Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

Science.gov (United States)

Chakravartty, Vandana

2012-01-01

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues

Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

Science.gov (United States)

Ali, Sikander; Nawaz, Wajeeha

2016-08-01

The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

DEFF Research Database (Denmark)

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster...... for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum....... growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant...... in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied...

Attenuation of the goose parvovirus strain B. Laboratory and field trials of the attenuated mutant for vaccination against Derzsy's disease.

Science.gov (United States)

Kisary, J; Derzsy, D; Meszaros, J

1978-07-01

Serial transfer of the goose parvovirus strain B, causal agent of Derzsy's gosling disease, in cultured goose-embryo fibroblast (GEF) resulted in a mutant (designated as Bav) apathogenic for both goose embryos and susceptible goslings. Goose embryos inoculated with the 38th or higher passages of strain B survived the infection, although the virus replicated in their organs. Susceptible goslings survived challenge with the Bav strain without showing symptoms, and developed normally. Only 4.2% of gosling progeny of parents vaccinated twice with strain Bav died after challenge with the virulent strain B goose parvovirus compared with 95% of gosling progeny of unvaccinated parents. Progeny of vaccinated and unvaccinated geese were placed on a farm on which Derzsy's disease was present. During the first month of life mortality was 7.7% in the progeny of vaccinated geese compared with 59.8% in the progeny of the unvaccinated geese. At 8 weeks of age the mean weight of the vaccinated goslings was 20% greater than for the unvaccinated goslings. These results indicate that the attenuated apathogenic Bav mutant is suitable for the immunisation of layers to protect their progeny by passive immunisation against Derzsy's disease.

Temperature-Sensitive Mutants of Mouse Hepatitis Virus Strain A59: Isolation, Characterization and Neuropathogenic Properties.

NARCIS (Netherlands)

M.J.M. Koolen (Marck); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert); M.C. Horzinek; B.A.M. van der Zeijst (Ben)

1983-01-01

textabstractTwenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the

Adaptive response in Drosophila melanogaster heat shock proteins mutant strains

International Nuclear Information System (INIS)

Shaposhnikov, M.V.; Moskalev, A.A.; Turysheva, E.V.

2007-01-01

Complete text of publication follows. The members of the heat shock proteins (Hsp) family function as molecular chaperones and assist intracellular folding of newly synthesized proteins. Also it is possible that molecular chaperones are induced during adaptive response to oxidative stress and radiation. The aim of our research was to exam the role of heat shock proteins in adaptive response to oxidative stress after low dose rate gamma-irradiation in Drosophila melanogaster. Drosophilamelanogaster strains were kindly provided by Bloomington Drosophila Stock Center (University of state of Indiana, Bloomington, USA). We used wild type strain (CS), heat shock protein mutant strains (Hsp22, Hsp70, Hsp83), and heat shock factor mutant strain (Hsf). Strains were chronically exposured to adaptive dose of gamma-irradiation in dose rate of 0.17 cGy/h during all stages of life history (from the embrional stage to the stage of matured imago). The rate of absorbed dose was 60 cGy. For oxidative-stress challenge twodays old flies were starved in empty vials for 6 h and then transferred to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival data were collected after 26 h of treatment. Dead flies were counted daily. The obtained data were subjected to survival analysis by Kaplan and Meier method and presented as survival curves. Statistical analysis was held by non-parametric methods. To test the significance of the difference between the two age distributions Kolmogorov-Smirnov test was applied. Gehan-Braslow- Wilcoxon and Cox-Mantel tests were used for estimation of median life span differences. In addition the minimal and maximal life span, time of 90% death, and mortality rate doubling time (MRDT) were estimated. The obtained results will be discussed in presentation.

Rat Strain Ontology: structured controlled vocabulary designed to facilitate access to strain data at RGD.

Science.gov (United States)

Nigam, Rajni; Munzenmaier, Diane H; Worthey, Elizabeth A; Dwinell, Melinda R; Shimoyama, Mary; Jacob, Howard J

2013-11-22

The Rat Genome Database (RGD) ( http://rgd.mcw.edu/) is the premier site for comprehensive data on the different strains of the laboratory rat (Rattus norvegicus). The strain data are collected from various publications, direct submissions from individual researchers, and rat providers worldwide. Rat strain, substrain designation and nomenclature follow the Guidelines for Nomenclature of Mouse and Rat Strains, instituted by the International Committee on Standardized Genetic Nomenclature for Mice. While symbols and names aid in identifying strains correctly, the flat nature of this information prohibits easy search and retrieval, as well as other data mining functions. In order to improve these functionalities, particularly in ontology-based tools, the Rat Strain Ontology (RS) was developed. The Rat Strain Ontology (RS) reflects the breeding history, parental background, and genetic manipulation of rat strains. This controlled vocabulary organizes strains by type: inbred, outbred, chromosome altered, congenic, mutant and so on. In addition, under the chromosome altered category, strains are organized by chromosome, and further by type of manipulations, such as mutant or congenic. This allows users to easily retrieve strains of interest with modifications in specific genomic regions. The ontology was developed using the Open Biological and Biomedical Ontology (OBO) file format, and is organized on the Directed Acyclic Graph (DAG) structure. Rat Strain Ontology IDs are included as part of the strain report (RS: ######). As rat researchers are often unaware of the number of substrains or altered strains within a breeding line, this vocabulary now provides an easy way to retrieve all substrains and accompanying information. Its usefulness is particularly evident in tools such as the PhenoMiner at RGD, where users can now easily retrieve phenotype measurement data for related strains, strains with similar backgrounds or those with similar introgressed regions. This

Mutant strains of Spirulina (Arthrospira) platensis to increase the efficiency of micro-ecological life support systems

Science.gov (United States)

Brown, Igor

The European Micro-Ecological Life Support System Alternative (MELiSSA) is an advanced idea for organizing a bioregenerative system for long term space flights and extraterrestrial settlements (Hendrickx, De Wever et al., 2005). Despite the hostility of both lunar and Martian environments to unprotected life, it seems possible to cultivate photosynthetic bacteria using closed bioreactors illuminated and heated by solar energy. Such reactors might be employed in critical processes, e.g. air revitalization, foodcaloric and protein source, as well as an immunomodulators production. The MELiSSA team suggested cyanobacterium Spirulina as most appropriate agent to revitalize air and produce a simple "fast" food. This is right suggestion because Spirulina was recently shown to be an oxygenic organism with the highest level of O2 production per unit mass (Ananyev et al., 2005). Chemical composition of Spirulina includes proteins (55Aiming to make Spirulina cultivation in life support systems like MELiSSA more efficient, we selected Spirulina mutant strains with increased fraction of methionine in the biomass of this cyanobacterium and compared the effect of parental wild strain of Spirulina and its mutants on the tendency of such experimental illnesses as radiationinduced lesions and hemolythic anemia. Results: It was found that mutant strains 198B and 27G contain higher quantities of total protein, essential amino acids, c-phycocyanin, allophycocyanin and chlorophyll a than parental wild strain of S. platensis. The strain 198B is also characterized with increased content of carotenoids. Revealed biochemical peculiarities of mutant strains suggest that these strains can serve as an additional source of essential amino acids as well as phycobiliproteins and carotenoids for the astronauts. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in deficient dehydrogenase activity and energy-rich phosphate levels

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

Science.gov (United States)

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F.; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

Directory of Open Access Journals (Sweden)

Annemarie Kramer

Full Text Available The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

Science.gov (United States)

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Comparative analysis on inactivation kinetics of between piezotolerant and piezosensitive mutant strains of Saccharomyces cerevisiae under combinations of high hydrostatic pressure and temperature.

Science.gov (United States)

Nomura, Kazuki; Kuwabara, Yuki; Kuwabara, Wataru; Takahashi, Hiroyuki; Nakajima, Kanako; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru

2017-12-01

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250MPa with 4 to 40°C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20°C, but did not show significant piezotolerance at 40°C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

Energy Technology Data Exchange (ETDEWEB)

Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

2005-09-30

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

International Nuclear Information System (INIS)

Chu, K.-W.; Chan, Shirley K.W.; Chow, King L.

2005-01-01

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring

Lipase production from a wild (LPF-5) and a mutant (HN1) strain of ...

African Journals Online (AJOL)

Lipase production from a wild (LPF-5) and a mutant (HN1) strain of Aspergillus niger. ... Several physical parameters (carbon source, nitrogen source, pH, ... for the development of industrial biotechnology for production of extracellular lipase.

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

Science.gov (United States)

Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

2017-09-01

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

Science.gov (United States)

Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

2015-11-01

Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

Mutant strain screening by 60Co γ-rays irradiation and its cellulase enzyme produce condition

International Nuclear Information System (INIS)

Song Andong; Su Lijuan; Xie Hui; Qu Yinbo; Yang Ming

2008-01-01

A mutant strain A50 with high cellulase activity was induced and isolated by using 60 Co γ-rays irradiation from the initial Penicillium decumbens A10. The optimum fermentation conditions of A50 were investigated through orthogonal designing experiment, the major carbon resource 5%, the ratio between wheat bran and corn straw 1:1, the concentration of glucose as supplemental carbon 0.1%, the concentration of (NH 4 ) 2 HPO 4 as supplemental nitrogen resource 0.2%, the initial pH of liquid medium 5.0, the inoculated amount for fermentation 10% and the concentration of Tween-80 0.1%, 30 ml initial media filled in the 300 ml flask with culture condition of 32 degree C and 200 r/min. Under the optimum conditions mentioned above, the highest activities of cellulase and filter paper enzyme were 27.28 and 1.98IU/ml at 60 h fermentation, respectively, which was 33.2% and 45.59% higher than those of the initial strain. (authors)

Induction of Aspergillus oryzae mutant strains producing increased levels of α-amylase by gamma-irradiation

International Nuclear Information System (INIS)

Ito, Hitoshi; Nessa, Azizun

1996-01-01

Spores of Aspergillus oryzae IAM 2630 were suspended in 0.067 m phosphate buffer and irradiated with gamma rays. Spores were incubated for 7 days and colony mutants counted by observing colour change compared to normal colours. α-amylase activities of the normal and mutant colonies were assayed. DNA assay of the spores was also carried out, after culture on different plating media. Enzyme production increased 2-5 times with increasing radiation dose. Increased spore size and DNA content was also observed in mutant strains with higher enzyme production suggesting that enzyme production is genetically controlled. Ultraviolet radiation did not appear to induce higher frequency of mutation. (UK)

Induction of Aspergillus oryzae mutant strains producing increased levels of {alpha}-amylase by gamma-irradiation

Energy Technology Data Exchange (ETDEWEB)

Ito, Hitoshi; Nessa, Azizun [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

1996-12-01

Spores of Aspergillus oryzae IAM 2630 were suspended in 0.067 m phosphate buffer and irradiated with gamma rays. Spores were incubated for 7 days and colony mutants counted by observing colour change compared to normal colours. {alpha}-amylase activities of the normal and mutant colonies were assayed. DNA assay of the spores was also carried out, after culture on different plating media. Enzyme production increased 2-5 times with increasing radiation dose. Increased spore size and DNA content was also observed in mutant strains with higher enzyme production suggesting that enzyme production is genetically controlled. Ultraviolet radiation did not appear to induce higher frequency of mutation. (UK).

The mutant strain of ZHJ6 degrading organophosphorous pesticide by 60Co-γ irradiation

International Nuclear Information System (INIS)

Zhao Renbang; Chi Jian; He Yi

2013-01-01

The strain of Penicillium oxalicum ZHJ6 that can degrade methamidophos was employed to obtain the mutant stain which has higher degradation rate than original strain by 60 Co-γ irradiation. Results showed that the Penicillium oxalicum ZHJ6 was sensitive to 60 Co-γ irradiation, and was easy to be killed by 60 Co-γ irradiation. Under the absorbed dose of 2.1 kGy, the survival rate of the strain was 0.04%. Two strains of A17 and A18 were obtained from the irradiated strains after first- and second- screening and the degradation rate of methamidophos of A17 and A18 strains were 10% higher than that of A0 strain (original stain). Moreover, the abilities to degrade folimat, phoxim and glyphosate were improved. Through 5 generations, the variation coefficient in degradation rate of methamidophos in the 6th day was 1.2%, showing that the new strains had hereditary stability. (authors)

Preliminary research on morphological differentiation of avilamycin high-yield mutant strain H15

International Nuclear Information System (INIS)

Liang Xinle; Jin Yingyan; Chen Ming; Zhang Hong

2010-01-01

Morphological differentiation characters such as colony, sporotrichial, and conidiophores of mutant H15, which was derived from Streptomyces viridochromogenes 4.1119 treated with 60 Co γ-rays irradiation, were investigated by scanning electron microscope and fluorescence microscope. The results showed that mutant H15 was remarkable variation from the strain 4.1119. Cultured on agar surface, H15 had a grayish-whitish-green colony, linear sporotrichial, smooth and round conidiophore without any spike, whereas strain 4.1119 had spiral sporotrichial and round conidiophore with spike on the surface. In the submerged cultures, differentiation process of mycelia pellet of H15 was also different. Spores germinated as a compartmentalized mycelium, the young compartmentalized mycelium started to form pellets which grew in a radial pattern. After apoptosis took place in the center of the pellets, the pellet diameter growth arrested. Compared with the strain 4.1119, H15 required a long developing course for hyphae clustering and pellets formation (at 48 h, φ 245 μm). The stage of pellet arrest or apoptosis in the pellet centre were extended, which would benefit the avilamycin accumulation since the antibiotic was mainly produced at the same time. These suggested that pellet formation kinetics, relational balance between pellet diameter enlargement and mycelia apoptosis in the pellet arrest stage were key factors to avilamyin accumulation in submerged cultures of Streptomyces viridoehrongenes H15. (authors)

Evaluation of symbiotic performance of some mutant lines of soybean inoculated with two bradyrhizobium japonicum strains using 15N technique

International Nuclear Information System (INIS)

Kurdali, F.; Mir-Ali, N.; Al-Nabulsi, I.

2002-11-01

A pot experiment was conducted to study the symbiotic performance of two soybean varieties and some of their mutants (that were obtained as a result of a previous mutation breeding program) with two bradyrhizobium japonicum strains (RG and FA3) using 15 N isotopic dilution method. Random amplified polymorphic DNA technique (RAPD) was used to study the genetic relationships among the soybean genotypes and to make sure that the two rhizobial strains are different. The 25 random primers used discriminated the different soybean genotypes and the dendrogram resultants from shared polymorphic fragments put each variety and its mutants in two separate clusters asserting that the mutants and their mother lines are different. Both strains of B. japonicum were able to form effective nodules on all soybean plants. However, number of nodules, dry matter yield and N-uptake from the available sources by soybeans were affected by both plant genotype and rhizobial strains. N 2 -fixation was affected to a large extent by different strain and plant genotype combinations. Percentage of fixed N 2 (N dfa) ranged between 35 and 49%; whereas, the actual amounts of fixed N 2 were between 105 and 210 mg N/pot. Amounts of N 2 -fixed by FA3 strain were higher than of RG in both soybean varieties, whereas, the latter strain showed higher performance in the mutant lines. The results showed that total plant N estimation may not be a sufficient indicator for high N 2 -fixation. the results also showed that it is very important to determine both the amount of nitrogen derived from N 2 -fixation and N derived from soil for evaluating the symbiotic performance ability. Moreover, the performance of symbiotic N 2 -fixation in soybean was shown to depend on both plant genotype and rhizobial strain and the amount of N 2 -fixation can be increased by combining the best plant genotypes and the most adapted strain. (author)

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Science.gov (United States)

Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

2016-07-01

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

Science.gov (United States)

Mitobe, Jiro; Sinha, Ritam; Mitra, Soma; Nag, Dhrubajyoti; Saito, Noriko; Shimuta, Ken; Koizumi, Nobuo; Koley, Hemanta

2017-07-01

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

Directory of Open Access Journals (Sweden)

Jiro Mitobe

2017-07-01

Full Text Available Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

Characterization and increment of amylase production in mutant strains of Iranian native Bacillus licheniformis

Directory of Open Access Journals (Sweden)

Mohsen Mobini-Dehkordi

2017-03-01

Results: In this study, two interesting mutant strains were isolated and named B.L.2.M.1 and B.L.2.M.2. Mutations caused many changes in bacteria such as cell growth speed and enzyme production content. Differences in cell growth, production of amylase and other characters were significant at 0.05 level (Pvalue

MUTANT STRAIN of Bacillus subtilis IFBG MC-1 WITH INCREASED TRYPTOPHAN SYNTHESIS

Directory of Open Access Journals (Sweden)

A. F. Tkachenko

2013-12-01

Full Text Available Scientific research of essential amino acids biotechnology is directed both to create optimum conditions for producer’s cultivation and economically viable raw materials selection for these technologies, so as breeding the more productive microorganisms strains capable of extracellular producing amino acids. For successful microbial synthesis it is necessary to have an excellent crop’s metabolism knowledge and ensure that the composition of growth medium have no repressing substances. Bacterial cultures from «Collection microorganism’s stains and plants line for food and agriculture biotechnology» from Institute of Food Biotechnology and Genomics of National Academy of Sciences of Ukraine have been studied. Tryptophan producer Bacillus subtilis have been selected, which accumulated the greatest amount of this amino acid in the cultivation liquid. The optimal culture producer conditions were selected. Using selection methods, namely mutagenesis with UV irradiation and sequential stepwise selection, mutant strain Bacillus subtilis IFBG MC-1 were obtained which produced nearly 50% more tryptophan (13.9 g/l than the parent strain.

Effect of varying temperature on growth, morphology and soluble protein content of div I and div II mutant strains of bacillus sub tills

International Nuclear Information System (INIS)

Ahmed, A.; Sabri, A.N.

2004-01-01

In B.subtilis, cell division is controlled by div-genes which have been mapped on its circular chromosome. In the present work, div-mutant strains 1A316(div II), 1A317 and 1A318 (div I) were studied. These strains exhibited temperature sensitive cell division mutations. Colony morphology, cell morphology, staining behavior, growth rate and protein content of PY79 (wild type) and div-mutant strains (1A316, 1A317, 1A318) was studied at different temperatures ( 25 deg. Centi grade and 42 deg. with varying incubation periods(4, 16, 24, 48, 72,96 hrs). div-mutants differ from wild type (PY79) in colony morphology. Colony margin in PY79 was entire while in the div strains it is undulate. Staining behavior of cells as well as cell morphology i.e., cell size, cell types, formation of filaments/minicells were affected by high temperature. At higher temperature (42 deg. Centi grade), div-mutants undergo more severe lysis and degeneration as compare to wild type (PY79). Defective spores were produced by div-mutants at 25 deg. Centi grade and 42 deg. Centi grade. Tetrazolium overlay test was performed at 37 deg. Centi grade and 42 deg. Centi grade to check the spore germination ability of wild type and div-mutants. In 1A318, defective spores were produced at 37 deg. Centi grade, div-mutant was checked after 24 and 96 hrs at different temperatures (25, 37 and 42 deg. Centi grade). At all temperatures protein content were more in PY79 as compare to div-mutants. Also at 25 and 42 deg. Centi grade, protein content was more as compare to 37 deg. Centi grade. Protein contents was reduced at sporulation stages. Thus cell division mutations affect cell morphology, sporulation and germination processes in B.subtilis and thus are multifaceted mutations. (author)

Flux control-based design of furfural-resistance strains of Saccharomyces cerevisiae for lignocellulosic biorefinery.

Science.gov (United States)

Unrean, Pornkamol

2017-04-01

We have previously developed a dynamic flux balance analysis of Saccharomyces cerevisiae for elucidation of genome-wide flux response to furfural perturbation (Unrean and Franzen, Biotechnol J 10(8):1248-1258, 2015). Herein, the dynamic flux distributions were analyzed by flux control analysis to identify target overexpressed genes for improved yeast robustness against furfural. The flux control coefficient (FCC) identified overexpressing isocitrate dehydrogenase (IDH1), a rate-controlling flux for ethanol fermentation, and dicarboxylate carrier (DIC1), a limiting flux for cell growth, as keys of furfural-resistance phenotype. Consistent with the model prediction, strain characterization showed 1.2- and 2.0-fold improvement in ethanol synthesis and furfural detoxification rates, respectively, by IDH1 overexpressed mutant compared to the control. DIC1 overexpressed mutant grew at 1.3-fold faster and reduced furfural at 1.4-fold faster than the control under the furfural challenge. This study hence demonstrated the FCC-based approach as an effective tool for guiding the design of robust yeast strains.

Improved hydrogen production by uptake hydrogenase deficient mutant strain of Rhodobacter sphaeroides O.U.001

Energy Technology Data Exchange (ETDEWEB)

Kars, Goekhan; Guenduez, Ufuk; Yuecel, Meral [Department of Biological Sciences, Middle East Technical University, 06531 Ankara (Turkey); Rakhely, Gabor; Kovacs, Kornel L. [Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged (Hungary); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, 06531 Ankara (Turkey)

2008-06-15

Rhodobacter sphaeroides O.U.001 is a purple non-sulfur bacterium producing hydrogen under photoheterotrophic conditions. Hydrogen is produced by Mo-nitrogenase enzyme and substantial amount of H{sub 2} is reoxidized by a membrane-bound uptake hydrogenase in the wild type strain. To improve the hydrogen producing capacity of the cells, a suicide vector containing a gentamicin cassette in the hupSL genes was introduced into R. sphaeroiodes O.U.001 and the uptake hydrogenase genes were destroyed by site directed mutagenesis. The correct integration of the construct was confirmed by uptake hydrogenase activity measurement, PCR and subsequent sequence analysis. The wild type and the mutant cells showed similar growth patterns but the total volume of hydrogen gas evolved by the mutant was 20% higher than that of the wild type strain. This result demonstrated that the hydrogen produced by the nitrogenase was not consumed by uptake hydrogenase leading to higher hydrogen production. (author)

Genetical, cytological and physiological studies on the induced mutants with special regard to effective methods for obtaining useful mutants in perennial woody plants

International Nuclear Information System (INIS)

Kukimura, H.; Ikeda, F.; Fujita, H.; Maeta, T.; Nakajima, K.; Katagiri, K.; Nakahira, K.; Somegou, M.

