Yoshie; Iwaki, Hiroaki [Kansai University (Japan); Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T. [Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald (Germany); Bourenkov, Gleb [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22607 Hamburg (Germany); Littlechild, Jennifer A., E-mail: j.a.littlechild@exeter.ac.uk [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom)

    2015-10-31

    The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

  20. The impact of succinate trace on pWW0 and ortho-cleavage pathway transcription in Pseudomonas putida mt-2 during toluene biodegradation.

    Science.gov (United States)

    Tsipa, Argyro; Koutinas, Michalis; Vernardis, Spyros I; Mantalaris, Athanasios

    2017-06-01

    Toluene is a pollutant catabolised through the interconnected pWW0 (TOL) and ortho-cleavage pathways of Pseudomonas putida mt-2, while upon succinate and toluene mixtures introduction in batch cultures grown on M9 medium, succinate was previously reported as non-repressing. The effect of a 40 times lower succinate concentration, as compared to literature values, was explored through systematic real-time qPCR monitoring of transcriptional kinetics of the key TOL Pu, Pm and ortho-cleavage PbenR, PbenA promoters in mixed-substrate experiments. Even succinate trace inhibited transcription leading to bi-modal promoters expression. Potential carbon catabolite repression mechanisms and novel expression patterns of promoters were unfolded. Lag phase was shortened and biomass growth levels increased compared to sole toluene biodegradation suggesting enhanced pollutant removal efficiency. The study stressed the noticeable effect of a preferred compound's left-over on the main route of a bioprocess, revealing the beneficiary supply of low preferred substrates concentrations to design optimal bioremediation strategies. Copyright © 2017 Elsevier Ltd. All rights reserved.