1975-01-01

The study was aimed at elucidating the biological aspects of artificially induced mutations in perennial tree crops and at promoting the utilization of such mutations in a practical breeding programme. A number of mutants obtained particularly in Cryptomeria and mulberry (Morus spp.) by means of gamma radiation were examined for their practical usefulness. Doses from 7.5 to 15.0 kR were used. In mulbery, some mutant strains showed increased shoot growth, and one mutant strain showed a remarkable increase also in rooting ability. Entire leaf mutants were investigated for their breeding behaviour. None of the mutant strains showed acquired disease resistance. Changes in the number of isozyme bands and different staining intensity was observed in all the mutant strains compared to the original strains

Methods of producing protoporphyrin IX and bacterial mutants therefor

Science.gov (United States)

Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

2016-03-01

The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

Science.gov (United States)

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

International Nuclear Information System (INIS)

Small, J.M.; Mitchell, T.G.

1986-01-01

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with 125 I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal

Restoration of growth by manganese in a mutant strain of Escherichia coli lacking most known iron and manganese uptake systems

DEFF Research Database (Denmark)

Taudte, Nadine; German, Nadezhda; Zhu, Yong-Guan

2016-01-01

The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron/manganese-uptake ......The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron...

Biochemical studies of the macromolecular matrix of long bones in the Op/Orl mutant rat strain

Energy Technology Data Exchange (ETDEWEB)

Moczar, E; Berenholc, S; Phan-Dinh-Tuy, B; Robert, A M

1978-01-01

The long bones of normal and Op/Orl mutant rats were incubated with /sup 14/C-glucose and fractionated by EDTA and urea extraction. The analytical results of the various extracts suggested an increase in structural glycoprotein content and a decrease in collagen solubility in the long bones of mutants. Significant differences were found in the organic matrix composition of male and female bones of the two strains. /sup 14/C-glucose incorporation was stronger in males than in females. The presence of a glycosaminoglycan different from the chondroitinesulfate was shown in males. Basic amino acid content (lysine, arginine, histidine) was clearly higher in the insoluble residue of male bones .

Biochemical studies of the macromolecular matrix of long bones in the Op/Orl mutant rat strain

International Nuclear Information System (INIS)

Moczar, E.; Berenholc, S.; Phan-Dinh-Tuy, B.; Robert, A.M.

1978-01-01

The long bones of normal and Op/Orl mutant rats were incubated with 14 C-glucose and fractionated by EDTA and urea extraction. The analytical results of the various extracts suggested an increase in structural glycoprotein content and a decrease in collagen solubility in the long bones of mutants. Significant differences were found in the organic matrix composition of male and female bones of the two strains. 14 C-glucose incorporation was stronger in males than in females. The presence of a glycosaminoglycan different from the chondroitinesulfate was shown in males. Basic amino acid content (lysine, arginine, histidine) was clearly higher in the insoluble residue of male bones

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

Directory of Open Access Journals (Sweden)

Muhammad Nauman Aftab

2012-03-01

Full Text Available The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1. Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG and Nitrous acid (HNO2 increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1 by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate, Yp/x (IU/g cells, Yx/s (g/g, Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

DEFF Research Database (Denmark)

Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

1985-01-01

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosin...

Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli

International Nuclear Information System (INIS)

Witkin, E.M.; Roegner-Maniscalco, V.; Sweasy, J.B.; McCall, J.O.

1987-01-01

Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC. Recovery of DNA synthesis after UV irradiation (induced replisome reactivation, or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein. IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants. In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present. IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation. We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein. We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis

Enhanced mucosal delivery of antigen with cell wall mutants of lactic acid bacteria.

Science.gov (United States)

Grangette, Corinne; Müller-Alouf, Heide; Hols, Pascal; Goudercourt, Denise; Delcour, Jean; Turneer, Mireille; Mercenier, Annick

2004-05-01

The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen. Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract. Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers. Recombinant wild-type and mutant strains of L. plantarum NCIMB8826 and L. lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments. Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used. However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L. plantarum mutant strain. Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract. The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.

Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

Science.gov (United States)

Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

2016-10-01

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

Establishment of pseudomonas putida strains for sensitive detection of heavy metals in effluents

International Nuclear Information System (INIS)

Genthe, B.

1987-09-01

The objective of this study was to isolate a mutant of Pseudomonas putida that is more sensitive to heavy metal toxicants in water than the wild type. P. putida was the organism chosen in this study as it occurs naturally in unpolluted waters, is nonpathogenic, aerobic and because it is commonly applied in bacterial toxicity assays due to its sensitivity to toxicants. Three methods of mutagenesis were employed, which included N-methyl-N'-nitro-N-nitrosoguanidine (NG) ; ultraviolet light and transposon-mediated mutagenesis in order to generate as wide a range of mutants as possible. Four mutants, which were more sensitive to mercury, copper, lead, zinc, cadmium and silver were isolated using the NG method of mutagenesis. These mutants were designated strains 53, 56, 60 and 61 and were characterized as P. putida strains on the basis of Gram staining, biochemical reactions and immunological properties. The sensitivity of the mutants to a variety of industrial effluents was compared to that of the parent strain using a bacterial growth test. Using industrial effluents, one of the mutants, namely strain 56 was found to be more sensitive than the parent strain on 71.4% of the tests. Strains 60 and 61 were also both more sensitive than the parent strain on 42.9% of the occasions using industrial effluents. The uptake rates of radioactive mercury were measured for the parent strain of P. putida and the mutants that were found to be more sensitive to mercury

Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

Science.gov (United States)

Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

2015-11-01

Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Characterization of a mutant of Escherichia coli B/R defective in mutation frequency decline

International Nuclear Information System (INIS)

George, D.L.

1974-01-01

A mutant of Escherichia coli B/r designated mfd is very deficient in the ability to exhibit mutation frequency decline (MFD), the characteristic loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with ultraviolet light (uv). This mutant is known to excise pyrimidine dimers very slowly, although it is as uv-resistant as its mfd + B/r parent strain. We have found that the mfd mutant performs the initial incision step of excision repair normally, but repairs the resulting single-strand breaks much more slowly than the mfd + strain. In spite of the slow dimer excision in the mfd mutant, single-strand DNA breaks do not accumulate during postirradiation incubation, implying that incision and excision are well corrdinated. the prolonged postirradiation lag in cell division and DNA synthesis which accompany slow excision in the mfd strain indicates that resumption of these processes of optimal rates is linked to the timing of excision repair. The normal uv-resistance of the mfd mutant also suggests such coordination and shows that the rate of excision repair is independent of its ultimate efficiency in the removal of potentially lethal uv-induced damage. (U.S.)

Protein nutrient value evaluation of mutant strain J5 fruitbody Agaricus bazei murrill by 60Co γ-irradiation

International Nuclear Information System (INIS)

Weng Boqi; Huang Tingjun

2004-01-01

Protein nutrient value evaluation of mutant strain J 5 fruitbody Agaricus bazei Murrill by 60 Co γ-irradiation was studied. The results showed its total content of amino acids Agaricus bazei murrill was 48.20%; chemical score 58.10; amino acids score 88.60; necessary amino acids index 89.94; biologic value 86.29; nutrient index 29.15. All the index above were higher than that in original strain J 1 , but the ratio score of amino acids of J 5 fruitbody (39.48) was lower than strain J 1 . The results indicated that nutrient value of protein in J 5 was higher than in J 1 . (authors)

Structural characterization of bioengineered α-D-glucans produced by mutant glucansucrase GTF180 enzymes of lactobacillus reuteri strain 180

NARCIS (Netherlands)

Leeuwen, S.S. van; Kralj, S.; Eeuwema, W.; Gerwig, G.J.; Dijkhuizen, L.; Kamerling, J.P.

2009-01-01

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith

Structural Characterization of Bioengineered alpha-D-Glucans Produced by Mutant Glucansucrase GTF180 Enzymes of Lactobacillus reuteri Strain 180

NARCIS (Netherlands)

van Leeuwen, Sander S.; Kralj, Slavko; Eeuwema, Wieger; Gerwig, Gerrit J.; Dijkhuizen, Lubbert; Kamerling, Johannis P.

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

Science.gov (United States)

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Microbial production of squalene by a nicotinic acid-resistant mutant derived from Fusarium sp. No.5-128B

International Nuclear Information System (INIS)

Ogawa, T.; Kojima, I.; Takeda, N.; Fukuda, H.

1994-01-01

A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no.5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The Na201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg-l(-1) of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O2 l(-1)h(-1)atm(-1) and the level of squalene in the mutant cells was 26 mg (g cell)(-1) on a dry weight basis. The greatest accumulation of squalene by the Na201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)(-1) on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v)

A yeast mutant specifically sensitive to bifunctional alkylation

International Nuclear Information System (INIS)

Ruhland, A.; Kircher, M.; Wilborn, F.; Brendel, M.

1981-01-01

A mutation that specifically confers sensitivity to bi- and tri-functional alkylating agents is presented. No or little cross-sensitivity to radiation or monofunctional agents could be detected. Sensitivity does not seem to be due to preferential alkylation of mutant DNA as parent and mutant strain exhibit the same amount of DNA alkylation and the same pattern of DNA lesions including interstrand crosslinks. The mutation is due to a defect in a nuclear gene which has been designated SNM1 (sensitive to nitrogen mustard); it may control an important step in the repair of DNA interstrand crosslinks (orig.(AJ)

Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing

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Muhammad Nadeem

2010-10-01

Full Text Available The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine and MMS (methyl methane sulfonate. After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1, Yp/s, Yp/x, Yx/s, q s, Qs, q p and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h at 40 º C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

Science.gov (United States)

Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhanced production of bacitracin by a mutant strain bacillus licheniformis UV-MN-HN-8 (enhanced bacitracin production by mutagenesis)

International Nuclear Information System (INIS)

Aftab, M.N.; Ikram-ul-Haq; Baig, S.

2010-01-01

The present study is focused on the improvement of Bacillus licheniformis through random mutagenesis to obtain mutant having enhanced production of bacitracin. Many isolates of Bacillus licheniformis were isolated and the isolate GP-40 produced maximum bacitracin production (16 +- 0.72 IU/mL). Treatment of Bacillus licheniformis GP-40 with ultraviolet (UV) radiations increased bacitracin production to 29 +- 0.69 IU/mL. Similarly, treatment of vegetative cells of GP-40 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO/sub 2/) increased bacitracin production to 35 +- 1.35 IU/mL and 29 +- 0.89 IU/mL respectively. Studies regarding the combined effect of UV and chemical treatment on parental cells exhibited significantly higher titers of bacitracin with maximum bacitracin production reached to 47.6 +- 0.92 IU/mL. An increase of 2.97 fold production of bacitracin in comparison to wild type was observed. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably mu (h-/sup 1/)max, Yp/x, qp, Qp and Qx mutant strain B. licheniformis UV-MN-HN-8 was found to be a hyper producer of bacitracin. (author)

Characterization and protective property of Brucella abortus cydC and looP mutants.

Science.gov (United States)

Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

2014-11-01

Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

UDP-N-acetylmuramic acid l-alanine ligase (MurC) inhibition in a tolC mutant Escherichia coli strain leads to cell death.

Science.gov (United States)

Humnabadkar, Vaishali; Prabhakar, K R; Narayan, Ashwini; Sharma, Sreevalli; Guptha, Supreeth; Manjrekar, Praveena; Chinnapattu, Murugan; Ramachandran, Vasanthi; Hameed, Shahul P; Ravishankar, Sudha; Chatterji, Monalisa

2014-10-01

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant

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Böttger Erik C

2008-07-01

Full Text Available Abstract Background The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy. Results As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia. Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA. Conclusion According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus

International Nuclear Information System (INIS)

Zolan, M.E.; Tremel, C.J.; Pukkila, P.J.

1988-01-01

We have isolated four gamma-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1;rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the pew viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants

Isolation of L-methionine-enriched mutant of a methylotrophic yeast, Candida boidinii No.2201

International Nuclear Information System (INIS)

Tani, Y.; Lim, W.J.; Yang, H.C.

1988-01-01

Six strains of methylotrophic yeast were examined for production of L-methionine-enriched cells. Candida boidinii (kloeckera sp.) No. 2201,which accumulated 0.54 mg/g-dry cell weight (DCW) of free L-methionine (pool methionine), was selected as the parental strain for breeding L-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradiation. A mutant strain, E500-78,which was resistant to 500 μg/ml of DL-ethionine, accumulated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were optimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine

Vph6 Mutants of Saccharomyces Cerevisiae Require Calcineurin for Growth and Are Defective in Vacuolar H(+)-Atpase Assembly

OpenAIRE

Hemenway, C. S.; Dolinski, K.; Cardenas, M. E.; Hiller, M. A.; Jones, E. W.; Heitman, J.

1995-01-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demo...

Restriction of phage T4 internal protein I mutants by a strain of Escherichia coli

International Nuclear Information System (INIS)

Black, L.W.; Abremski, K.

1974-01-01

Phage T4 internal protein I(IPI), a small (ca, 10,000 MW), basic protein injected into the host with the phage DNA, is not required for infection of most hosts, but mutants defective in IPI are restricted by at least one naturally occurring strain of Escherichia coli, CT 596 (CT). Phages lacking IPI (IPI - ) appear to inject their DNA and bind it to the membrane of CT cells as well as wild-type phage T4 does, but shutoff of host protein synthesis, initiation of T4 protein synthesis, and cell killing are abnormal in the IPI - mutant infected CT host. The injection of IPI appears to be important in allowing T4 DNA to carry out early steps involved in takeover of this host. Restriction of IPI - phage growth by CT cells appears to be due, at least in part, to a defective prophage it harbors which renders the host resistant to successful infection by phage T4 which lack IPI or rII functions. Bacteria cured of this prophage can be infected by mutants defective in these functions. The resistance of CT cells to other coliphages, and the question of T-even phage internal protein diversity are discussed. (U.S.)

Evaluation of the G145R Mutant of the Hepatitis B Virus as a Minor Strain in Mother-to-Child Transmission.

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Haruki Komatsu

Full Text Available The role of the hepatitis B virus (HBV mutant G145R, with a single change in amino acid 145 of the surface protein, as a minor population remains unknown in mother-to-child transmission. The minor strain as well as the major strain of the G145R mutant were evaluated in three cohorts using a locked nucleic acid probe-based real-time PCR. The breakthrough cohort consisted of children who were born to HBV carrier mothers and became HBV carriers despite immnoprophylaxis (n = 25. The control cohort consisted of HBV carriers who had no history of receiving the hepatitis B vaccine, hepatitis B immunoglobulin or antiviral treatment (n = 126. The pregnant cohort comprised pregnant women with chronic HBV infection (n = 31. In the breakthrough cohort, 6 showed positive PCR results (major, 2; minor, 4. In the control cohort, 13 showed positive PCR results (major, 0; minor, 13. HBeAg-positive patients were prone to have the G145R mutant as a minor population. Deep sequencing was performed in a total of 32 children (PCR positive, n = 13; negative, n = 19. In the breakthrough cohort, the frequency of the G145R mutant ranged from 0.54% to 6.58%. In the control cohort, the frequency of the G145R mutant ranged from 0.42% to 4.1%. Of the 31 pregnant women, 4 showed positive PCR results (major, n = 0; minor, n = 4. All of the pregnant women were positive for HBeAg and showed a high viral load. Three babies born to 3 pregnant women with the G145R mutant were evaluated. After the completion of immunoprophylaxis, 2 infants became negative for HBsAg. The remaining infant became negative for HBsAg after the first dose of HB vaccine. G145R was detected in one-fourth of the children with immunoprophylaxis failure. However, the pre-existence of the G145R mutant as a minor population in pregnant women does not always cause breakthrough infection in infants.

Strain improvement in dye decolourising mutants of Mucor mucedo ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... M. mucedo {MMM1-U.V. irradiated mutant and MMM2-EMS (ethyl methyl sulfonate) treated ... tions were induced and two positive mutants (MMM1, .... yeast biofilter for the treatment of a Nigerian fertilizer plant effluent. World J.

EXTRACTION AND PURIFICATION OF EXTRACELLULAR LACCASE FROM WILD, MUTANTS AND HYBRID STRAINS OF TWO WHITE-ROT FUNGUS AND ITS APPLICATIONS IN DECOLOURIZATION AND LIGNINOLYSIS

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Olusola Majolagbe

2012-12-01

Full Text Available Extracellular laccases were extracted from a 5-day old submerge cultures of the wild, mutants and hybrid of Lentinus subnudus. Mutants were generated by exposure of the wild strain of L. subnudus to ultraviolet radiation (ג = 280 nm at specific time intervals while the hybrid was produced by cross-breeding L. subnudus with L. edodes. The crude enzyme was fractionated with 80% ammonium sulphate and further purified on DEAE column. The laccase has a molecular weight of about 45 KDa. Purification yield on DEAE column gave the highest purification yield of 23.25% in SWT and least in SHT (5.29%. Its potentials in decolourization of 2, 6-dichlorophenol-indophenol dye at different pH conditions were investigated. Five out of the six fungal strains tested gave significant (P<0.05 percentage decolourization (≥43.94% at pH 8. The fungus was further studied for their ability in degrading wheat and paddy straws. The solid substrate fermentation was inoculated with two pieces (0.6cm diameter mycelial agar blocks of each of the fungal strains, supplemented with 30mg/100g sucrose, 24mg/100g KNO3 and 60mg/100g CaCO3. The periodic reduction in weight of the solid substrate medium and enzymatic activity of laccase for each of the fungal strains was assessed. Therefore, the ability of the wild, mutants and hybrid of L subnudus strains to produce laccase enzyme shows their significant potential in textile industry, especially in decolourization of dye and bioconversion of lignocellulosic wastes.

Symbiotic N fixation of several soybean varieties and mutants

International Nuclear Information System (INIS)

Soertini, G.; Hendratno

1988-01-01

Symbiotic N fixation of several soybean varieties and mutants. Research activities comprising of three experiments were carried out to screen several soybean varieties and mutants for symbiotic N fixation potential. The first two experiments involved screening of seven rhizobium strains/isolate for effective N fixation. Depending on the medium used, plant response to strains was different. In sterile medium, rhizobium strain USDA 136, 142 and TAL 102 showed a high nitrogen fixation potential. In soil only rhizobium strain USDA 110 had better performance and proved to be competitive to the native strains. Nitrogen-15 dilution method was used to screen nitrogen fixing ability of several soybean varieties and mutants. Guntur variety showed a better response to high dose of N fertilizer without disturbance in its fixing ability. This variety then was considered good to be introduced in the cropping system. (author). 8 refs

Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

International Nuclear Information System (INIS)

Lee, Young Keun; Murugesan, Senthilkumar

2009-01-01

Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg -1 protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg -1 protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg -1 protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg -1 protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg -1 protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase

Energy Technology Data Exchange (ETDEWEB)

Schreferl, G.; Kubicek, C.P.; Roehr, M.

1986-03-01

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn/sup 2 +/ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Science.gov (United States)

Shima, Jun; Hino, Akihiro; Yamada-Iyo, Chie; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Mori, Katsumi; Takano, Hiroyuki

1999-01-01

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough. PMID:10388673

Isolation and characterization of radioresistant mutants in Bacillus subtilis and Bacillus thuringiensis

International Nuclear Information System (INIS)

Kalinin, V.L.; Petrov, V.N.; Petrova, T.M.

1981-01-01

Vegetative cells of Bac. thuringiensis var. galleriae (the wild-type strain 351) are much more sensitive to lethal effects of UV light and 60 Co-γ-rays than those of Bac. subtilis (the wild-type strain 168). This difference is less pronounced for spores of these strains. By means of repeated γ-irradiation-regrowth cycles radioresistant mutants Bac. thuringiensis Gamsup(r) 14 and Bac. subtilis Gamsup(r) 9 were selected. The vegetative cells of these mutants are correspondingly 19 times and 3.9 times more resistant to lethal effects of γ-radiation than the cells of the parental strains. The resistance of the Gamsup(r) mutant cells to lethal effects of UV light and H 2 O 2 is also increased. The spores of the Gamsup(r) 14 mutant are 1.5-1.7 times more resistant to γ-radiation and UV light than the wild-type spores. The radioresistant mutants and the parental strains do not vary in their capacity for host-cell reactivation of UV- or γ-irradiated phages Tg13 and 105

Optimisation of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of Aspergillus oryzae EMS-6.

Science.gov (United States)

Ali, Sikander; Nawaz, Wajeeha

2017-02-01

The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 μg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 μg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.

Metabolite Profiling to Characterize Disease-related Bacteria GLUCONATE EXCRETION BY PSEUDOMONAS AERUGINOSA MUTANTS AND CLINICAL ISOLATES FROM CYSTIC FIBROSIS PATIENTS

OpenAIRE

Behrends, V; Bell, TJ; Liebeke, M; Cordes-Blauert, A; Ashraf, SN; Nair, C; Zlosnik, JEA; Williams, HD; Bundy, JG

2013-01-01

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component sy...

Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

Energy Technology Data Exchange (ETDEWEB)

Lee, Young Keun; Murugesan, Senthilkumar [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2009-06-15

Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg{sup -1} protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg{sup -1} protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg{sup -1} protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg{sup -1} protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg{sup -1} protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions.

Morphological and physiological investigations on mutants of Fusarium monoliforme IM

International Nuclear Information System (INIS)

Gancheva, V.

1996-01-01

High-producing mutants of Fusarium moniliforme IM are obtained as a result of gamma irradiation. The cultural characteristics of mutant strains 3284, 3211 and 76 following incubation of the producers for 14 days on potato-glucose agar are described. The colour of the aerial and substrate mycelium and the ability of the mutant strains to form conidiae and pigments are discussed in detail. The differences in the ability of mutants to assimilate different carbon and nitrogen sources are of specific importance for modelling nutrient media for submerged cultivation of F. moniliforme. 2 tabs., 2 figs. 7 refs

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

Science.gov (United States)

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Lack of chemically induced mutation in repair-deficient mutants of yeast

International Nuclear Information System (INIS)

Prakash, L.

1974-01-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), β-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents. (auth)

Lack of chemically induced mutation in repair-deficient mutants of yeast.

Science.gov (United States)

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis

International Nuclear Information System (INIS)

Garber, E.D.; Baird, M.L.; Chapman, D.J.

1975-01-01

Wild-type strains of Ustilago violacea produce pink colonies on laboratory medium and yield white, orange, pumpkin, and yellow colonies after uv mutagenesis. The wild-type strains contain neurosporene and lycopene; one orange mutant, γ-carotene; and one yellow mutant, β-carotene. One white mutant had no detectable carotenoids. Diploid colonies heterozygous for wild type and orange, pumpkin, yellow, or white are phenotypically wild type. Diploid colonies heterozygous for yellow and orange are also phenotypically wild type. Diploid colonies heterozygous for white and orange; white and yellow; and white, yellow, and orange are phenotypically light orange, light yellow, and orange-yellow, respectively. The white mutants give a circular complementation map; the color mutants fit a linear complementation map. We propose a multienzyme of four identical dehydrogenases and one or two identical cyclases for carotenogenesis in this species. The white and color mutants represent structural mutations altering the conformation of the dehydrogenase or cyclase, respectively. Furthermore, cyclases may or may not aggregate in association with the dehydrogenase aggregate to form the multienzyme aggregate responsible for the color mutants

Study of the UV-sensitivity of the morphological Salmonella typhimurium mutant

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Sakanyan, V A; Dombrovskii, A M; Belokrysenko, S S; Levashev, V S [Vtoroj Moskovskij Gosudarstvennyj Meditsinskij Inst. (USSR)

1975-05-01

As regards sensitivity to ultraviolet radiation, the morphological mutant S. typhimurium LT2 WT ED 143 is similar to the ion-mutants E. coli K12. Data are presented on the sensitivity of the mutant and initial strains to ultraviolet radiation at various phases of growth, on the capacity for restoring the bacteriophages P22 and Felix O after irradiation and on the influence of various treatments after ultraviolet irradiation (incubation in minimum media and at 42/sup 0/ C) on the irradiated strains. The results of densitometry of the membrane proteins of the initial and mutant strains point to a connection between unusual morphology, the disruption of division and the enhanced sensitivity to ultraviolet radiation on one hand and the state of the membrane components of the bacterial cell on the other.

Cellulase production by two mutant strain of Trichoderma longibrachiatum Qm9414 and Rut C30

International Nuclear Information System (INIS)

Blanco, M.J.

1991-01-01

Native or pretreated biomass from Onopordum nervosum boiss, has been examined as candidate feedstock for cellulase production by two mutant strain of trichoderma longibrachiatum QM9414 and Rut C30. Batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka floc). Batch cultivation of T. longibrachiatum Rut C30 on 55% (W/V) acid pretreated O. nervosum biomass yielded enzyme productivities and activities comparable to those obtained on Solka floc. However, the overall enzyme production performance was lower than on Solka floc at comparable cellulose concentrations. This fact may be due to the accumulation of pretreated by products and lignin in the fermentor.(author)

Studies on reduced height mutants in rice

International Nuclear Information System (INIS)

Narahari, P.; Bhagwat, S.G.

1984-01-01

Two cross-bred derivatives of the mutant TR5xTR17 and TR21 continued to show promise and were advanced to wider scale testing. TR5 was found to carry a semi-dwarfing gene different from that in IR8. New semi-dwarf mutants were screened from M 2 through M 4 from two separate radiation experiments. The gibberellin response of seedlings of mutant and tester strains was evaluated and crosses of tester stocks and mutant semi-dwarfs were made for genetic analyses. (author)

Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

Science.gov (United States)

Rawat, Mamta; Newton, Gerald L.; Ko, Mary; Martinez, Gladys J.; Fahey, Robert C.; Av-Gay, Yossef

2002-01-01

Mycothiol (MSH; 1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1d-myo-inosityl 2-deoxy-α-d-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase (MshC) activities of the three mutant strains were ≤2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. PMID:12384335

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

Science.gov (United States)

Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi

2016-10-01

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Competitive Interactions Between Incompatible Mutants of the Social Bacterium Myxococcus xanthus DK1622

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Ya Gong

2018-06-01

Full Text Available Due to the high similarity in their requirements for space and food, close bacterial relatives may be each other's strongest competitors. Close bacterial relatives often form visible boundaries to separate their swarming colonies, a phenomenon termed colony-merger incompatibility. While bacterial species are known to have many incompatible strains, it is largely unclear which traits lead to multiple incompatibilities and the interactions between multiple incompatible siblings. To investigate the competitive interactions of closely related incompatible strains, we mutated Myxococcus xanthus DK1622, a predatory bacterium with complex social behavior. From 3392 random transposon mutations, we obtained 11 self-identification (SI deficient mutants that formed unmerged colony boundaries with the ancestral strain. The mutations were at nine loci with unknown functions and formed nine independent SI mutants. Compared with their ancestral strain, most of the SI mutants showed reduced growth, swarming and development abilities, but some remained unchanged from their monocultures. When pairwise mixed with their ancestral strain for co-cultivation, these mutants exhibited improved, reduced or unchanged competitive abilities compared with the ancestral strain. The sporulation efficiencies were affected by the DK1622 partner, ranging from almost complete inhibition to 360% stimulation. The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies. However, the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures. We propose that the colony-merger incompatibility in M. xanthus is associated with multiple independent genetic loci, and the incompatible strains hold competitive interaction abilities, which probably determine the complex relationships between multiple incompatible M. xanthus strains and

UV and gamma-ray sensitivity of meiosis-deficient mutants in Podospora anserina

International Nuclear Information System (INIS)

Simonet, J.M.

1976-01-01

Two mutants, mei1 and mei2, were isolated by screening for deficiencies occurring in the meiotic process. The sensitivity of mei1 and mei2 mutant strains to UV irradiation showed a significant increase as compared with that of the wild-type stock, hwhereas the sensitivity to γ-rays remained unchanged. The double-mutant strains were no more sensitive than each single mutant. The data indicate that both mei1 and mei2 loci are probably involved in the same pathway of excision-repair of UV-induced lesions

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

Science.gov (United States)

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

Mutant E. coli strain with increased succinic acid production

Science.gov (United States)

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

2001-09-25

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Mutant E. coli strain with increased succinic acid production

Science.gov (United States)

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

1998-01-01

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Isolation and genetic analysis of Aspergillus niger mutants with reduced extracellular glucoamylase

International Nuclear Information System (INIS)

Valent, G.U.; Calil, M.R.; Bonatelli Junior, R.

1992-01-01

Mutants with impaired production of extracellular glucoamylase were isolated at a high frequency (2% of survivors) from an Aspergillus niger strain treated with UV light. These were designated as low glucoamylase producers (lgp, up to 30% of the parental yield) and medium producers (mgp, a 35 to 50% decrease in enzyme level). All the mutants were shown to be recessive; one strain segregated two unlinked genes. Complementation tests, and segregation from heterozygous diploid, suggested at least three to four unlinked genes, each able to impair glucoamylase production. There is evidence of a single structural gene for glucoamylase in A. niger. Therefore, as production of extracellular enzymes is normally the final result of several steps at intracellular and membrane levels, including regulation of enzyme synthesis, we suggest intergenic interaction that controls extracellular enzyme accumulation and that mutation in any of these genes would result in impaired production. (author)

Characterization of a bacteriophage T4 mutant lacking DNA-dependent ATPase

International Nuclear Information System (INIS)

Behme, M.T.; Ebisuzaki, K.

1975-01-01

A DNA-dependent ATPase has previously been purified from bacteriophage T4-infected Escherichia coli. A mutant phage strain lacking this enzyme has been isolated and characterized. Although the mutant strain produced no detectable DNA-dependent ATPase, growth properties were not affected. Burst sizes were similar for the mutant phage and T4D in polAl, recB, recC, uvrA, uvrB, uvrC, and various DNA-negative E. coli. UV sensitivity and genetic recombination were normal in a variety of E. coli hosts. Mapping data indicate that the genetic locus controlling the mutant occurs near gene 56. The nonessential nature of this gene is discussed

Cellulase production by two mutant strain of Trichoderma longibranchiatum QM 9414 and Rut C30

International Nuclear Information System (INIS)

Blanco, M. J.

1991-01-01

Native or pretreated biomass from Onopordum nervosum Boiss, has been examined as candidate feedstock for cellulase production by two mutant strain of Trichoderma Ionqibrachiatum QM9414 and Rut C30. Batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka floc). Batch cultivation of T. Ionqibrachiatum Rut C30 on 5% (w/v) acid pretreated O. nervosum biomass yielded enzyme productivities and activities comparable to those obtained on Solka floc. However, the overall enzyme production performance was lower than on Solka floc at comparable cellulose concentrations. This fact may be due to the accumulation of pretreated by products and lignin in the ferment. (Author) 40 refs

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants.

Science.gov (United States)

Viljoen, Albertus; Gutiérrez, Ana Victoria; Dupont, Christian; Ghigo, Eric; Kremer, Laurent

2018-01-01

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus , increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus ( M. abscessus ) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus , testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus , we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a , necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near

Breeding L(+)-lactic acid high productive mutant from xylose by nitrogen ions

International Nuclear Information System (INIS)

Yang Yingge; Li Wen; Liu Dan; Fan Yonghong; Wang Dongmei; Zheng Zhiming; Yu Zengliang

2007-01-01

In order to obtain higher L(+)-lactic acid yield strain fermentating from xylose, the original strain Rhizopus oryzae RLC41-6 was mutated by 10keV N + ion implantation. A mutant strain RQ4012 was obtained. After 72h shake-flask cultivation, the concentration of L(+)-lactic acid reached 74.37g/L, and the productivity was 1.03g/(L.h). Its lactic acid yield was 160% higher than that of the original one, and the mutant strain has high genetic stability. (authors)

Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

Science.gov (United States)

Gregoriou, M; Brown, P R; Tata, R

1977-11-01

Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.

[Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation].

Science.gov (United States)

Hua, Ying; Sun, Qi; Wang, Xiangyu; DU, Yanli; Shao, Na; Zhang, Qiwei; Zhao, Wei; Wan, Chengsong

2015-11-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation. A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain. We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.

Comparative production of cellulases by mutants of Trichoderma parceramosume PTCC5140

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Hoda Nouri

2017-06-01

Discussion and conclusion: Evaluation of cellulase production in mutant strains of Trichoderma parceramosume PTCC 5140 showed that use of chemical mutagenesis with 2 to 11 fold increasing in enzyme activity is a potent method to improve cellulase complex activity. In the current study, obtained mutant strains could be introduced as a potent cellulase producer for further studies in bioconversion processes.

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

International Nuclear Information System (INIS)

Kaefer, E.; Mayor, O.

1986-01-01

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or γ-rays. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. (Auth.)

Behaviour of UV-sensitive mutants of Proteus mirabilis to repair incision breaks

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

In U.V.-sensitive mutants of P. mirabilis with the phenotype HCR, REC and EXR single-strand breaks appeared immediately after UV-irradiation. The behaviour of REC- and EXR-mutants was similar to the wildtype. The number of incision breaks observed by sedimentation analysis in these strains was very low. They could be joined during the excision repair process. From the ability of REC- and EXR-strains to rejoin most of the induced single-strand breaks it can be concluded that these strains have approximately the same capacity for excision repair as the wildtype. HCR-mutants of P. mirabilis produced single-strand breaks after UV-irradiation in contrast to HCR-mutants of E. coli. Therefore we suggest that HCR-mutants of P. mirabilis are not completely inhibited in the incision step. The single-strand breaks introduced in the DNA at the beginning of the repair process were not rejoined during further incubation. Experiments with toluenized cells led to the same results. The newly synthesized daughter DNA-strands of UV-irradiated HCR-mutants were of low molecular weight in comparison with those from unirradiated control cells during the repair period. This result is in agreement with the incapability of HCR-mutants to remove the pyrimidine dimers from the parental template strand. (author)

A new strain of Claviceps purpurea accumulating tetracyclic clavine alkaloids.

Science.gov (United States)

Schumann, B; Erge, D; Maier, W; Gröger, D

1982-05-01

A new strain of Claviceps was isolated from a blokked mutant of Claviceps purpurea. This strain accumulates substantial amounts of clavine alkaloids (2 g/l). The alkaloid fraction is composed of chanoclavine-I ( approximately 10%) and a mixture of agroclavine/elymoclavine (90%). Most suitable for alkaloid production in submerged culture is an ammoncitrate/sucrose medium. The genealogy of the new strain, designated Pepty 695/ch-I is the following one: Pepty 695/S (ergotoxine producer) --> Pepty 695/ch (secoergoline producer) --> Pepty 695/ch-I (tetracyclic clavine producer).

Studies on auxotrophic mutants of Auricularia auricula and Auricularia fuscosucinea induced by irradiation

International Nuclear Information System (INIS)

Han Xincai; Yang Xinmei

1991-01-01

The induction of auxotrophic mutants of Auricularia auricula and Auricularia fuscosucinea from monokaryotic basidiospore by means of 60 Co-γ ray irradiation was reported. Under the irradiations of 10 krad-200 krad, 9 auxotrophic mutant strains were obtained, including 8 strains of A. auricula and 1 strain of A. fuscosucinea. The frequency of mutagenesis was 2.38 x 10 -3 -44.4 x 10 -3 . It was found that the optimum irradiation dose for A. auricula was 200 krad and for A. fuscosucinea was 10 krad. Biochemical and physiological researches indicated that the colony morphology, the hyphae growth speed, the contents of amino acid and the pattern of esterase isozyme of the mutants were different from those of the prototrophic strains

Construction of acetoin high-producing Bacillus subtilis strain

Directory of Open Access Journals (Sweden)

Yanjun Tian

2016-07-01

Full Text Available This paper describes the construction and selection of a high-producing mutant, Bacillus subtilis HB-32, with enhanced acetoin yield and productivity. The mutant was obtained by the protoplast fusion of a Bacillus subtilis mutant TH-49 (Val− producing acetoin and Bacillus licheniformis AD-30 producing α-acetolactate decarboxylase, with the fusogen polyethylene glycol and after the regeneration and selection, etc. of the fusant. The acetoin production reached 49.64 g/L, which is an increase of 61.8% compared to that of B. subtilis strain TH-49. Random amplified polymorphic DNA analysis was performed to determine the mutagenic and protoplast fusion effects and the genomic changes in the acetoin high-producing strain compared to the parent strains at the molecular level. The constructed strain was shown to be promising for large-scale acetoin production. Future studies should focus on the application of the mutant strain in practice.

Mutant prevention concentrations of four carbapenems against gram-negative rods.

Science.gov (United States)

Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C

2010-06-01

We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ss-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to > or =16. The MPC/MIC ratios for beta-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 microg/ml) than those for ss-lactamase-negative strains.

Kinetic modeling of batch fermentation for Populus hydrolysate tolerant mutant and wild type strains of Clostridium thermocellum.

Science.gov (United States)

Linville, Jessica L; Rodriguez, Miguel; Mielenz, Jonathan R; Cox, Chris D

2013-11-01

The extent of inhibition of two strains of Clostridium thermocellum by a Populus hydrolysate was investigated. A Monod-based model of wild type (WT) and Populus hydrolysate tolerant mutant (PM) strains of the cellulolytic bacterium C. thermocellum was developed to quantify growth kinetics in standard media and the extent of inhibition to a Populus hydrolysate. The PM was characterized by a higher growth rate (μmax=1.223 vs. 0.571 h(-1)) and less inhibition (KI,gen=0.991 vs. 0.757) in 10% v/v Populus hydrolysate compared to the WT. In 17.5% v/v Populus hydrolysate inhibition of PM increased slightly (KI,gen=0.888), whereas the WT was strongly inhibited and did not grow in a reproducible manner. Of the individual inhibitors tested, 4-hydroxybenzoic acid was the most inhibitory, followed by galacturonic acid. The PM did not have a greater ability to detoxify the hydrolysate than the WT. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

Science.gov (United States)

Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

2016-01-10

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). Copyright © 2015 Elsevier B.V. All rights reserved.

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid: Growth profiles and catalase activities in relation to microbody proliferation

OpenAIRE

Klei, Ida J. van der; Rytka, Joanna; Kunau, Wolf H.; Veenhuis, Marten

1990-01-01

The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T-), catalase A (A-T+) or both catalases (A-T-), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A+-strains (A+T+ and A+T-) high catalase activities were found; catalase activity invariably remained low in the A-T+ strain and was never detected in the A-T- strain. The levels of β-...

Enhancing the Production of D-Mannitol by an Artificial Mutant of Penicillium sp. T2-M10.

Science.gov (United States)

Duan, Rongting; Li, Hongtao; Li, Hongyu; Tang, Linhuan; Zhou, Hao; Yang, Xueqiong; Yang, Yabin; Ding, Zhongtao

2018-05-26

D-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient D-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial D-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of D-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24 g/L glucose for 9 days. The results showed that the production of D-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that D-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a D-mannitol-producing strain.

Comparative Study of Nonautolytic Mutant and Wild-Type Strains of Coprinopsis cinerea Supports an Important Role of Glucanases in Fruiting Body Autolysis.

Science.gov (United States)

Liu, Zhonghua; Niu, Xin; Wang, Jun; Zhang, Wenming; Yang, Mingmei; Liu, Cuicui; Xiong, Yuanjing; Zhao, Yan; Pei, Siyu; Qin, Qin; Zhang, Yu; Yu, Yuan; Yuan, Sheng

2015-11-04

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection.

Science.gov (United States)

Guo, Yuanyuan; Xun, Zhe; Coffman, Stephanie R; Chen, Feng

2017-01-01

The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host-virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 ( ne219 ) strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 ( ne219 ) mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 ( ne219 ) mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection

Directory of Open Access Journals (Sweden)

Yuanyuan Guo

2017-05-01

Full Text Available The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host–virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 (ne219 strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 (ne219 mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 (ne219 mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.

Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization

International Nuclear Information System (INIS)

Almond, J.W.; McGeoch, D.; Barry, R.D.

1979-01-01

Forty-nine temperature-sensitive mutants of fowl plague virus (FPV) strain Rostock and four ts mutants of FPV-strain Dobson were isolated by utilizing two methods of plaque screening, after either spontaneous or chemically induced mutagenesis. Twenty-nine of the FPV-Rostock mutants were further characterized by genetic recombination studies and were found to fall into six high frequency recombination groups. The genome segment carrying the ts mutation in each group was identified by analyzing the gene composition of ts + recombinants generated from crosses between representatives of each group and ts mutants of FPV-Dobson. It was concluded that the six groups correspond to mutations in six different genome segments, namely, those coding for the P 1 , P 2 , P 3 , HA, NP, and NS proteins

Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

Science.gov (United States)

Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

2000-01-01

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

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Dedeke Rockx-Brouwer

Full Text Available Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes.

Science.gov (United States)

Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

2012-02-01

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens.

Science.gov (United States)

Kawai, E; Akatsuka, H; Sakurai, N; Idei, A; Matsumae, H; Shibatani, T; Komatsubara, S; Omori, K

2001-01-01

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

Mutant breeding of Aspergillus niger irradiated by 12C6+ for hyper citric acid

International Nuclear Information System (INIS)

Hu Wei; Li Wenjian; Chen Jihong; Liu Jing; Wang Shuyang; Wang Jufang; Lu Dong

2014-01-01

In this study, strains of Aspergillus niger No.4 for hyper citric acid were irradiated to different doses by 80 MeV/u 12 C 6+ ion beams. Seven mutant strains showed marked citric acid over-production records and faster productivity than initial Aspergillus niger No.4 by shaking flash fermentation. The maximum product yield was 132.8 gL -1 (the H4002 strain) being a 8.8% increase to the initial strain. The scale-up experiment was carried out in a 100 L bioreactor. The mutant H4002 can accumulate 187gL -1 product yield of citric acid from starch liquefying supernatant. The productivity of citric acid was 2.75 g L -1 h -1 . So, the mutant H4002 possesses rapid sugar katabolism for producing citric acid. Meanwhile, the pellet morphology kept compact and round during the whole submerged fermentation, which was suited to produce citric acid. The results indicate that mutant H4002 has potential ability to produce citric acid rapidly. (authors)

Comparisons of radiosensitivity and damage repair potential between mutants from the Saccharomyces cerevisiae strain of yeast and laboratory-bred wild yeasts with particular attention being given to giant cell formation after X-radiation

International Nuclear Information System (INIS)

Heinen, A.

1988-01-01

Yeast cells were exposed to X-rays at dose levels up to 10 kGy to induce damage to the DNA and investigate its effects on cellular growth patterns. For this purpose, comparisons were carried out between one diploid strain and six haploid strains of the Saccharomyces uvarum and Saccharomyces cerevisiae species, which permitted the individual recovery and damage repair pathways to be described in more detail. The laboratory-bred wild strains ATCC 9080, 211 and 706 were judged to have unimpaired repair mechanisms as compared to the auxotrophs, which fact was evident from the higher radiosensitivity of the latter. A further parameter in this evaluation of growth behaviours was giant cell formation. The results here provided evidence in confirmation of deviations between wild strains and mutants. Even though the ceiling values for the formation of giant cells were similarly high in all strains, impairments of cell division and initial development were observed for the mutants already at considerably lower dose levels. (orig./MG) [de

The Effect of Vitamin E on the Survival Rate of unc-13 Caenorhabditis elegans mutants under Oxidative Stress

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Jessica Porcelan

2012-01-01

Full Text Available Caenorhabditis elegans unc-13 mutants express decreased neuronal activity and thus are a good model strain for examining defective nervous systems. These unc-13 mutants as well as wild type N2 strains, show rapid mortality when under oxidative stress. However, the antioxidant vitamin E may prolong survival in unc-13 mutant and N2 strains under oxidative stress. The addition of vitamin E to organisms under oxidative stress has a protective effect in both N2 and unc-13 C. elegans strains. Interestingly, vitamin E resulted in a greater increase in survival rate in N2 worms than with unc-13 mutant worms. While both strains displayed lower mortality rates with the addition of vitamin E, this finding suggests that vitamin E more efficiently increases survival rates of C. elegans with typical nervous system function. The efficacy of vitamin E implies that use of antioxidants may lessen the damage caused by oxidative stress in both N2 and mutant worms.

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

Science.gov (United States)

Field, H J; Wildy, P

1978-10-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.

Recombination-deficient mutants of Bacillus subtilis

International Nuclear Information System (INIS)

Sadaie, Y.; Kada, T.

1976-01-01

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (uv), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation, SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr + capacity for uv-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 tranduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The uv impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene product whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strains NIG43 and NIG45 were not inducible, indicating involvement of rec-43 + or rec-45 + gene product in the development of SPO2 prophage to a vegetative form. The uv-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains

Immunization by intrabronchial administration to 1-week-old foals of an unmarked double gene disruption strain of Rhodococcus equi strain 103+.

Science.gov (United States)

Pei, Yanlong; Nicholson, Vivian; Woods, Katharine; Prescott, John F

2007-11-15

Rhodococcus equi causes fatal granulomatous pneumonia in foals and immunocompromised animals and humans. However, there is no effective vaccine against this infection. In this study, the chromosomal genes isocitrate lyase (icl) and cholesterol oxidase (choE) were chosen as targets for mutation and assessment of the double mutant as an intrabronchial vaccine in 1-week-old foals. Using a modification of a suicide plasmid previously developed in this laboratory, we developed a choE-icl unmarked deletion mutant of R. equi strain 103+. Five 1-week-old foals were infected intrabronchially with the mutant and challenged intrabronchially with the parent, virulent, strain 2 weeks later. Three of the foals were protected against pneumonia caused by the virulent strain, but the other two foals developed pneumonia caused by the mutant strain during the post-challenge period. Since infection of 3-week-old foals by an icl mutant in an earlier study had shown complete attenuation of the strain, we conclude that a proportion of foals in the 1st week or so of life are predisposed to developing R. equi pneumonia because of an inability to mount an effective immune response. This has been suspected previously but this is the first time that this has been demonstrated experimentally.

Phanerochaete mutants with enhanced ligninolytic activity

International Nuclear Information System (INIS)

Kakar, S.N.; Perez, A.; Gonzales, J.

1994-01-01

In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organo pollutants in soils and aqueous media. Most of the organic compounds are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, bio pulping, bio bleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated, or are hyper producers or super secretors of key enzymes under enriched conditions. Through UV-light and γ-ray mutagenesis, we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants, 76UV, produced 272 U of lignin peroxidases enzyme activity/L after 9 d under high nitrogen (although the parent strain does not produce this enzyme under these conditions). The mutant and the parent strains produced up to 54 and 62 U/L, respectively, of the enzyme activity under low nitrogen growth conditions during this period. In some experiments, the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 d

Inactivation of cephapirin sodium by the radiation-resistant strain micrococcus roseus

International Nuclear Information System (INIS)

Tawfik, Z.S.

1991-01-01

The susceptibility of the radioresistant mutants B. firmus, B.megaterium, B, laterosporus, M. roseus and M. luteus to the betalactam antibiotic cephapirin sodium was estimated using the microbiological assay technique. All the studied species were found to be sensitive to the concerned antibiotic except the radioresistant mutant M. rosues. Accordingly, the inactivation of betalactam, antibiotic cephapirin sodium, by this mutant strain was interesting to be investigated. A microbiological assay was used to determine the potency of the studied antibiotic and its degraded compound produced after its incubation with the above mentioned mutant strain for different periods of time in basal salt mineral medium.Results obtained for antibiotic samples extracted after 7-day incubation with the mutant strain indicated that the antibiotic was metabolized by this mutant strain to inactive products. These results were confirmed by chromatograms of the antibiotic samples, extracted from cultures with the mutant incubated for zero, 7 and 14 days. Degraded products were eluted at retention time values different from those observed for the noninucubated antibiotic samples. The inactivation of the antibiotic by the studied mutant starin seems to be due to extracellular enzymes in the surrounding medium.1 tab

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

Science.gov (United States)

Monforte, Anna Maria; De Luca, Laura; Buemi, Maria Rosa; Agharbaoui, Fatima E; Pannecouque, Christophe; Ferro, Stefania

2018-02-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Un Nuevo Enfoque en el Estudio de la Esporotricosis: Mutantes de Sporothrix schenckii

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Haydee Torres-Guerrero

2012-02-01

Full Text Available Una cepa silvestre y cepas mutantes de Sporothrix schenckii, se han estudiado como un modelo experimental de los procesos de diferenciación y desarrollo que se presentan al ser invadidas las células huésped y causar la esporotricosis. Las cepas mutantes de S. schenckii fueron obtenidas por exposición a la luz ultravioleta y Nitrosoguanidina. Las mutantes morfológicas M-III y M-V fueron seleccionadas. Estas mutantes muestran una alteración colonial y un mayor desarrollo que las cepas silvestres. Además, las mutantes presentan mayor adhesión al sustrato. El análisis de componentes de la pared celular y la distribución de núcleos, indican que no existen diferencias significativas que implique un daño por la mutación. Los resultados indican que en las mutantes morfológicas existe una alteración en el patrón de crecimiento y su regulación. Son necesarios, estudios bioquímicos e inmunológicos, relacionados con la virulencia S. schenckii que puedan ser útiles en el diagnóstico y en un futuro contribuyan a medidas preventivas para la esporotricosis. A wild-type strain and mutant strain of Sporothrix schenckii were studied as an experimental model in the process of differentiation and development which occurs when the host cell is invaded causing sporotrichosis. The mutant strains of S. schenckii were obtained by exposure to ultraviolet light and Nitrosoguanidine. The morphological mutants M-III and M-V were selected. These mutants showed a colonial alteration and a higher growth rate than the wild-type strains. Moreover, the mutants showed greater adhesion to the substratum. An analysis of the components of the cell wall and the distribution of nuclei indicate that significant differences do not exist which involve damage by mutation. The results suggest that in morphological mutants there is an alteration of growth and its regulation in the host cell. Biochemical and immunological studies related to the virulence of S. schenkii are

RobOKoD: microbial strain design for (over)production of target compounds.

Science.gov (United States)

Stanford, Natalie J; Millard, Pierre; Swainston, Neil

2015-01-01

Sustainable production of target compounds such as biofuels and high-value chemicals for pharmaceutical, agrochemical, and chemical industries is becoming an increasing priority given their current dependency upon diminishing petrochemical resources. Designing these strains is difficult, with current methods focusing primarily on knocking-out genes, dismissing other vital steps of strain design including the overexpression and dampening of genes. The design predictions from current methods also do not translate well-into successful strains in the laboratory. Here, we introduce RobOKoD (Robust, Overexpression, Knockout and Dampening), a method for predicting strain designs for overproduction of targets. The method uses flux variability analysis to profile each reaction within the system under differing production percentages of target-compound and biomass. Using these profiles, reactions are identified as potential knockout, overexpression, or dampening targets. The identified reactions are ranked according to their suitability, providing flexibility in strain design for users. The software was tested by designing a butanol-producing Escherichia coli strain, and was compared against the popular OptKnock and RobustKnock methods. RobOKoD shows favorable design predictions, when predictions from these methods are compared to a successful butanol-producing experimentally-validated strain. Overall RobOKoD provides users with rankings of predicted beneficial genetic interventions with which to support optimized strain design.

Microbial Fe (III) reduction and hydrogen production by a transposon-mutagenized strain of Pantoea agglomerans BH18

International Nuclear Information System (INIS)

Liu, Hongyan; Wang, Guangce

2015-01-01

Based on the transposon-mutagenized library of Pantoea agglomerans BH18, mutant screens were conducted to obtain the strain with the highest Fe (III) reduction and hydrogen production. Of these transposon-mutagenized mutants, the mutant strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. The PCR amplification and kanamycin resistance selection results indicated that the transposon insertion of the mutant strain TB230 was stable. Hydrogen production of the mutant strain TB230 was (2.21 ± 0.34) mol H 2 /mol glucose, which increased hydrogen production by over 40% compared with that of the wild type strain. The accumulation concentration of Fe (II) in the medium of the mutant strain TB230 with Fe (OH) 3 as the sole electron acceptor was (7.39 ± 0.49) mmol/l, which was approximately 3-fold greater than that of the wild type strain. The mutant strain TB230 showed high Fe (III)-reducing activity and hydrogen production by adopting glucose and pyruvate as the carbon source. In addition, the mutant strain TB230 was capable of Fe (III) reduction and hydrogen production under fresh or marine conditions. This result indicates that the mutant strain with high microbial Fe (III) reduction and hydrogen production is beneficial for the improvement of anaerobic performance. - Highlights: • The mutant strain TB230 was a transposon-mutagenized strain of Pantoea agglomerans BH18. • Strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. • H 2 yield and Fe (III)-reducing activity were 2.21 ± 0.34 and 7.39 ± 0.49 in marine condition. • Strain TB230 was capable of Fe (III) reduction and hydrogen production in fresh or marine condition

Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin, and gemifloxacin against pneumococci.

Science.gov (United States)

Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A; Appelbaum, Peter C

2010-02-01

We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P<0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 microg/ml) within the susceptible range.

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Nygaard, Per

1982-01-01

, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

Establishment and characterization of a hypocatalasemic mouse cell strain

International Nuclear Information System (INIS)

Utsumi, Hiroshi; Tano, Keizo; Hashimoto, Mitsumasa W.; Kodama, Seiji; Watanabe, Hiromitsu

1998-01-01

Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Cs b mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Cs b mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Cs a cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cis-diamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress. (author)

Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

Directory of Open Access Journals (Sweden)

Hassan Abdel-Latif A. Mohamed

2006-06-01

Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of enterotoxigenic Escherichia coli and Cfae-R181A mutant.

Science.gov (United States)

Liaqat, Iram; Sakellaris, Harry

2012-07-01

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacI (q)/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacI (q)/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

Mutants of Streptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites

International Nuclear Information System (INIS)

Blumauerova, M.; Stajner, K.; Pokorny, V.; Hostalek, Z.; Vanek, Z.

1978-01-01

Mutants of Streptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, γ-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described in Streptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, zeta-rhodomycinone and glycosides of epsilon-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases. (author)

Mutant Prevention Concentrations of Four Carbapenems against Gram-Negative Rods▿ †

Science.gov (United States)

Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C.

2010-01-01

We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ß-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to ≥16. The MPC/MIC ratios for β-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 μg/ml) than those for ß-lactamase-negative strains. PMID:20308376

Generation and characterization of pigment mutants of ...

African Journals Online (AJOL)

acer

2014-01-08

Jan 8, 2014 ... aquatic ecosystems were studied. In the present ... logy and photosynthesis research (Stolbov, 1995;. Pedersen ... Microalgal strain and cultivation conditions ..... evaluated for their ecotoxicological effects using 124y-1 mutant.

The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

Science.gov (United States)

Lawal, Abidat; Kirtley, Michelle L; van Lier, Christina J; Erova, Tatiana E; Kozlova, Elena V; Sha, Jian; Chopra, Ashok K; Rosenzweig, Jason A

2013-09-01

Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

Metabolism of the chemo antibiotic combination sulphamethoxazol and trimethoprim by some radioresistant strains

International Nuclear Information System (INIS)

Zahiera, T.S.

1990-01-01

The radioresistant mutants B. Firmus, B. Laterosporus, B. Megaterium, M. Roseus and M. Luteus were tested microbiologically for their sensitivity to a drug consisting of the chemo antibiotic combination of sulphamethoxazole (SMZ) and trimethoprim (TMP) in a ratio of 20:1. All the studied mutant strains were found to be insensitive to this combination of antibiotics except the radioresistant mutant strain of M.roseus. The mechanism of the inactivation of both SMZ and TMP by the radioresistant mutants of B. Laterosporus, B. Firmus, and M.Luteus was studied. The drug was incubated with each of these mutants for different periods of time to study its degradation. High performance liquid chromatography and microbiological assay techniques were used to determine the activity and the pathway of the consumption of the drug after its incubation with these three mutants for time. The results indicated that the drug acts in a synergistic effect on all the studied mutants. Only a part of both molecules of SMZ and TMP was utilized by the studied mutant strains. The ratio of the left over of SMZ/TMP were found to be 6,6 and 8 after seven days incubation periods and 12, 7.5 and 19 after two weeks incubation periods with B. Laterosporus, B. Firmus, and M. Luteus mutants respectively. The microbial activity of the extracted drug for bacilli strains isolated from clean environment had not changed significantly due to incubation with the mutants, but it was decreased for M. Luteus strain. The drug inactivation by the studied mutant strains seemed to be due to decreased affinities to SMZ and TMP, and to formation of an altered 7,8 dihydropteroate synthetase and dihydrofolate reductase

Reduction of FR900525 using an S-(2-aminoethyl) l-cysteine-resistant mutant.

Science.gov (United States)

Shimizu, Shiho; Futase, Ayako; Yokoyama, Tatsuya; Ueda, Satoshi; Honda, Hiroyuki

2017-06-01

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S. tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mutagenesis of Xanthomonas campestris and selection of strains with enhanced Xanthan production

International Nuclear Information System (INIS)

Kamal, F.; Mehrgan, H.; Mazaheri, M.; Mortazavi, A. R.

2003-01-01

Xanthan gum is microbial polysaccharide of great commercial importance as it has been unusual rheological properties in solution and consequent range of applications. In this study, a series of mutants were isolated from Xanthomonas PTSS 1473 by ethyl methanesulfonate mutagenesis. The polysaccharide yield of one mutant, XC1473E 2 , was 30% better than that of the parent strain. It also showed higher xanthan formation of glucose consumption rates compared to the parent strain. xanthan produced by the mutant and enhanced viscosity, higher pseudo plasticity and larger molecular weight. Since mutant XC1473E 2 appeared white on agar plates, it underwent pigment extraction with methanol. Contrary to the parent strain, the mutant showed no absorption at 443 nm, i.e. the wavelength related to yellow pigment. This finding suggested that yellow pigmentation and normal xanthan biosynthesis are not necessarily concurrent. In general, mutant ZC1473e 2 seems to be a strain with interesting characteristics for use in commercial production of Xanthan

Genome-scale metabolic models as platforms for strain design and biological discovery.

Science.gov (United States)

Mienda, Bashir Sajo

2017-07-01

Genome-scale metabolic models (GEMs) have been developed and used in guiding systems' metabolic engineering strategies for strain design and development. This strategy has been used in fermentative production of bio-based industrial chemicals and fuels from alternative carbon sources. However, computer-aided hypotheses building using established algorithms and software platforms for biological discovery can be integrated into the pipeline for strain design strategy to create superior strains of microorganisms for targeted biosynthetic goals. Here, I described an integrated workflow strategy using GEMs for strain design and biological discovery. Specific case studies of strain design and biological discovery using Escherichia coli genome-scale model are presented and discussed. The integrated workflow presented herein, when applied carefully would help guide future design strategies for high-performance microbial strains that have existing and forthcoming genome-scale metabolic models.

Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

Directory of Open Access Journals (Sweden)

Komatsu Haruki

2012-01-01

Full Text Available Abstract Background Hepatitis B virus (HBV can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145 is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted. Methods The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years; group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years. To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products. Results By mutant-specific real-time PCR, one subject (5.6% was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6% of the group 1 subjects and 10 (9.3% of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects of 12 subjects who had overlapped peaks at nt 587 in the electropherogram. Conclusions The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in

Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Morohoshi, F.; Munakata, N.

1985-03-01

Three mutant strains exhibiting hyper-sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, but not to methyl methanesulfonate, were selected by a replica method from mutagenized spores of Bacillus subtilis. All three were totally deficient in the adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine with regard to both lethality and mutagenesis. The activity to destroy O/sup 6/-methylguanine residues in the methylated DNA was not elevated in the mutant cells by the pretreatment with sublethal concentrations of N-methyl-N-nitro-N-nitrosoguanidine. This deficiency corresponded to the persistance of O/sup 6/-methylguanine residues in the DNA of both control and pretreated mutant cells challenged with the drug. The lethal and mutagenic sensitivity of the mutant strains were observed only for methyl- or ethyl-nitroso compounds that are thought to be active as inducers and are also active in O-alkylation. Except for the insensitivity to methyl methanesulfonate, the phenotypes of these mutants look very similar to those of ada mutants isolated previously in Escherichia coli.

Mutation induction of pleurotus ferulae by low-energy N+ ion implantation and characters of the selected mutant

International Nuclear Information System (INIS)

Chen Henglei; Wan Honggui; Zhang Jun; Zeng Xianxian

2008-01-01

In order to obtain Pleurotus ferulae with high temperature tolerance, mycelium mono-cells of wild type strain ACK was treated by nitrogen ion (5-30 keV, 1.5x10 15 -1.5x10 16 cm -2 ) implantation, and mutant CGMCC1762 was selected through auxotrophy screening method, which was Lys, VB6 auxotrophy stress with high temperature. We found that during riper period the surface layer mycelium of the mutant was not aging neither grew tegument even above 30 degree C. The mycelium endurable temperature of the mutant was increased 7 degree C compared with that of the wild type strain. The fruiting bodies growth temperature of the mutant was 16-20 degree C in daytime and was 6-12 degree C at night. The highest growth temperature of fruiting bodies of the mutant was increased by 5 degree C than that of original strain. Through three generation investigation, we found that the mutant CGMCC1762 was stable with high temperature tolerance. (authors)

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

OpenAIRE

Sakai, Yasuyoshi; Sawai, Tohru; Tani, Yoshiki

1987-01-01

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initia...

Fibre qualities of bolls developed under different day and night temperatures in various Pakistani cotton varieties and mutant strains

International Nuclear Information System (INIS)

Bandesha, A.A.; Aslam, M.; Ishaque, W.; Haq, M.A.

2004-01-01

Four commercial cotton varieties NIAB-78, B-557, SLH-41, MNH-93 and four advanced mutants strains N-82, L-21, L-25 and M-626 were used to study the effect of temperature on fibre quality during boll developing stage. The results showed that varieties differed significantly in all fibre quality parameters. There was significant increase in fibre length under medium temperature range while significant increase in fibre strength and highly significant increase in Micronaire values and maturity index under high temperature conditions. The medium temperature range (24.5 to 30.6 C) seemed to be ideal for cotton fibre development. (author)

Stress-tolerant mutants induced by heavy-ion beams

International Nuclear Information System (INIS)

Abe, Tomoko; Yoshida, Shigeo; Bae, Chang-Hyu; Ozaki, Takuo

2000-01-01

Comparative study was made on mutagenesis in tobacco embryo induced by exposure to EMS (ethyl methane-sulfonate) ion beams during the fertilization cycle. Tobacco embryo cells immediately after pollination were exposed to heavy ion beam and the sensitivity to the irradiation was assessed in each developmental stage and compared with the effects of EMS, a chemical mutagen. Morphologically abnormality such as chlorophyll deficiency was used as a marker. A total of 17 salt-tolerant plants were selected from 3447 M 1 seeds. A cell line showed salt resistance. The cell growth and chlorophyll content were each two times higher than that of WT cells in the medium containing 154 mM NaCl. Seven strains of M 3 progeny of 17 salt-tolerant plants, showed strong resistance, but no salt tolerant progeny were obtained from Xanthi or Ne-ion irradiation. This shows that the sensitivity of plant embryo to this irradiation technique may vary among species. When exposed to 14 N ion beam for 24-108 hours after pollination, various morphological mutants appeared at 18% in M 1 progeny and herbicide tolerant and salt tolerant mutants were obtained. A strong Co-tolerant strain was obtained in two of 17 salt-tolerant strains and a total of 46 tolerant strains (0.2%) were obtained from 22,272 grains of M 1 seeds. In these tolerant strains, the absorption of Co was slightly decreased, but those of Mg and Mn were increased. Mutants induced with ion-beam irradiation have potential not only for practical use in the breeding of stress-tolerant plants but also for gene analysis that will surely facilitate the molecular understanding of the tolerance mechanisms. (M.N.)

2-deoxyglucose as a selective agent for derepressed mutants of Pichia stipitis

Science.gov (United States)

Hassan K. Sreenath; Thomas W. Jeffries

1998-01-01

The glucose analog 2-deoxyglucose (2-DOG) has been used to obtain mutants derepressed for pentose metabolism. Some researchers have used 2-DOG alone whereas others have used it in the presence of a glucoserepressible carbon source. We examined both methods and screened mutant strains for improved use of xylose in the presence of glucose. Pichia stipitis mutants...

Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant.

Science.gov (United States)

Ando, Akira; Nakamura, Toshihide

2016-10-01

γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

International Nuclear Information System (INIS)

Wang Hua; Zhang Jian; Wang Kai; Liu Bing-Bing; Zou Bo; Zou Guang-Tian; Yang Fan; Shen Si-Le

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively. (general)

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

Science.gov (United States)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

Fatigue and rutting strain analysis of flexible pavements designed ...

African Journals Online (AJOL)

ELO

The study was carried out with the layered elastic analysis software EVERSTRESS. Key words: Layered elastic analysis, fatigue, rutting, strains, design, flexible pavement, CBR. ..... Numerical Computational Stresses and Strains in Multi-Layer ...

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

Science.gov (United States)

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

OpenAIRE

Hillyard, D R; Edlund, M; Hughes, K T; Marsh, M; Higgins, N P

1990-01-01

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient...

Isolation and partial characterization of carotenoid underproducing and overproducing mutants from an extremely thermophilic Thermus thermophilus HB27

International Nuclear Information System (INIS)

Hoshino, T.; Yoshino, Y.; Guevarra, E.D.; Ishida, S.; Hiruta, T.; Fujii, R.; Nakahara, T.

1994-01-01

Twenty-two carotenoid underproducing and thirteen overproducing mutants were obtained from Thermus thermophilus HB27. The strain HB27 was found to produce at least seven colored carotenoids, believed to be identical to those produced by Thermus aquaticus YT1. Based on the results of the genetic analyses performed on twelve carotenoid underproducing mutants, they were classified into three groups; groups 1, 2 and 3. No colored carotenoid was extracted from the cells of mutants belonging to groups 2 and 3, although the accumulation of phytoene, a colorless carotenoid, was observed in group 2 strains. Group 1 was subdivided into groups 1-a and 1-b, where 1-a stains produced neither colored carotenoids nor phytoene and 1-b strains produced two polar colored carotenoids. All of the overproducing mutants produced about twelve times as much seven colored carotenoid mixtures as the parental strain. The mutation loci among all the overproducing mutants were very close to one another, possibly in the same gene. Carotenoid overproducing mutants showed an extensive resistance to UV-irradiation and showed poorer growth at higher temperatures. Carotenoid underproducing mutants were slightly more UV-sensitive but they grew almost normally at higher temperatures. These results suggest that carotenoids are secondary metabolites which are not essential for growth of T. thermophilus

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kistler, M.; Eckardt, F.; Summer, K.-H.

1986-01-01

Glutathione-deficient (gsh - ) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh - :GSH + phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh - mutants belong to one complementation group. (Auth.)

Study on breeding of daptomycin-producing strains by nitrogen ion implantation

International Nuclear Information System (INIS)

Zhou Jian; Liu Ying; Fang Dongsheng; Jiang Hong; Zhang Yin; Gao Wuyan

2008-01-01

Streptomyces roseosporus C20, the bacteria used in production of daptomycin, were implanted with (15-200)x10 13 /cm 2 of 20keV N + ions. Survival rate of the bacteria at different absorbed doses was investigated, and mutagenic effects of the microbe were studied. After breeding under the selection pressure of resistance to streptomycin (the lethal concentration is 1.2μg/mL), several mutant strains with higher yields of daptomycin have been obtained. One of mutant strains, N3-36, can increase up to 126% compared to the original strain. It also shows that the mutant strains have high genetic stability. (authors)

Comparative Study of the Mutant Prevention Concentrations of Moxifloxacin, Levofloxacin, and Gemifloxacin against Pneumococci▿ †

Science.gov (United States)

Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A.; Appelbaum, Peter C.

2010-01-01

We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P < 0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 μg/ml) within the susceptible range. PMID:20008781

Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae

Directory of Open Access Journals (Sweden)

Olga Gavrilova

2017-03-01

Full Text Available Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported.

Genetic studies with morphological mutants of Aspergillus niger

International Nuclear Information System (INIS)

Roy, Ponty; Das, Arati

1979-01-01

Three classes of coloured mutations, viz., fawn, yellow and green, occurred recurrently among the population following UV- and γ-radiation from Co 60 of a wild Aspergillus niger strain 350. Ten mutants were picked up and complementation tests were performed by growing them in pairwise combinations. In two cases, allelic mutants of the same colour were observed. All these mutants were again grown in pairwise crosses with a brown A. niger mutant of different lineage. A poor heterokaryotic growth was, however, observed in one combination which later produced a diploid heterozygous nucleus. It segregated spontaneously to develop a large variety of colonies ranging from haploidy to diploidy including aneuploids. These have been analysed genetically and the possible explanations have been given. (auth.)

Investigation of isochronous stress-strain formulations for elevated temperature structural design

International Nuclear Information System (INIS)

Koo, Gyeong Hoi; Kim, Jong Bum

2012-01-01

For elevated temperature design evaluations by the ASME-NH rules, the most important material data is the isochronous stress-strain curves, which can provide design creep information. The main purpose of this paper is to investigate appropriate formulations to be able to generate the isochronous stress-strain curves and implement it to the computer program which is coded the ASME-NH design evaluation procedures. To do this, formulations by the strain-time relationship are investigated in detail and the sensitivity studies for rapid initial transient creep contributions, slower and longer transient creep contribution, and secondary creep contributions are carried out for type 316 austenitic stainless steel. From the results of this study, it is found that the strain-time relationship formulations can well describe the isochronous stress-strain curves with the transient creep contributions

UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in a tolC Mutant Escherichia coli Strain Leads to Cell Death

OpenAIRE

Humnabadkar, Vaishali; Prabhakar, K. R.; Narayan, Ashwini; Sharma, Sreevalli; Guptha, Supreeth; Manjrekar, Praveena; Chinnapattu, Murugan; Ramachandran, Vasanthi; Hameed, Shahul P.; Ravishankar, Sudha; Chatterji, Monalisa

2014-01-01

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tol...

Isolation and partial characterization of a mutant of Bacillus thuringiensis producing melanin Isolamento e caracterização parcial de um mutante de Bacillus thuringiensis produtor de melanina

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Gislayne T. Vilas-Bôas

2005-09-01

Full Text Available A mutant (407-P of Bacillus thuringiensis subsp. thuringiensis strain 407 producing a melanin was obtained after treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological and biochemical properties of the two strains were analyzed and the results were similar. The mutant 407-P was also incorporated into non-sterilized soil samples, recovered, easily identified, and quantified, what enables its use in ecology of B. thuringiensis.Um mutante (407-P da linhagem Bacillus thuringiensis subsp. thuringiensis 407 produtor de melanina foi obtido após tratamento com o agente mutagênico etil-metano-sulfonato. Diversas propriedades microbiológicas e bioquímicas das duas linhagens foram analisadas e os resultados foram similares. O mutante 407-P foi incorporado em amostras de solo não esterilizado, recuperado, facilmente identificado e quantificado, possibilitando seu uso em estudos de ecologia de B. thuringiensis.

Photoproduct formation and repair capacity in a mutant of Bacillus cereus 569 producing UV-sensitive spores

International Nuclear Information System (INIS)

Weinberger, S.; Evenchik, Z.; Hertman, I.; Bar-Ilan Univ., Ramat-Gan

1982-01-01

A mutant of Bacillus cereus 569 UV sensitive in both vegetative and sporal stages was isolated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis followed by selection on mitomycin C. The UV-sensitive mutant designated as B. cereus 2422 exhibited normal content of dipicolinic acid (DPA) and resistance to X-rays and ethyl methanesulphonate. The photoproduct type and amount, induced by a given UV dose, was similar in either cells or spores of both the mutant 2422 and the wild-type ancestor. The mutant 2422 excised cyclobutane thymine dimers only to a limited extent (20%) as compared with 80% removal in the wild type. Removal of a spore-specific photoproduct (TDHT) during germination proceeded to a similar extent in B. cereus 2422 and the wild-type parent. However, under growing conditions, an additional removal of the TDHT was observed only in the wild-type strain. Liquid holding recovery occurred in irradiated wild-type cells, but not in mutant cells. Spontaneous revertants were isolated that regained UV resistance simultaneously in both the vegetative and sporal stage. (orig./AJ)

A wireless sensor network design and evaluation for large structural strain field monitoring

International Nuclear Information System (INIS)

Qiu, Zixue; Wu, Jian; Yuan, Shenfang

2011-01-01

Structural strain changes under external environmental or mechanical loads are the main monitoring parameters in structural health monitoring or mechanical property tests. This paper presents a wireless sensor network designed for monitoring large structural strain field variation. First of all, a precision strain sensor node is designed for multi-channel strain gauge signal conditioning and wireless monitoring. In order to establish a synchronous strain data acquisition network, the cluster-star network synchronization method is designed in detail. To verify the functionality of the designed wireless network for strain field monitoring capability, a multi-point network evaluation system is developed for an experimental aluminum plate structure for load variation monitoring. Based on the precision wireless strain nodes, the wireless data acquisition network is deployed to synchronously gather, process and transmit strain gauge signals and monitor results under concentrated loads. This paper shows the efficiency of the wireless sensor network for large structural strain field monitoring

The mutagenesis and breeding of high productive strains of streptomyces jingyangensis '5406'

International Nuclear Information System (INIS)

Qi Hongyan; Yin Xinyun

1988-03-01

The purpose of these experiments is to explore the mutagenesis rhythm and breed high productive strains of actinomycete '5406'. The single colony agar pieces of strain F 358 were treated with fast neutron and 60 Co-γ ray irradiation Two mutants have been selected from 20025 treated single colonies. The output of cytokinins from them is higher than from strain F 358 . The original strain 'Mu-Tan-al' rejuvenated by freezing was treated with several physical and chemical mutagens. The mutagenesis rhythm has been summed up tentatively. Eight mutants obtained from 93014 treated single colonies produced more '5406' antibiotics than that of strain 'Mu-Tan-al,. The effect of mutant 'N2-10-Ra3' was the best

Development mutants of anabaena doliolum defective in repair of UV-damage

International Nuclear Information System (INIS)

Tiwari, D.N.; Singh, C.B.

1980-01-01

Nitrosoguanidine induced 'blue' pigment mutants of the blue-green alga anabaena doliolum were isolated. The blue-mutants on further characterization were grouped into three developmental phenotypes - (i) those forming doli-form blue-spores of heterogenous size i.e., Ad 011, (ii) those forming spheroidal cells in the stationary phase, some of which behave like spores on transfer to fresh medium i.e., Ad 012, and (iii) those showing no sporulation and conditionally producing abnormal cells in the presence of combined nitrogen only i.e., Ad 007. The former two classes of mutants showed the formation of abnormal cells irrespective of the presence or absence of combined nitrogen sources in the medium. The formation of abnormal cells in the filaments of the above mutants were distinguished by their larger size and irregular mode of division leading to true-branch formation. The comparative characterization of these mutant strains with the parental one showed sluggish growth, increased UV-sensitivity, almost unchanged photorepair capacity, a marked change in the pigment composition and relative resistance to nitrosoguanidine. Irregular cell division in both space and time in the mutant strains and their increased sensitivity to ultraviolet irradiation indicate the possible involvement of dark repair system in maintaining the precision of cell cylce in this alga. (orig.) 891 AJ/orig. 892 HIS

Stress-tolerant mutants induced by heavy-ion beams

Energy Technology Data Exchange (ETDEWEB)

Abe, Tomoko; Yoshida, Shigeo [Institute of Physical and Chemical Research, Wako, Saitama (Japan); Bae, Chang-Hyu [Sunchon National University, Sunchon (Korea); Ozaki, Takuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment; Wang, Jing Ming [Akita Prefectural Univ. (Japan)

2000-07-01

Comparative study was made on mutagenesis in tobacco embryo induced by exposure to EMS (ethyl methane-sulfonate) ion beams during the fertilization cycle. Tobacco embryo cells immediately after pollination were exposed to heavy ion beam and the sensitivity to the irradiation was assessed in each developmental stage and compared with the effects of EMS, a chemical mutagen. Morphologically abnormality such as chlorophyll deficiency was used as a marker. A total of 17 salt-tolerant plants were selected from 3447 M{sub 1} seeds. A cell line showed salt resistance. The cell growth and chlorophyll content were each two times higher than that of WT cells in the medium containing 154 mM NaCl. Seven strains of M{sub 3} progeny of 17 salt-tolerant plants, showed strong resistance, but no salt tolerant progeny were obtained from Xanthi or Ne-ion irradiation. This shows that the sensitivity of plant embryo to this irradiation technique may vary among species. When exposed to {sup 14}N ion beam for 24-108 hours after pollination, various morphological mutants appeared at 18% in M{sub 1} progeny and herbicide tolerant and salt tolerant mutants were obtained. A strong Co-tolerant strain was obtained in two of 17 salt-tolerant strains and a total of 46 tolerant strains (0.2%) were obtained from 22,272 grains of M{sub 1} seeds. In these tolerant strains, the absorption of Co was slightly decreased, but those of Mg and Mn were increased. Mutants induced with ion-beam irradiation have potential not only for practical use in the breeding of stress-tolerant plants but also for gene analysis that will surely facilitate the molecular understanding of the tolerance mechanisms. (M.N.)

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

Science.gov (United States)

Brokaw, C J; Luck, D J

1985-01-01

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways

International Nuclear Information System (INIS)

Moseley, B.E.B.; Evans, D.M.

1983-01-01

A mutant of Deinococcus (formerly Micrococcus) radiodurans sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers. The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively. When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant. On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles. Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302. (author)

Strain and culture medium optimization for production enhancement of prodiginines from marine-derived Streptomyces sp. GQQ-10

Science.gov (United States)

Li, Xueping; Zhang, Guojian; Zhu, Tianjiao; Li, Dehai; Gu, Qianqun

2012-09-01

A mutant (GQQ-M6) of a Sponge-Derived streptomyces sp. GQQ-10 obtained by UV-induced mutation was used for producing prodiginines (PGs). Single factor experiments and orthogonal array design (OAD) methods were employed for medium optimization. In the single factor method, the effects of soluble starch, glucose, soybean flour, yeast extract and sodium acetate on PGs production were investigated individually. In the subsequent OAD experiments, the concentrations of these 5 key nutritional components combined with salinity were further adjusted. The mutant strain GQQ-M6 gave a 2.2-fold higher PGs production than that of the parent strain; OAD experiments offered a PGs yield of 61mg L-1, which was 10 times higher than that of the initial GQQ-10 strain under the original cultivation mode.

Selection of the Mutants with High Hydroquinone Degradation Ability of Serratia Marcesscen by Plasma Mutation

International Nuclear Information System (INIS)

Yao Risheng; You Qidong; He Weijing; Zhu Huixia

2009-01-01

In this study, an efficient way by plasma induced mutation was applied to improve the hydroquinone degradation capacity of Serratia marcescens AB 90027 (SM27). The results showed that combined with the selection of hydroquinone tolerance, the mutant with high hydroquinone degradation ability induced by plasma could be achieved. The best dose for plasma mutation was 15 s, which showed a 47.0% higher positive mutation ratio. Besides, the aimed mutant was markedly different from the parent strain (SM27) in colonial traits while cultivated on Kings media. Finally, the hydroquinone degradation ratio reached 70.5% using the induced mutant strain with 1500 mg/L hydroquinone (HQ) after 15 days of cultivation as the selective conditions; however, it was only 46.7% for SM27. The improvement of the degradation capacity by the induced mutant with a high concentration of HQ selection was attributed to its faster growth and higher hydroquinone tolerance compared with that of the parent strain.

High yielding and early maturing mutants in mungbean (Vigna radiata (L) Wilczek)

International Nuclear Information System (INIS)

Malik, I.A.

1988-01-01

Mungbean in Pakistan is grown on about 79 thousand hectares with an annual production of around 39600 t. The poor yield of cultivars may be largely due to their indeterminate excessive vegetative growth, low harvest index, and susceptibility to various diseases. Lack of synchrony in maturity and pod shattering are also limiting factors. Mutation breeding of mungbean at NIAB has the object of evolving early and uniform maturing high yielding mutants. Seeds of mungbean strains Pak-22 and RC71-27 were irradiated with 60 Co gamma rays (5 kR to 80 kR) in 1977. After selecting mutants in the M 2 , further selections were made in M 3 for earliness, uniform maturity, short plant stature and larger number of pods/plant. In the M 4 , 62 selections were subjected to micro plot yield trials and seed protein analysis. Selection was continued in the advanced generations and performance was studied in multilocational trials arranged through the Department of Agriculture. The important characteristics of two mutants namely NM19-19 (derivative of strain Pak 22 at 40 kR) and NM121-25 (derivative of strain RC71-27 at 20 kR) are listed and their field performance is summarized. Both the mutants are short statured and have erect determinate growth habit. They mature early by a margin of 16 days and yield higher. The high harvest index of the mutants indicates their efficiency in partitioning photosynthates towards grain formation. Because of their synchrony in maturity and top fruit bearing habit the mutants are amenable to mechanized harvesting. The early maturity in mutants also makes them more suitable for intercropping practices. The mutants possess greater degree of tolerance to yellow mosaic disease and have shown wide adaptability and stability when grown under different agroclimatic conditions. Both the mutants have been released in 1986, by the Punjab Seed Council as commercial varieties under the names of 'NIAB Mung 121-25' and 'NIAB Mung 19-19' respectively

Mutation Induction In White Oyster Mushroom (Pleurotus Ostreatus) Using GAMMA Irradiation And Analyzing Genetic Diversity Of Induced Mutants By RAPD.PCR

International Nuclear Information System (INIS)

Mohammed, H.M; Soliman, S.S.A; Shawky, A.S.H; Mahgoub, E.M.I

2013-01-01

The present study aimed to understand the effect of gamma rays on three strains of white oyster mushroom Pleurotus ostreatus and induction of new benefit mutants. Five different doses, i.e., 0.25,0.5,0.75, and 1.00 KGy of gamma rays were used. Twelve morphological criteria were measured. The result confirmed the existence of different response of three strains to different doses of radiation. The 21 strain as a control, the first flush was misshapen, but the second flush gave normal fruit, while mutant Po 21-3 gave excellent growth performance for the shape, colour, fruit diameter and stem length, while slightly decrease in wet and dry total matter per bag and flushes numbers were found.The po 21-2 mutant considered as important mutant because slightly increased in wet and dry total matter than the control, and had short stem length. This mutant Po 21 -2 gave spores, while the control was sporeless. Control of strain 22 possessed good performance for fruit characterization but it forms fruits less than its primordial it formed (semi maturation less strain) per bag, While po 22-l and Po 22-2 appeared good performance, in addition high yielding as wet and dry total matter and faster flushing. The control of strain 66 had a good growth performance, and the four mutants for this strain is very good too they had excellent growth performance; Po 66-1 and Po 66-3 mutants had significant and highly significant values for wet matter than the control (21.04, 21.64 and 12.27) for (Po 66-1, Po 66-4, control). As well as more important criteria there were taken as short fruit stem length and fruit number/bag. These results confirmed the important of Po 66-4 followed by po 66-1 in next breeding and production programs for white oyster mushroom (Pleurotus ostreatus).the RAPD PCR results showed the oligonucleotide OPB-10, OPA-05 and OPC-02 presented the highest percentage of RAPD polymorphism (80⁒, 80⁒, 66.6⁒). The pattern obtained with OPB-10 oligonucleotide for strains

Quantitative measures of mutagenicity and multability based on mutant yield data

International Nuclear Information System (INIS)

Eckhardt, F.; Haynes, R.H.

1980-01-01

We describe, how mutant yield data (mutants per cell treated) can be used both to compare the mutagenenicity of different mutagens, and to characterize the mutability of different cell types. Yield curves reveal the net effect of the lethal and genetic actions of mutagens on cells. Normally, yields are the quantities measured in assays for mutagenesis, and rectilinear plots of such data baldly reveal the amount of experimental error and the extent of actual mutant induction above the background level. Plots of yield versus lethal hits can be used to quantify the relative mutagenenic efficiency (RME) of agents whose physical exposure doses otherwise would be incommensurable, as well as the relative mutability (Rmt) of different strains to the same mutagen. Plots of yield versus log dose provide an unambiguous way of assessing the relative mutational sensitivities (Rms) and mutational resolutions (Rmr) of different strains against a given mutagen. Such analysis is important for evaluation of the relative merits of excision-proficient and excision-deficient strains of the same organism as mutagen-testing systems. The mathematical approach outlined here is applied, by way of example, to measurements of UV and 4-NQO induced mutagenesis in both repair-deficient and repair-proficient haploid strains of the yeast Sacccharomyces cerevsiae. (orig.)

Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

International Nuclear Information System (INIS)

Kalayanamitr, A.; Bhumiratana, A.; Flegel, T.W.; Glinsukon, T.; Shinmyo, A.

1987-01-01

A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production

Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis

International Nuclear Information System (INIS)

Hahn, J.; Albano, M.; Dubnau, D.

1987-01-01

The authors isolated 28 mutants of Bacillus subtilis deficient in the development of competence by using the transposon Tn917lacZ as a mutagen. The mutant strains were poorly transformable with plasmid and chromosomal DNAs but were normally transducible and exhibited wild-type resistance to DNA-damaging agents. The mutations were genetically mapped, and the mutants were characterized with respect to their abilities to bind and take up radiolabeled DNA. All were defective in uptake, and some failed to bind significantly amounts of DNA. The abilities of the mutant strains to resolve into two buoyant density classes on Renografin gradients were studied. Most resolved normally, but several banded in Renografin only at the buoyant density of noncompetent cells. The genetic mapping studies and the other analyses suggested that the mutations define a minimum of seven distinct com genes

Synthesis and biological properties of novel 2-aminopyrimidin-4(3H)-ones highly potent against HIV-1 mutant strains.

Science.gov (United States)

Mai, Antonello; Artico, Marino; Rotili, Dante; Tarantino, Domenico; Clotet-Codina, Imma; Armand-Ugón, Mercedes; Ragno, Rino; Simeoni, Silvia; Sbardella, Gianluca; Nawrozkij, Maxim B; Samuele, Alberta; Maga, Giovanni; Esté, José A

2007-11-01

Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs. The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the higher inhibitory effect exerted by the new molecules against the mutated RTs.

[Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

Science.gov (United States)

Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I

2015-01-01

Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

Phenotypic and genomic comparisons of highly vancomycin-resistant Staphylococcus aureus strains developed from multiple clinical MRSA strains by in vitro mutagenesis.

Science.gov (United States)

Ishii, Kenichi; Tabuchi, Fumiaki; Matsuo, Miki; Tatsuno, Keita; Sato, Tomoaki; Okazaki, Mitsuhiro; Hamamoto, Hiroshi; Matsumoto, Yasuhiko; Kaito, Chikara; Aoyagi, Tetsuji; Hiramatsu, Keiichi; Kaku, Mitsuo; Moriya, Kyoji; Sekimizu, Kazuhisa

2015-11-25

The development of vancomycin (VCM) resistance in Staphylococcus aureus threatens global health. Studies of the VCM-resistance mechanism and alternative therapeutic strategies are urgently needed. We mutagenized S. aureus laboratory strains and methicillin-resistant S. aureus (MRSA) with ethyl methanesulfonate, and isolated mutants that exhibited high resistance to VCM (minimum inhibitory concentration = 32 μg/ml). These VCM-resistant strains were sensitive to linezolid and rifampicin, and partly to arbekacin and daptomycin. Beta-lactams had synergistic effects with VCM against these mutants. VCM-resistant strains exhibited a 2-fold increase in the cell wall thickness. Several genes were commonly mutated among the highly VCM-resistant mutants. These findings suggest that MRSA has a potential to develop high VCM resistance with cell wall thickening by the accumulation of mutations.

Design of Strain-Compensated Epitaxial Layers Using an Electrical Circuit Model

Science.gov (United States)

Kujofsa, Tedi; Ayers, John E.

2017-12-01

The design of heterostructures that exhibit desired strain characteristics is critical for the realization of semiconductor devices with improved performance and reliability. The control of strain and dislocation dynamics requires an understanding of the relaxation processes associated with mismatched epitaxy, and the starting point for this analysis is the equilibrium strain profile, because the difference between the actual strain and the equilibrium value determines the driving force for dislocation glide and relaxation. Previously, we developed an electrical circuit model approach for the equilibrium analysis of semiconductor heterostructures, in which an epitaxial layer may be represented by a stack of subcircuits, each of which involves an independent current source, a resistor, an independent voltage source, and an ideal diode. In this work, we have applied the electrical circuit model to study the strain compensation mechanism and show that, for a given compositionally uniform device layer with fixed mismatch and layer thickness, a buffer layer may be designed (in terms of thickness and mismatch) to tailor the strain in the device layer. A special case is that in which the device layer will exhibit zero residual strain in equilibrium (complete strain compensation). In addition, the application of the electrical circuit analogy enables the determination of exact expressions for the residual strain characteristics of both the buffer and device layers in the general case where the device layer may exhibit partial strain compensation. On the basis of this framework, it is possible to develop design equations for the tailoring of the strain in a device layer grown on a uniform composition buffer.

Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

Directory of Open Access Journals (Sweden)

Antje Blumenthal

2010-12-01

Full Text Available Mycobacterium tuberculosis (Mtb represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL, the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen

Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne

1989-01-01

A DNA fragment encoding kanamycin resistance was inserted in vitro into a plasmid-borne prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. The resulting plasmids were subsequently transferred to the chromosome by homologous recombination and the haploid strains prs-3::Kan......R and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show...

Comparison of expression of key sporulation, solventogenic and acetogenic genes in C. beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A.

Science.gov (United States)

Kolek, J; Diallo, M; Vasylkivska, M; Branska, B; Sedlar, K; López-Contreras, A M; Patakova, P

2017-11-01

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

Studies on Drosophila radiosensitivity strains

International Nuclear Information System (INIS)

Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.U.

1985-01-01

Fertility of radiosensitive mutant drosophila female strain rad (2) 201 61 after irradiation and frequency of dominant lethal mutations (DLM), induced by γ-radiation for 0-5 h and 5-7 days, are investigated. It is shown, that oocytes of the mutant strain are more radiosensitive as compared with cells of mongrel flies as to criterion of DLM appearance over the period of maturing. Early oocytes of stages 2-7 are the most sensitive, i.e. at the stages, corresponding to the manifestation of previously established recombination-defective properties of mutations rad (2) 201 61 . It is also sown, that doses of γ-rays, exceeding 10 Gy produce a strong sterilizing effect on mutant females due to destruction and resorption of egg chambers, irradiated at the stages of previtellogenetic growth of oocytes. In females, carrying mutation of radiosensitivity there is no direct correlation betwen sensitivity of oocytes proper to DLM induction and sensitivity of egg folleicles to resorbing effect of γ-rays. The ways of possible involvement of mutant locus studied into genetic processes in various specialized cells of drosophila

Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

Science.gov (United States)

Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

2016-01-01

The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid : Growth profiles and catalase activities in relation to microbody proliferation

NARCIS (Netherlands)

Klei, Ida J. van der; Rytka, Joanna; Kunau, Wolf H.; Veenhuis, Marten

The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T-), catalase A (A-T+) or both catalases (A-T-), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two

Trehalose, glycogen and ethanol metabolism in the gcr1 mutant of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Seker, Tamay; Hamamci, H.

2003-01-01

Since Gcr1p is pivotal in controlling the transcription of glycolytic enzymes and trehalose metabolism seems to be one of the control points of glycolysis, we examined trehalose and glycogen synthesis in response to 2 % glucose pulse during batch growth in gcr1 (glucose regulation-1) mutant lacking...... fully functional glycolytic pathway and in the wild-type strain. An increase in both trehalose and glycogen stores was observed 1 and 2 h after the pulse followed by a steady decrease in both the wild-type and the gcr1 mutant. The accumulation was faster while the following degradation was slower in gcr......1 cells compared to wild-type ones. Although there was no distinct glucose consumption in the mutant cells it seemed that the glucose repression mechanism is similar in gcr1 mutant and in wild-type strain at least with respect to trehalose and glycogen metabolism....

Enhancement of Echinocandin B Production by a UV- and Microwave-Induced Mutant of Aspergillus nidulans with Precursor- and Biotin-Supplying Strategy.

Science.gov (United States)

Hu, Zhong-Ce; Peng, Li-Yuan; Zheng, Yu-Guo

2016-08-01

Echinocandin B belongs to lipopeptide antifungal antibiotic bearing five types of direct precursor amino acids including proline, ornithine, tyrosine, threonine, and leucine. The objective of this study is to screen over-producing mutant in order to improve echinocandin B production; a stable mutant Aspergillus nidulans ZJB12073, which can use fructose as optimal carbon source instead of expensive mannitol, was selected from thousand isolates after several cycles of UV and microwave irradiation in turn. The results showed that mutant strain ZJB12073 exhibited 1.9-fold improvement in echinocandin B production to 1656.3 ± 40.3 mg/L when compared with the parent strain. Furthermore, the effects of precursor amino acids and some chemicals on echinocandin B biosynthesis in A. nidulans were investigated, respectively. Tyrosine, leucine, and biotin were selected as key factors to optimize the medium employing uniform design method. The results showed that the optimized fermentation medium provided another 63.1 % increase to 2701.6 ± 31.7 mg/L in final echinocandin B concentration compared to that of unoptimized medium.

Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants

International Nuclear Information System (INIS)

Lawrence, C.W.; Christensen, R.B.

1979-01-01

Dominant mutations at two newly identified loci, designated SRS1 and SRS2, that metabolically suppress the trimethoprim sensitivity of rad6 and rad18 strains, have been isolated from trimethorprim-resistant mutants arising spontaneously in rad6-1 rad18-2 strains of the yeast Saccharomyces cerevisiae. The SRS2 mutations also efficiently suppress the ultraviolet light sensitivity of the parent strains. They do not, however, suppress their sensitivity to ionizing radiation or their deficiency with respect to induced mutagenesis and sporulation. Such observations support the hypothesis that RAD6-dependent activities can be separated into two functionally distinct groups: a group of error-free repair activities that are responsible for a large amount of the radiation resistance of wild-type strains and also for their resistance to trimethoprim, and a group of error-prone activities that are responsible for induced mutagenesis and are also important in sporulation, but which account at best for only a very small amount of wild-type recovery

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

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Deepali Bisht

2013-01-01

Full Text Available Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100. The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1. The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R² value (0.9987. The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

Science.gov (United States)

Bisht, Deepali; Yadav, Santosh Kumar; Darmwal, Nandan Singh

2013-01-01

Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization. PMID:24159311

Protein nutrient value of Agaricus bazei murrill mutant J3 induced by 60Co γ-irradiation in different generations

International Nuclear Information System (INIS)

Jiang Zhihe; Lin Yong; Xiao Shuxia

2004-01-01

Protein nutritional values of Agaricus bazei Murrill mutant J 3 and original strain J 1 were compared by non-biological evaluation methods. The results showed that five protein indexes in the fruitbodies of M 1 and M 6 generations of Agaricus bazei Murrill mutant J 3 were higher than that of original strain J 1 ; four protein indexes in M 4 and M 5 generations were higher than that of original strain J 1 ; and six protein indexes in M 2 and M 3 generations were higher than that of original strain J 1 . It was concluded that the protein nutritional values of Agaricus bazei Murrill mutant J 3 was better than that of original strain J 1 . Except the ratio scores of amino acids had little change in the different generations, the other indexes in strain J 3 Agaricus bazei Murrill showed that the heredity efficiencies of the proteins was rather stable. (authors)

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Science.gov (United States)

Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki; Takai, Yuki; Okumura, Yoshiko; Baba, Miki; Datsenko, Kirill A; Tomita, Masaru; Wanner, Barry L; Mori, Hirotada

2006-01-01

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

DEFF Research Database (Denmark)

Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Jers, Carsten

2017-01-01

were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate......, that computational studies of trans-glycosylation can be a valuable input in the design of novel trans-glycosidases, but also highlight the importance of experimental validation in order to assess the performance. In conclusion, two of the seven mutants displayed a dramatic switch in specificity from hydrolysis...

Design and Construction of Strain Gauge Interface Pressure ...

African Journals Online (AJOL)

Design and Construction of Strain Gauge Interface Pressure Transducer for Measurement of Static and Dynamic Interface Pressure Applied by Pressure Garments and its Relationship to Deep Vein Thrombosis.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

Science.gov (United States)

Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

2016-01-01

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

A New Strain Collection for Improved Expression of Outer Membrane Proteins

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Ina Meuskens

2017-11-01

Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

Use of X-ray and gamma-induced mutants of lactic acid bacteria in the manufacture of dairy products

Energy Technology Data Exchange (ETDEWEB)

Dilanian, Z; Makarian, K; Chuprina, D [Erevan Zootechnical and Veterinary Inst. (USSR). Chair of Dairying

1976-04-01

With the aid of X-ray and gamma irradiation were got mutants of lactic acid bacteria, which steadily retain acquired properties. Use of proteolytically active mutant strains in the production of armianski and sovetski cheeses shortened the time of their ripening and increased their quality. Gamma-mutant strain L. lactis 1621/I-M with high phenolstability was received and antibiotic activity with respect to some representatives of pathogenic microflora of the bowels. Use of this mutant in starters for sour milk products will raise their therapeutic effect against intestinal diseases. Deep morphological changes are taking place in lactic acid bacteria under the influence of ionizing radiation.

Use of X-ray and gamma-induced mutants of lactic acid bacteria in the manufacture of dairy products

International Nuclear Information System (INIS)

Dilanian, Z.; Makarian, K.; Chuprina, D.

1976-01-01

With the aid of X-ray and gamma irradiation were got mutants of lactic acid bacteria, which steadily retain acquired properties. Use of proteolytically active mutant strains in the production of armianski and sovetski cheeses shortened the time of their ripening and increased their quality. Gamma-mutant strain L. lactis 1621/I-M with high phenolstability was received and antibiotic activity with respect to some representatives of pathogenic microflora of the bowels. Use of this mutant in starters for sour milk products will raise their therapeutic effect against intestinal diseases. Deep morphological changes are taking place in lactic acid bacteria under the influence of ionizing radiation. (orig.) [de

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

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Eliton da Silva Vasconcelos

2013-12-01

Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

Science.gov (United States)

da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

2013-12-01

Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

Energy Technology Data Exchange (ETDEWEB)

Unrau, P.

1977-01-01

The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

International Nuclear Information System (INIS)

Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E.

1991-01-01

The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus

Design and Validation of a Cyclic Strain Bioreactor to Condition Spatially-Selective Scaffolds in Dual Strain Regimes

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J. Matthew Goodhart

2014-03-01

Full Text Available The objective of this study was to design and validate a unique bioreactor design for applying spatially selective, linear, cyclic strain to degradable and non-degradable polymeric fabric scaffolds. This system uses a novel three-clamp design to apply cyclic strain via a computer controlled linear actuator to a specified zone of a scaffold while isolating the remainder of the scaffold from strain. Image analysis of polyethylene terephthalate (PET woven scaffolds subjected to a 3% mechanical stretch demonstrated that the stretched portion of the scaffold experienced 2.97% ± 0.13% strain (mean ± standard deviation while the unstretched portion experienced 0.02% ± 0.18% strain. NIH-3T3 fibroblast cells were cultured on the PET scaffolds and half of each scaffold was stretched 5% at 0.5 Hz for one hour per day for 14 days in the bioreactor. Cells were checked for viability and proliferation at the end of the 14 day period and levels of glycosaminoglycan (GAG and collagen (hydroxyproline were measured as indicators of extracellular matrix production. Scaffolds in the bioreactor showed a seven-fold increase in cell number over scaffolds cultured statically in tissue culture plastic petri dishes (control. Bioreactor scaffolds showed a lower concentration of GAG deposition per cell as compared to the control scaffolds largely due to the great increase in cell number. A 75% increase in hydroxyproline concentration per cell was seen in the bioreactor stretched scaffolds as compared to the control scaffolds. Surprisingly, little differences were experienced between the stretched and unstretched portions of the scaffolds for this study. This was largely attributed to the conditioned and shared media effect. Results indicate that the bioreactor system is capable of applying spatially-selective, linear, cyclic strain to cells growing on polymeric fabric scaffolds and evaluating the cellular and matrix responses to the applied strains.

Nuclear magnetic resonance probe head design for precision strain control

International Nuclear Information System (INIS)

Kissikov, T.; Sarkar, R.; Bush, B. T.; Lawson, M.; Canfield, P. C.; Curro, N. J.

2017-01-01

Here, we present the design and construction of an NMR probe to investigate single crystals under strain at cryogenic temperatures. The probe head incorporates a piezoelectric-based apparatus from Razorbill Instruments that enables both compressive and tensile strain tuning up to strain values on the order of 0.3% with a precision of 0.001%. 75 As NMR in BaFe 2 As 2 reveals large changes to the electric field gradient and indicates that the strain is homogeneous to within 16% over the volume of the NMR coil.

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

International Nuclear Information System (INIS)

Liu Qingmei; Yuan Hang; Wang Jun; Gong Guohong; Zhou Wei; Fan Yonghong; Wang Li; Yao Jianming; Yu Zengliang

2006-01-01

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope (SEM). The optimum condition of ions implantation, 20 keV of energy and 2.6x10 15 N + /cm 2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum (ESI/MS) and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain

Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

Science.gov (United States)

Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

2016-01-01

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

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Tiffany M Mott

Full Text Available In this study, a Burkholderia mallei tonB mutant (TMM001 deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001.Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

Production of the bioactive compounds violacein and indolmycin is conditional in a maeA mutant of Pseudoalteromonas luteoviolacea S4054 lacking the malic enzyme

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Mariane S. Thøgersen

2016-09-01

Full Text Available It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level or even more pronounced as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wildtype strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wildtype. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s may be play a role in the antibacterial activity of P. luteoviolacea.

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant.

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Cutzu, Raffaela; Coi, Annalisa; Rosso, Fulvia; Bardi, Laura; Ciani, Maurizio; Budroni, Marilena; Zara, Giacomo; Zara, Severino; Mannazzu, Ilaria

2013-06-01

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2) = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l(-1)) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

Unusual Δ7,12,19 C35:3 Alkenone Produced by the Mutant Emiliania huxleyi strain CCMP2758 in Culture

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Zheng, Y.; Huang, Y.; Zhang, Y.; Dillon, J. T.

2015-12-01

Alkenones with chain length ranging from C37 to C40 are highly specific biomarkers for certain haptophyte algae in ocean and lake sediments and have been widely used for paleoclimate studies. Short chain alkenones (e.g., C35 and C36) have been found in environmental and culture samples but the origin and structures of these compounds are not fully understood. The benchmark marine alkenone producer, Emiliania huxleyi CCMP2758 strain (the mutant of strain CCMP1742, NEPCC55a) was reported to make 35:2 alkenone when cultured at 15 °C (Prahl et al., 2006). Here we show, when this strain is cultured at lower temperatures (e.g., 4°C), CCMP2758 produces large amount of 35:3 alkenone with unusual double bond positions of Δ7,12,19. We determined the double bond positions of the C35:3 methyl ketonee based on GC-MS analysis of cyclobutylimine derivatives and dimethyl disulfide derivatives respectively, and provide the first temperature calibrations based on the unsaturation ratios of C35 alkenones. Previous studies have found 35:2 alkenone with three methylene interruption in the Black Sea sediment, but it is the first time that an alkenone with a mixed three and five methylene interruption is found. The discovery of short chain alkenones with unusual double bond positions may shed new light to alkenone biosynthesis.

Low Dose Vaccination with Attenuated Francisella tularensis Strain SchuS4 Mutants Protects against Tularemia Independent of the Route of Vaccination

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Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D.; Child, Robert; Crane, Deborah D.

2012-01-01

Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia. PMID:22662210

Ultraviolet light-induced mutants of Streptococcus lactis subspecies diacetylactis with enhanced acid- or flavor-producing abilities

International Nuclear Information System (INIS)

Kuila, R.K.; Ranganathan, B.

1978-01-01

A strain of Streptococcus lactis subspecies diacetylactis S 1 isolated from fresh milk was exposed to 7200 ergs/mm 2 of ultraviolet radiation. Over 8100 colonies surviving from 7.4 x 10 6 cells exposed to radiation were screened on citrate agar for detection and isolation of mutants with increased flavor and/or acid production. Of the survivors, 960 were type-I mutants that exhibited clear zone on citrate agar after 18 h (presumed to be high diacetyl producers), and 288 were type-II mutants which did not exhibit clear zones on citrate agar for up to 72 h (high acid producers). Type-II mutants produced an average .93 percent titratable acidity which was 34 percent more than the .69 percent of the parent. Reduction in titratable acidity (56 percent less) was considerable in type-I mutants, compared with the parent culture. Diacetyl + acetoin production by type-I mutants was 137.9 ppM which has 4.5 times more than that of the parental strain. Acetaldehyde production in the mutants varied from 1.5 to 34.5 ppM (parent culture 3.0 ppM). The mutants with increased acid and high acetoin plus diacetyl production were stable after 50 subcultures in milk

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion.

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Hatvani, Lóránt; Manczinger, László; Kredics, László; Szekeres, András; Antal, Zsuzsanna; Vágvölgyi, Csaba

2006-01-01

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T. atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 microg/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 microg/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T. atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.

Strain improvement of Gluconacetobacter xylinus NCIM 2526 for ...

African Journals Online (AJOL)

The present investigation demonstrates the effectiveness of ultraviolet (UV) radiation and ethyl methanesulfonate (EMS) in strain improvement for enhanced cellulose production by Gluconacetobacter xylinus NCIM 2526. The mutants were compared with wild type for cellulose production. UV mutants GHUV3, GHUV4, and ...

Crystallization of mutants of Turnip yellow mosaic virus protease/ubiquitin hydrolase designed to prevent protease self-recognition.

Science.gov (United States)

Ayach, Maya; Bressanelli, Stéphane

2015-04-01

Processing of the polyprotein of Turnip yellow mosaic virus is mediated by the protease PRO. PRO cleaves at two places, one of which is at the C-terminus of the PRO domain of another polyprotein molecule. In addition to this processing activity, PRO possesses an ubiquitin hydrolase (DUB) activity. The crystal structure of PRO has previously been reported in its polyprotein-processing mode with the C-terminus of one PRO inserted into the catalytic site of the next PRO, generating PRO polymers in the crystal packing of the trigonal space group. Here, two mutants designed to disrupt specific PRO-PRO interactions were generated, produced and purified. Crystalline plates were obtained by seeding and cross-seeding from initial `sea urchin'-like microcrystals of one mutant. The plates diffracted to beyond 2 Å resolution at a synchrotron source and complete data sets were collected for the two mutants. Data processing and analysis indicated that both mutant crystals belonged to the same monoclinic space group, with two molecules of PRO in the asymmetric unit.

ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

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Kavita Rajesh Pandey

2016-09-01

Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

Improved ethanol fermentation of a yeast mutant by C-12 ion beam irradiation

International Nuclear Information System (INIS)

Lu Dong; Liu Qingfang; Wu Xin; Wang Ying; Wang Jufang; Ma Shuang; Li Wenjian

2010-01-01

The yeast Saccharomyces cerevisiae YY was irradiated with 100 MeV/u 12 C 6+ ion beams. After screening,we obtained the mutant strain C03A of high ethanol yield. The influence of fermentation temperature, pH and concentration of sugar on ethanol fermentation were studied. The range analysis and analysis of variance were applied for the result of orthogonal experiments. The optimal ethanol fermentation conditions are: fermentation temperature 35 degree C, pH value 5.0, and sugar concentration 24%. The results of fermentation in the 10 L bioreactor showed that the ethanol fermentation of the mutant strain could be completed in 36 hours, the production of ethanol was to 13.2%(V/V), which means 12 hours faster and 1.6%(V /V) ethanol yield higher than original strain. (authors)

Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

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Victoria A Lawson

Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

[Mutant prevention concentrations of antibacterial agents to ocular pathogenic bacteria].

Science.gov (United States)

Liang, Qing-Feng; Wang, Zhi-Qun; Li, Ran; Luo, Shi-Yun; Deng, Shi-Jing; Sun, Xu-Guang

2009-01-01

To establish a method to measure mutant prevention concentration (MPC) in vitro, and to measure MPC of antibacterial agents for ocular bacteria caused keratitis. It was an experimental study. Forty strains of ocular bacteria were separated from cornea in Beijing Institute of Ophthalmology, which included 8 strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae respectively. The minimal inhibitory concentration (MIC) of the levofloxacin (LVF), ofloxacin (OFL), ciprofloxacin (CIP), norfloxacin (NFL), tobramycin (TOB) and chloromycetin (CHL) were determined by agar dilution method from National Committee of Clinical Laboratory Standard (NCCLS). The MPC were measured by accumulate-bacterial methods with bacterial population inoculated more than 1.2 x 10(10) colony forming units per milliliter with Mueller-Hinton broth and tryptic soy agar plate. With the software of SPSS 11.0, the datum such as the range of MIC, MPC, MIC90 and MPC90 were calculated, and the selection index (MPC90/ MI90) and mutant selection window (MSW) were obtained. The MI90 of LVF and TOB (4 mg/L) to Staphylococcus aureus strains were the lowest. CIP showed the lowest MIC90 (0.25 mg/L) to Pseudomonas aeruginosa among six kinds of antibacterial agents. The MIC90 of LVF to Staphylococcus epidermidis (256 mg/L), Streptococcus pneumoniae (1 mg/L) and Klebsiella pneumoniae (0.25 mg/L) were lower than other antibacterial agents. The MPC90, MSW and the MPC90/MIC90 of levofloxacin showed lower values compared with other antibacterial medicines. From all the datum, the MIC90 of CHL was the highest and the activity was the weakest. Although the activity of LVF was higher to every kind of bacteria, CIP had the highest activity antibacterial to Pseudomonas aeruginosa. The capacity of CHL and TOB was weaker than Quinolones for restricting resistant mutants on ocular bacteria. LVF had the strongest capacity for restricting resistant

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

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Joel Bozue

Full Text Available Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5'----3' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII

International Nuclear Information System (INIS)

Chase, J.W.; Masker, W.E.

1977-01-01

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants

Characterization of MMS-sensitive mutants of Neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

DeLange, A.M.; Mishra, N.C.

1982-01-01

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways. On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. On the basis of data presented, the MMS sensitivity of the first group mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-rays, high frequencies of spontaneous mutation and defects in meiotic behavior.

Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of zymomonas mobilis

Energy Technology Data Exchange (ETDEWEB)

Park, S.C.; Baratti, J.C. (Univ. de Provence, Marseille (France). Centre National de la Recherche Scientifique)

1992-01-25

In conventional alcohol fermentation processes using yeast species, the substrate cost represents a major fraction of the total production cost. Therefore, it may be very attractive to use the bacterium Zymomonas mobilis, since it has shown higher ethanol yields than yeasts when grown on a glucose-based medium. A report is made on the use of mutant strain of Zymomonas mobilis for ethanol production from hydrolyzed sugar beet molasses in a two-stage continuous culture which showed high ethanol yield and an ethanol concentration sufficiently high for economical recovery. A single stage continuous culture was first operated in an attempt to reduce the formation of sorbitol. Further on, a second fermentor was added with additional substrate feeding to increase the effluent ethanol concentration. An ethanol concentration of 59.9g/l was obtained at 97% sugar conversion and at high ethanol yield. The volumetric ethanol productivity was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the ethanol concentration was increased to a level acceptable for economical recovery. 18 refs., 3 figs., 5 tabs.

Characterisation of a radiation-resistant strain of bacillus thuringiensis subsp. Aizawai with improved toxicity to larval plutella xylostella

International Nuclear Information System (INIS)

Mahadi, N.M.; Boo, J.M.L.; Jangi, M.S.

1989-01-01

A radiation-resistant strain of Bacillus thuringiensis subsp. Aizawai which was previously shown to be more toxic against larval Plutell xylostella was further characterized. Some of the growth characteristics of the mutant strain were quite different from those of the parent strain. In shake flask culture, its lag period was shorter and its cell yield was lower. The growth rate, however, was the same as that of the parent. Electron microscope studies show that the insecticidal parasporal crystals from the mutant strain are significantly bigger than those produced by the parent strain. The average length and width of the crystals were 1.25 and 0.53 um respectively whereas those of the parent were 0.87 and 0.35 um, respectively. The crystals from the mutant strain were also more toxic. The LC 50 was 0.30 ug crystal protein per ml as against 0.66 ug crystal protein per ml for those from the parent strain. Protein profile of the crystals obtained with SDS-PA gel electrophoresis showed that the mutant strain produced an additional polypeptide of 143 KDa polypeptide. The mutant strain also has an additional high molecular weight plasmid. The improved toxicity may have been brought about by a number of factors including an alteration in the regulatory mechanism that control the synthesis of the polypeptides that make up the crystals. (Auth.). 5 figs.; 21 refs.; 2 tabs

Ultra-violet-resistant mutants of Bacillus thuringiensis

Energy Technology Data Exchange (ETDEWEB)

Jones, D R; Karunakaran, V [Polytechnic of Central London (UK). Faculty of Engineering and Science, School of Biological and Health Sciences; Burges, H D [Institute of Horticultural Research, Littlehampton (UK); Hacking, A J [Reading Univ. (UK). Dextra Labs.Ltd.

1991-06-01

One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author).

Ultra-violet-resistant mutants of Bacillus thuringiensis

International Nuclear Information System (INIS)

Jones, D.R.; Karunakaran, V.; Hacking, A.J.

1991-01-01

One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author)

Isolation and characterization of Candida albicans morphological mutants derepressed for the formation of filamentous hypha-type structures

International Nuclear Information System (INIS)

Gil, C.; Pomes, R.; Nombela, C.

1990-01-01

Several Candida albicans morphological mutants were obtained by a procedure based on a combined treatment with nitrous acid plus UV irradiation and a double-enrichment step to increase the proportion of mutants growing as long filamentous structures. Altered cell morphogenesis in these mutants correlated with an altered colonial phenotype. Two of these mutants, C. albicans NEL102 and NEL103, were selected and characterized. Mutant blastoconidia initiated budding but eventually gave rise to filamentous hypha-type formations. These filaments were long and septate, and they branched very regularly at positions near septa. Calcofluor white (which is known to bind chitin-rich areas) stained septa, branching zones, and filament tips very intensely, as observed under the fluorescence microscope. Wild-type hybrids were obtained by fusing protoplasts of strain NEL102 with B14, another morphological mutant previously described as being permanently pseudomycelial, indicating that genetic determinants responsible for the two altered phenotypes are different. The mutants characterized in this work seemed to sequentially express the morphogenic characteristics of C. albicans, from blastoconidia to hyphae, in the absence of any inducer. Further characterization of these strains could be relevant to gain understanding of the genetic control of dimorphism in this species

[Mutant alleles associated to chloroquine and sulfadoxine-pyrimethanime resistance in Plasmodium falciparum of the Ecuador-Peru and Ecuador-Colombia borders].

Science.gov (United States)

Arróspide, Nancy; Hijar-Guerra, Gisely; de Mora, Doménica; Diaz-Cortéz, César Eduardo; Veloz-Perez, Raúl; Gutierrez, Sonia; Cabezas-Sánchez, César

2014-04-01

The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.

X-ray-sensitive mutants of Chinese hamster ovary cell line

International Nuclear Information System (INIS)

Jeggo, P.A.; Kemp, L.M.

1983-01-01

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D 10 values 5-10-fold of wild-type D 10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D 10 values less than 2-fold of wild-type D 10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks. (Auth.)

Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

International Nuclear Information System (INIS)

Bame, K.J.; Kiser, C.S.; Esko, J.D.

1987-01-01

The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate 35 SO 4 into acid-precipitable material. Some mutants did not incorporate 35 SO 4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects

Association of methionine requirement with methyl mercury resistant mutants of yeast

Energy Technology Data Exchange (ETDEWEB)

Singh, A.; Sherman, F.

1974-01-25

It has been known for several years that strains resistant to mercury can be obtained in several bacterial species. Soon after the correlation between resistance to antibiotics and to mercury was recognized, it was established that genetic elements conferring resistance to antibiotics, mercury and other heavy metals in Escherichia coli and Samonella typhimurium and Staphylococcus aureus reside on extrachromosomal resistance transfer factors or plasmids. Among fungi, mercury resistant strains of Botrytis cinerea, Penicillium notatum, Sclerotinia fructicola, Stemphylium sarcinaeforme, and Saccharomyces cerevisiae have been reported. In most cases, this was accomplished by training the normal strains for growth on media supplemented with successively increasing concentrations of mercury compounds, and in some cases the resistance was lost when subcultured on mercury-free media. It is noteworthy that in none of the mercury-adapted strains of fungi has the genetic basis of resistance been determined. In this report we describe a method of isolation and characterization of methyl mercury resistant mutants of S. cerevisiae. This study was undertaken with the view that the examination of physiological changes associated with genetically defined resistant mutants will be useful in studying the mechanisms of cellular detoxification of organic mercurials.

Kinetics, improved activity and thermostability of endoglucanase and beta glucosidase from a mutant-derivative of aspergillus niger ms82

International Nuclear Information System (INIS)

Sohail, M.; Ahmad, A.; Khan, S.A.; Uddin, F.

2013-01-01

A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and beta-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (Tm and T1/2) showed that the wild type and mutant endoglucanase was more heat-resistant compared to beta-glucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and beta-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant beta-glucosidase was substantially lower than the parent enzyme while Ea of mutant endoglucanase was slightly less than the parent. The lowered Ea values can be attributed to the improved beta-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82. (author)

Improved vanillin production in baker's yeast through in silico design.

Science.gov (United States)

Brochado, Ana Rita; Matos, Claudia; Møller, Birger L; Hansen, Jørgen; Mortensen, Uffe H; Patil, Kiran Raosaheb

2010-11-08

Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

Science.gov (United States)

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

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Chen Bo-Ruei

2012-05-01

Full Text Available Abstract Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning

A strain-isolation design for stretchable electronics

Science.gov (United States)

Wu, Jian; Li, Ming; Chen, Wei-Qiu; Kim, Dae-Hyeong; Kim, Yun-Soung; Huang, Yong-Gang; Hwang, Keh-Chih; Kang, Zhan; Rogers, John A.

2010-12-01

Stretchable electronics represents a direction of recent development in next-generation semiconductor devices. Such systems have the potential to offer the performance of conventional wafer-based technologies, but they can be stretched like a rubber band, twisted like a rope, bent over a pencil, and folded like a piece of paper. Isolating the active devices from strains associated with such deformations is an important aspect of design. One strategy involves the shielding of the electronics from deformation of the substrate through insertion of a compliant adhesive layer. This paper establishes a simple, analytical model and validates the results by the finite element method. The results show that a relatively thick, compliant adhesive is effective to reduce the strain in the electronics, as is a relatively short film.

Experiences with strain based limit state design in The Netherlands

NARCIS (Netherlands)

Gresnigt, A.M.; Foeken, R.J. van

1996-01-01

Limit state design differs from conventional design methods in that each failure mode is specifically addressed (e.g. burst, collapse, local buckling, fracture due to insufficient strain capacity of the pipe wall, fatigue). Based on an extensive theoretical and experimental research programme,

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 3

International Nuclear Information System (INIS)

Schuepbach, M.E.; Serres, F.J. de

1981-01-01

γ-ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV- sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivy to γ-ray-induced inactivation, with the relative sensitivy at 37% survival being uvs-6 > upr-1 > uvs-2 UE uvs-3 > wild-type > uvs-5 > uvs-4. Studies on the induction of ad-3 mutants by γ-rays show that when the dose-response curves (expressed in terms of ad-3 mutants among the surving colonies) of the UV-sensitive strains are compared with wild-type, the excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type. (orig.)

Characterization of new radiation-sensitive mutant, Escherichia coli K-12 radC102

International Nuclear Information System (INIS)

Felzenszwalb, I.; Sargentini, N.J.; Smith, K.C.

1984-01-01

A new radiation-sensitive mutant, radC, has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for γ radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amount of X- and uv-radiation-induced DNA degradation, and it wasapprox. =60% deficient in recombination ability. The radC strain was normal for host cell reactivation of γ and uv-irradiated bacteriophage the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, it is suggested that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation

Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains of Bacillus subtilis in Submerged and Solid State Fermentation

Science.gov (United States)

Haq, Ikramul

2013-01-01

The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine) of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh) was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions. PMID:24294129

Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains of Bacillus subtilis in Submerged and Solid State Fermentation

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Hamid Mukhtar

2013-01-01

Full Text Available The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions.

Mutant strain screening and its enzyme production conditions of cellulase

International Nuclear Information System (INIS)

Dong Zhiyang; Zhu Lingxiang; Yu Wei

2001-01-01

Trichoderma koeningii T-801, which can produce relatively high cellulase, was isolated. The ability of producing cellulase of mutant T-801 had increased 1.77 times after treated with nitrous guanide and γ-ray and was higher than that of Trichoderma QM9414. The medium with straw powder as carbon source and peptone as nitrogen source is optimal and the maximum cellulase activity is reached at 30 degree C and pH 5.0 when cultured for 5 days

Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity.

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Christian Nyffenegger

Full Text Available A sialidase (EC 3.2.1.18 from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P to form the so-called TrSA5mut enzyme. By computational and hypothesis driven approaches additional mutations enhancing the trans-sialidase activity have been suggested. In the present work, we made a systematic combination of these mutations leading to seven new variants of the T. rangeli sialidase, having 6-16 targeted amino acid mutations. The resulting enzyme variants were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate, that computational studies of trans-glycosylation can be a valuable input in the design of novel trans-glycosidases, but also highlight the importance of experimental validation in order to assess the performance. In conclusion, two of the seven mutants displayed a dramatic switch in specificity from hydrolysis towards trans-sialylation and constitute the most potent trans-sialidase mutants of TrSA described in literature to date.

Global Phenotypic Characterization of Effects of Fluoroquinolone Resistance Selection on the Metabolic Activities and Drug Susceptibilities of Clostridium perfringens Strains

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Miseon Park

2014-01-01

Full Text Available Fluoroquinolone resistance affects toxin production of Clostridium perfringens strains differently. To investigate the effect of fluoroquinolone resistance selection on global changes in metabolic activities and drug susceptibilities, four C. perfringens strains and their norfloxacin-, ciprofloxacin-, and gatifloxacin-resistant mutants were compared in nearly 2000 assays, using phenotype microarray plates. Variations among mutant strains resulting from resistance selection were observed in all aspects of metabolism. Carbon utilization, pH range, osmotic tolerance, and chemical sensitivity of resistant strains were affected differently in the resistant mutants depending on both the bacterial genotype and the fluoroquinolone to which the bacterium was resistant. The susceptibilities to gentamicin and erythromycin of all resistant mutants except one increased, but some resistant strains were less susceptible to amoxicillin, cefoxitin, ceftriaxone, chloramphenicol, and metronidazole than their wild types. Sensitivity to ethidium bromide decreased in some resistant mutants and increased in others. Microarray analysis of two gatifloxacin-resistant mutants showed changes in metabolic activities that were correlated with altered expression of various genes. Both the chemical structures of fluoroquinolones and the genomic makeup of the wild types influenced the changes found in resistant mutants, which may explain some inconsistent reports of the effects of therapeutic use of fluoroquinolones on clinical isolates of bacteria.

A detailed study of gerJ mutants of Bacillus subtilis.

Science.gov (United States)

Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O

1986-08-01

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.

Visible laser and UV-A radiation impact on a PNP degrading Moraxella strain and its rpoS mutant.

Science.gov (United States)

Nandakumar, Kanavillil; Keeler, Werden; Schraft, Heidi; Leung, Kam T

2006-07-05

The role of stationary phase sigma factor gene (rpoS) in the stress response of Moraxella strain when exposed to radiation was determined by comparing the stress responses of the wild-type (WT) and its rpoS knockout (KO) mutant. The rpoS was turned on by starving the WT cultures for 24 h in minimal salt medium. Under non-starved condition, both WT and KO planktonic Moraxella cells showed an increase in mortality with the increase in duration of irradiation. In the planktonic non-starved Moraxella, for the power intensity tested, UV radiation caused a substantially higher mortality rate than did by the visible laser light (the mortality rate observed for 15-min laser radiation was 53.4 +/- 10.5 and 48.7 +/- 8.9 for WT and KO, respectively, and 97.6 +/- 0 and 98.5 +/- 0 for 25 s of UV irradiation in WT and KO, respectively). However, the mortality rate decreased significantly in the starved WT when exposed to these two radiations. In comparison, rpoS protected the WT against the visible laser light more effectively than it did for the UV radiation. The WT and KO strains of Moraxella formed distinctly different types of biofilms on stainless steel coupons. The KO strain formed a denser biofilm than did the WT. Visible laser light removed biofilms from the surfaces more effectively than did the UV. This was true when comparing the mortality of bacteria in the biofilms as well. The inability of UV radiation to penetrate biofilms due to greater rates of surface absorption is considered to be the major reason for the weaker removal of biofilms in comparison to that of the visible laser light. This result suggests that high power visible laser light might be an effective tool for the removal of biofilms. (c) 2006 Wiley Periodicals, Inc.

A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

Energy Technology Data Exchange (ETDEWEB)

Lindberg, P.; Lindblad, P. [Uppsala Univ. (Sweden). Dept. of Physiological Botany; Schuetz, K.; Happe, T. [Universitaet Bonn (Germany). Botanisches Inst.

2002-12-01

The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL{sup -} strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H{sub 2} evolution under air was observed using an H{sub 2} electrode. (Author)

Study on yeast mutant with high alcohol yield fermented in sweet sorghum juice using carbon ion irradiation

International Nuclear Information System (INIS)

Yan Yaping; Lu Dong; Wang Jufang; Dong Xicun; Gao Feng; Ma Liang; Li Wenjian

2009-01-01

Five mutants with high ability of producing alcohol were selected out by using TTC as an indicator after irradiation of the alcohol yeast with 100 MeV/u carbon ions. The fermentation experiment in sweet sorghum juice showed that the alcohol production ability of mutant T4 strain increased 18.6% compared to the control strain. The residual sugar content in the juice was decreased too. After that,the optimum fermentation conditions of the T4 strain in sweet sorghum juice were investigated. The results showed that the optimum temperature and pH value for fermentation were 30 degree C and 4.5, respectively. The verification experiment was fermented in a 10 l bio-reactor and the obtained data indicated that the fermentative rate and the ability of producing alcohol in T4 strain was higher than that in the control strain under the same fermentation condition. (authors)

Strain improvement in Streptomyces galilaeus, a producer of anthracycline antibiotics galirubins

International Nuclear Information System (INIS)

Kralovcova, E.; Blumauerova, M.; Vanek, Z.

1977-01-01

The production of epsilon-pyrromycinone glycosides in Streptomyces galilaeus increased 12-fold, with respect to the wild strain, as a result of a sequential procedure including both natural selection and treatment with mutagens (nitrous acid, UV light and γ irradiation). Nitrous acid exhibited the highest mutagenic effect, both in increasing the productivity and in inducing blocked mutants. A mutant strain blocked in the biosynthesis of glycosides and accumulating free epsilon-pyrromycinone as the principal metabolite was obtained. (author)

Lethal effect of short-wave (254 nm) UV-radiation on cells of Chlamidomonas reinhardii strains with different carotenoid content

International Nuclear Information System (INIS)

Kamchatova, I.E.; Chunaev, A.S.; Bronnikov, V.A.

1987-01-01

In experiments on related Chlamidomonas reinhardii strains of similar mating type a study was made of sensitivity of cells with different carotenoid content to UV-radiation of 254 nm. Mutants having a lower, as opposed to the wild type strain, content of carotenoids exhibited an increased radiosensitivity. A carotenoid-free mutant was found to possess a higher sensitivity to UV-radiation which was typical of the strain with the impaired excision repair system. The studied subclone of the UV-radiosensitive strain CC-888 was unable to photoreactivate the UV-induced damages which was typical of the wild-type strain. The content of carotenoids in cells of this subnuclone exceeded that in cells of mutants with the reduced pigmentation

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.

Science.gov (United States)

Field, H J; Darby, G; Wildy, P

1980-07-01

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

Phase-Change Memory Materials by Design: A Strain Engineering Approach.

Science.gov (United States)

Zhou, Xilin; Kalikka, Janne; Ji, Xinglong; Wu, Liangcai; Song, Zhitang; Simpson, Robert E

2016-04-20

Van der Waals heterostructure superlattices of Sb2 Te1 and GeTe are strain-engineered to promote switchable atomic disordering, which is confined to the GeTe layer. Careful control of the strain in the structures presents a new degree of freedom to design the properties of functional superlattice structures for data storage and photonics applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

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Anja Wartenberg

2014-12-01

Full Text Available Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

Excision-repair in mutants of Escherichia coli deficient in DNA polymerase I and/or its associated 5'. -->. 3' exonuclease

Energy Technology Data Exchange (ETDEWEB)

Cooper, P [Stanford Univ., Calif. (USA). Dept. of Biological Sciences

1977-01-01

The UV sensitivity of E.coli mutants deficient in the 5'..-->..3' exonuclease activity of DNA polymerase I is intermediate between that of pol/sup +/ strains and mutants which are deficient in the polymerizing activity of pol I (polA1). Like polA1 mutants, the 5'-econuclease deficient mutants exhibit increased UV-induced DNA degradation and increased repair synthesis compared to a pol/sup +/ strain, although the increase is not as great as in polA1 or in the conditionally lethal mutant BT4113ts deficient in both polymerase I activities. When dimer excision was measured at UV doses low enough to avoid interference from extensive DNA degradation, all three classes of polymerase I deficient mutants were found to remove dimers efficiently from their DNA. We conclude that enzymes alternative to polymerase I can operate in both the excision and resynthesis steps of excision repair and that substitution for either of the polymerase I functions results in longer patches of repair. A model is proposed detailing the possible events in the alternative pathways.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

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Sara eAmorim-Vaz

2015-05-01

Full Text Available The aim of the present study was to identify C. albicans transcription factors (TF involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens quantified in kidneys. Mutants of unannotated genes which generated a kidney fungal burden significantly different from that of wild-type were selected and individually examined in G. mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25 % of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects, a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching fungal burden phenotypes were observed in 50 % of the cases, highlighting the bias due to host effects. In contrast, 33.4 % concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the pool effect. After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adaptation.

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 6

International Nuclear Information System (INIS)

De Serres, F.J.; Inoue, H.; Schuepbach, M.E.

1983-01-01

Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, #betta#-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair. (orig./AJ)

Improved vanillin production in baker's yeast through in silico design

Science.gov (United States)

2010-01-01

Background Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Conclusion Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories. PMID:21059201

Improved vanillin production in baker's yeast through in silico design

Directory of Open Access Journals (Sweden)

Hansen Jørgen

2010-11-01

Full Text Available Abstract Background Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1 were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Conclusion Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

Improvement of selected strains through gamma irradiation for enhanced lipolytic potential

International Nuclear Information System (INIS)

Iftikhar, T.; Mubashir, N.; Hussain, Y.; Abbas, S.Q.; Ashraf, I.

2010-01-01

The purpose of the present investigation was to enhance the production of industrially important enzyme lipase by subjecting the wild lipase producing fungal strains i.e. Aspergillus niger, Rhizopus microsporus and Penicillium atrovenetum to various doses of gamma irradiation (20, 40, 60, 80, 100, 120, 140 and 160 Gy). The isolation and lipolytic activity of selected mutant derived strains is described in this paper. Among all the mutants tested, MBL-5 obtained at 140Gy of Aspergillus niger strain showed highest extracellular lipase activity (13.75 +- 0.15 U mL/sup -1/) while MBL-1 Rhizopus microsporus at the rate 20Gy showed the lowest activity i.e., 1.06 +- 0.11 U mL/sup -1/. A range of pH 3, 5, 7, 9 and 11 was used to check the lipolytic potential of various mutants along with their wild type. It was observed that MBL-5 (Aspergillus niger) and MBL-2 (Rhizopus microsporus) showed enhanced extracellular lipase activity at pH 11 while MBL-3 (Penicillium atrovenetum) showed the highest extracellular lipase activity 22.53 +- 0.21 U mL/sup -1/ at pH 9. It indicates a possible role for the MBL-2, MBL-3 and MBL-5 mutant strains in the detergent industry for the development of eco-friendly technologies. (author)

BIOUTILIZATION OF GRAPE WASTE FOR EXOPOLYSACCHARIDE PRODUCTION USING ALTERNARIA ALTERNATA NON-PIGMENT STRAIN

International Nuclear Information System (INIS)

MELEIGY, S.A.

2009-01-01

In the present investigation, five mutant strains from A. alternate were isolated after exposure to gamma irradiation at dose level 8 kGy. The mutant isolated strains (MIS) were non-producing dark pigment and producing polysaccharide. The mutant isolated strain (MIS) belong to the Alternaria alternata MIS (1-5). In shake flask experiments, the exopolysaccharide (EPS) production was 2.90-5.24 g/l and biomass 5.8-8.31g/l. The effect of some fermentation conditions on grape wastes by A. alternata were investigated to produce EPS using gamma irradiation and non-pigment strain. The economical optimum fermentation condition for the highest EPS production by MIS4 when grown on grape waste containing 150 g/l sugar, incubation temperature 28 o C, pH 7 with addition of both 2 % yeast extract and 1.5 % surfactant triton x-100 achieved 14.68 g/l EPS and 6.22 g/l biomass

Reduced host cell invasiveness and oxidative stress tolerance in double and triple csp gene family deletion mutants of Listeria monocytogenes.

Science.gov (United States)

Loepfe, Chantal; Raimann, Eveline; Stephan, Roger; Tasara, Taurai

2010-07-01

The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and

Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains

Energy Technology Data Exchange (ETDEWEB)

OEztuerk, Yavuz; Yuecel, Meral; Guenduez, Ufuk [Department of Biology, Middle East Technical University, Ankara (Turkey); Daldal, Fevzi [Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104-6018 (United States); Mandaci, Sevnur [TUEBITAK Research Institute for Genetic Engineering and Biotechnology, Gebze Kocaeli 41470 (Turkey); Tuerker, Lemi [Department of Chemistry, Middle East Technical University, Ankara (Turkey); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, Ankara (Turkey)

2006-09-15

In Rhodobacter capsulatus excess reducing equivalents generated by organic acid oxidation is consumed to reduce protons into hydrogen by the activity of nitrogenase. Nitrogenase serves as a redox-balancing tool and is activated by the RegB/RegA global regulatory system during photosynthetic growth. The terminal cytochrome cbb{sub 3} oxidase and the redox state of the cyclic photosynthetic electron transfer chain serve redox signaling to the RegB/RegA regulatory systems in Rhodobacter. In this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes or lacking the cyt cbb{sub 3} oxidase or the quinol oxidase were compared with the wild type. The results indicated that hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3- to 4-fold, but the rate of hydrogen production increased significantly in a cbb{sub 3}{sup -} mutant. Moreover, hydrogen production efficiency of various R. capsulatus strains further increased by inactivation of uptake hydrogenase genes. (author)

Dictyostelium discoideum: mutants in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides

International Nuclear Information System (INIS)

Freeze, H.; Willies, L.; Hamilton, S.

1986-01-01

The lysosomal enzymes of Dictyostelium discoideum share highly immunogenic oligosaccharides which contain multiple Man-6-SO 4 residues. Two mutant strains which lack the shared antigenic determinant were analyzed in an attempt to identify the primary defect in each. [ 3 H]Man labelled N-linked oligosaccharides of secreted glycoproteins were released by Endo/PNGaseF digestion and analyzed. Both of the mutant strains produced smaller, less sulfated oligosaccharides compared to the wild-type, yet both still contained considerable amounts of Man-6-SO 4 . The size of the precursor lipid-linked oligosaccharide from the wild-type is consistent with a Glc 3 Man 9 GlcNAc 2 structure, while those from both of the mutants have an oligosaccharide the size of Man 5 GlcNAc 2 . The authors conclude that both of the mutants are defective in the biosynthesis of the precursor oligosaccharide. Both oligosaccharides from the mutants contain a tri-mannosyl core and are not glucosylated. Two of the five Man residues are released by a 1,2 specific α mannosidase. Based on the size and mannosidase digestions the authors conclude that 4/5 of the Man residues on the α1,6 branch of the β-linked Man residues are missing. Thus, these residues must be required to define the shared antigenic determinant

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

Science.gov (United States)

Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi

2015-06-01

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

DEFF Research Database (Denmark)

Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

2002-01-01

as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition...... activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid. FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well...

Anti-H-Y responses of H-2b mutant mice.

Science.gov (United States)

Simpson, E; Gordon, R D; Chandler, P R; Bailey, D

1978-10-01

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

Protein design in systems metabolic engineering for industrial strain development.

Science.gov (United States)

Chen, Zhen; Zeng, An-Ping

2013-05-01

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere

DEFF Research Database (Denmark)

Kristensen, K.E.; Jacobsen, C.S.; Hansen, L.H.

2006-01-01

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker...... into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected...... for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain...

Mutation of purD and purF genes further attenuates Brucella abortus strain RB51.

Science.gov (United States)

Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Watarai, Masahisa; Hahn, Tae-Wook

2015-02-01

In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

Science.gov (United States)

Davenport, K. D.; Williams, K. E.; Ullmann, B. D.; Gustin, M. C.; McIntire, L. V. (Principal Investigator)

1999-01-01

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

Kinetics Studies on citric acid production by gamma ray induced mutant of Aspergillus niger

International Nuclear Information System (INIS)

Begum, A.A.; Choudhury, N.; Islam, M.S.

1991-01-01

Effect of cultural pH and incubation temperature on citric acid yield and kinetic patterns of citric acid fermentation by a natural isolate of aspergillus niger as CA16 and one of its gamma ray induced mutants were studied using cane molasses as growth and fermentation substrate. Mutant strain, 277/30 gave maximum citric acid yield of 85 g/l at pH 3.5 and 28 degree centigrade in molasses medium adjusted to 16% sugar and 25% prescott salt in the medium. Parent strain, CA16 gave a maximum yield of 34 g/l at pH 4.0 and 26 degree centigrade in molasses medium adjusted to 16% sugar and 100% prescott salt in the medium. In kinetic studies, strains showed combination kinetics of citric acid fermentation where product formation is directly related to growth and cell mass and indirectly related to carbohydrate uptake

New rifamycins produced by a recombinant strain of Nocardia mediterranei.

Science.gov (United States)

Schupp, T; Traxler, P; Auden, J A

1981-08-01

A recombinant strain of Nocardia mediterranei was found to produce a number of new rifamycins which are structurally related to rifamycin S, rifamycin W and rifamycin G. This strain was derived from two Nocardia mediterranei mutants by intraspecific recombination.

Jeast (Saccharomyces cerevisial) mutants with enhanced induced mutagenesis

International Nuclear Information System (INIS)

Ivanov, E.L.; Koval'tsova, S.V.; Korolev, V.G.

1987-01-01

The influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae has been. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adeine-dependent mutations (ade, ade2) were induced more frequently (1.5-2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed than him1-1, him2-1, and himX mutations increase specifically the yield of transitions (AT-GC and GC→AT), whereas in the him3-1, strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction

Candidacidal Activity of a Novel Killer Toxin from Wickerhamomyces anomalus against Fluconazole-Susceptible and -Resistant Strains

Directory of Open Access Journals (Sweden)

Laura Giovati

2018-02-01

Full Text Available The isolation and characterization from the sand fly Phlebotomus perniciosus of a Wickerhamomyces anomalus yeast strain (Wa1F1 displaying the killer phenotype was recently reported. In the present work, the killer toxin (KT produced by Wa1F1 was purified and characterized, and its antimicrobial activity in vitro was investigated against fluconazole- susceptible and -resistant clinical isolates and laboratory strains of Candida albicans and C. glabrata displaying known mutations. Wa1F1-KT showed a differential killing ability against different mutant strains of the same species. The results may be useful for the design of therapeutic molecules based on Wa1F1-KT and the study of yeast resistance mechanisms.

[Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts].

Science.gov (United States)

Zakharov, I A; Kasinova, G V; Koval'tsova, S V

1983-01-01

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

Science.gov (United States)

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

Science.gov (United States)

Jernejc, Katarina; Bencina, Mojca

2003-08-29

Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

Characterization of Emericella nidulans RodA and DewA hydrophobin mutants

DEFF Research Database (Denmark)

Jensen, Britt Guillaume; Nielsen, Jakob Blæsbjerg; Pedersen, Mona Højgaard

Hydrophobins are small amphiphilic proteins containing an eight cysteine pattern only found in filamentous fungi. They are involved in the attachment of hyphae to hydrophobic structures and the formation of aerial structures. Five Emericella nidulans mutant strains were examined to study the two...

Evolution of a recombinant (gucoamylase-producing) strain of Fusarium venenatum A3/5 in chemostat culture.

Science.gov (United States)

Wiebe, M G; Robson, G D; Shuster, J; Trinci, A P

2001-04-20

Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. Copyright 2001 John Wiley & Sons, Inc.

Inactivation of normal and mutant Neurospora crassa conidia by visible light and near-UV: role of 1O2, carotenoid composition and sensitizer location

International Nuclear Information System (INIS)

Thomas, S.A.; Sargent, M.L.; Tuveson, R.W.

1981-01-01

Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-ultraviolet light was investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV was virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, 1 O 2 is not involved in any substantial way in the formation of lethal lesions. Carotenoid deficient strains were similar to wild-type strains in sensitivity to near-UV inactivation which is consistent with 1 O 2 not being involved. Photodynamic inactivation of conidia by visible light occurred in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains were more sensitive to such inactivation only when MB and TB were used. This suggests that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids. A newly isolated, lemon-yellow mutant exhibited sensitivities to photodynamic inactivation similar to other pure-white mutants. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation of the two coloured carotenoids produced by the mutant. (author)

Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

International Nuclear Information System (INIS)

Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

2003-01-01

We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

Mutante espontâneo de Bacillus licheniformis bloqueado no estágio I da esporogênese, possuidor de metabolismo respiratório aumentado A spontaneous mutant of Bacillus licheniformis with increased respiratory metabolism, blocked in stage I of sporogenesis

Directory of Open Access Journals (Sweden)

Leon Rabinovitch

1976-01-01

Full Text Available Um mutante espontâneo de Bacillus licheniformis, derivado da amostra esporogênica 2390, foi estudado com vistas ao reconhecimento do estágio da evolução para esporo em que o mesmo se encontrava bloqueado. Eletronmicrografias sugeriram que as células desse mutante, colhidas durante a fase estacionária da curva de crescimento, não ultrapassaram o estágio I da esporogênese (i.e., permaneceram com o nucleóide disposto como filamento axial, enquanto a produção de antibiótico (bacitracina e a atividade proteolítica foram francamente detectadas. A linhagem mutante, designada Spolp-72, nas condições experimentais empregadas não biossintetizou esporos por estarvação em solução de sais inorgãnicos, mas evidenciou uma frequência de esporulação menor que 10*-7, após crescimento vegetativo em meio de cultura favorável á esporogênese. A amostra Spolp-72 externa um crescimento vegetativo inicial restringido, quando comparada com a amostra 2390, enquanto que, inversamente, sua atividade respiratória é significativamente mais elevada. Este último comportamento foi confirmado no presente trabalho, contrastando, nesse particular, com outros tipos de mutantes de esporulação já descritos, os quais se encontram bloqueados nos primeiros estágios da via esporogenética.A spontaneous mutant strain derived from the sporogenic B. licheniformis 2390 was studied with a view to determining at what developmental stage toward sporulation it was blocked. Electronmicrographs suggested that the mutant cells harvested during the stationary phase of the growth curve were unable to go beyond stage I of sporogenesis (i. e., their nucleoid remained as an axial filament. On the other hand, antibiotic production (bacitracin and proteolytic activity were easily detected. Under the present experimental conditions the mutant strain, named Spolp-72, did not synthesize spores by starvation in a solution of inorganic salts, in contrast with the parental

Characterization of Cell Surface and EPS Remodeling of Azospirillum brasilense Chemotaxis-like 1 Signal Transduction Pathway mutants by Atomic Force Microscopy

Energy Technology Data Exchange (ETDEWEB)

Billings, Amanda N [ORNL; Siuti, Piro [ORNL; Bible, Amber [University of Tennessee, Knoxville (UTK); Alexandre, Gladys [University of Tennessee, Knoxville (UTK); Retterer, Scott T [ORNL; Doktycz, Mitchel John [ORNL; Morrell-Falvey, Jennifer L [ORNL

2011-01-01

To compete in complex microbial communities, bacteria must quickly sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the modulation of multiple cellular responses, including motility, EPS production, and cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from Azospirillum brasilense was shown to modulate flocculation. In A. brasilense, cell surface properties, including EPS production, are thought to play a direct role in promoting flocculation. Using atomic force microscopy (AFM), we have detected distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains that are absent in the wild type strain. Whereas the wild type strain produces a smooth mucosal extracellular matrix, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition and lectin-binding assays suggest that the composition of EPS components in the extracellular matrix differs between the cheA1 and cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that mutations in the Che1 pathway that result in increased flocculation are correlated with distinctive changes in the extracellular matrix structure produced by the mutants, including likely changes in the EPS structure and/or composition.

Kinetics of formation of induced mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Chepurnoj, A.I.; Levkovich, N.V.; Mikhova-Tsenova, N.; Mel'nikova, L.A.

1990-01-01

UV and γ-radiation mutagenic effect an various strains of Saccharomyces cerevisiae was studied by analyzing formation kinetics of induced mutants at the period of postirradiation incubation. Mechanisms of induced reverse formation was suggested. The presented analysis is considered to be differential taking account of more subtle aspects of induced mutagenesis. 8 refs.; 10 figs.; 3 tabs

Production of alpha amylase from a randomly induced mutant strain of bacillus amyloliquefaciens and its application as a desizer in textile industry

International Nuclear Information System (INIS)

Haq, I.; Ali, S.; Javed, M.M.; Hameed, U.; Saleem, A.; Adnan, F.; Qadeer, M.A.

2010-01-01

The present study is concerned with the improvement of Bacillus amyloliquefaciens strain UNG-16 for alpha amylase production. The bacterial culture was exposed to UV irradiation at 1.6X102 J/m2/S for 15-60 min. However, UV induced viables did not give improved alpha amylase production; therefore chemical mutation using ethyl methane sulphonate (EMS 50-300 mu l/ml) was undertaken for 10-60 min. The mutant B. amyloliquefaciens EMS-6 gave 102.78+-2.22 U/ml/min enzyme activity which was 1.4 fold higher than the parental strain. In stirred fermentor, the incubation period was reduced from 72 to 48 h after inoculation. The production of alpha amylase was found to be maximal when the 60% volume, 2.0 vvm air supply and 400 rpm agitation rate was maintained during the fermentation period. The incubation temperature (37 deg. C) and size of inoculum (8.0 %) were also optimized. A 100% desizing of grey fabric (or starch removal) was obtained with 200-250 enzyme units at pH 6.5 at 60 deg. C in 1 h. (author)

Phenotypic, fermentation characterization, and resistance mechanism analysis of bacteriophage-resistant mutants of Lactobacillus delbrueckii ssp. bulgaricus isolated from traditional Chinese dairy products.

Science.gov (United States)

Deng, Kaibo; Fang, Wei; Zheng, Baodong; Miao, Song; Huo, Guicheng

2018-03-01

Bacteriophage infection is a large factor in dairy industrial production failure on the basis of pure inoculation fermentation, and developing good commercial starter cultures from wild dairy products and improving the environmental vigor of starter cultures by enhancing their phage resistance are still the most effective solutions. Here we used a spontaneous isolation method to obtain bacteriophage-resistant mutants of Lactobacillus delbrueckii ssp. bulgaricus strains that are used in traditional Chinese fermented dairy products. We analyzed their phenotypes, fermentation characteristics, and resistance mechanisms. The results showed that bacteriophage-insensitive mutants (BIM) BIM8 and BIM12 had high bacteriophage resistance while exhibiting fermentation and coagulation attributes that were as satisfying as those of their respective parent strains KLDS1.1016 and KLDS1.1028. According to the attachment receptor detection, mutants BIM8 and BIM12 exhibited reduced absorption to bacteriophage phiLdb compared with their respective bacteriophage-sensitive parent strains because of changes to the polysaccharides or teichoic acids connected to their peptidoglycan layer. Additionally, genes, including HSDR, HSDM, and HSDS, encoding 3 subunits of a type I restriction-modification system were identified in their respective parent strains. We also discovered that HSDR and HSDM were highly conserved but that HSDS was variable because it is responsible for the DNA specificity of the complex. The late lysis that occurred only in strain KLDS1.1016 and not in strain KLDS1.1028 suggests that the former and its mutant BIM8 also may have an activatable restriction-modification mechanism. We conclude that the L. bulgaricus BIM8 and BIM12 mutants have great potential in the dairy industry as starter cultures, and their phage-resistance mechanism was effective mainly due to the adsorption interference and restriction-modification system. Copyright © 2018 American Dairy Science

A mycobacterial smc null mutant is proficient in DNA repair and long-term survival.

Science.gov (United States)

Güthlein, Carolin; Wanner, Roger M; Sander, Peter; Böttger, Erik C; Springer, Burkhard

2008-01-01

SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for smc null mutants in other species.

A Mycobacterial smc Null Mutant Is Proficient in DNA Repair and Long-Term Survival▿

OpenAIRE

Güthlein, Carolin; Wanner, Roger M.; Sander, Peter; Böttger, Erik C.; Springer, Burkhard

2007-01-01

SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for smc null mutants in other species.

Strain-Based Design Methodology of Large Diameter Grade X80 Linepipe

Energy Technology Data Exchange (ETDEWEB)

Lower, Mark D. [ORNL

2014-04-01

Continuous growth in energy demand is driving oil and natural gas production to areas that are often located far from major markets where the terrain is prone to earthquakes, landslides, and other types of ground motion. Transmission pipelines that cross this type of terrain can experience large longitudinal strains and plastic circumferential elongation as the pipeline experiences alignment changes resulting from differential ground movement. Such displacements can potentially impact pipeline safety by adversely affecting structural capacity and leak tight integrity of the linepipe steel. Planning for new long-distance transmission pipelines usually involves consideration of higher strength linepipe steels because their use allows pipeline operators to reduce the overall cost of pipeline construction and increase pipeline throughput by increasing the operating pressure. The design trend for new pipelines in areas prone to ground movement has evolved over the last 10 years from a stress-based design approach to a strain-based design (SBD) approach to further realize the cost benefits from using higher strength linepipe steels. This report presents an overview of SBD for pipelines subjected to large longitudinal strain and high internal pressure with emphasis on the tensile strain capacity of high-strength microalloyed linepipe steel. The technical basis for this report involved engineering analysis and examination of the mechanical behavior of Grade X80 linepipe steel in both the longitudinal and circumferential directions. Testing was conducted to assess effects on material processing including as-rolled, expanded, and heat treatment processing intended to simulate coating application. Elastic-plastic and low-cycle fatigue analyses were also performed with varying internal pressures. Proposed SBD models discussed in this report are based on classical plasticity theory and account for material anisotropy, triaxial strain, and microstructural damage effects

UV-induced mitotic recombination and its dependence on photoreactivation and liquid holding in the rad6-1 mutant of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Haladus, E.; Zuk, J.

1980-01-01

Spontaneous and UV-induced mitotic recombination was compared in diploids homozygous for rad6-1 mutation and in the wild-type strain carrying heterozygous markers for detecting gene conversion (hom 2-1, hom 2-2) and crossing over (ade 1, ade 2). Diploids homozygous for rad6-1 mutation were characterised by an elevated level of spontaneous and UV-induced mitotic recombination, particularly the intergenic events. Exposure of UV-irradiated strains to visible light resulted in an increased survival and decreased level of mitotic recombination. Liquid holding (LH) differentially affected frequency of mitotic intergenic and intragenic recombination in mutant and wild-type strains, being without any significant effect on cell survival. In a mutant strain intragenic recombination is significantly increased, intergenic only slightly. In the wild-type strain intragenic recombination is slightly decreased but intergenic is not changed by LH. Visible light applied after LH had no effect on survival and mitotic recombination in the wild type, while in the mutant strain photoreactivability of survival was fully preserved and accompanied by a decrease in the frequency of intragenic and intergenic recombination. The results suggest that metabolic pathways responsible for restoring cell survival are independent of or only partly overlapping with those concerning recombination events. (orig.) [de

Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

DEFF Research Database (Denmark)

Jensen, Lise Torp; Ladefoged, Søren; Birkelund, Svend

1995-01-01

monoclonal antibody (MAb) 552. The epitope for MAb 552 was localized at the repeated part of the protein. The gene encoding Lmp1 is part of a transcriptional complex that contains 9.5 direct repeats of 471 bp each. Pure cultures of mutant strains were obtained by subcloning, and three mutants were...

An induced early mutant in finger millet Eleusin coranaca Gaertn

International Nuclear Information System (INIS)

Shivashankar, G.; Kempanna, C.; Viswanatha, S.R.

1975-01-01

Among the several collections of ragi (Eleusine coracana. Gaertn.) from all over the world, the strain 'H.E.S.927' has been found to be the highest yielder compared to other cultivars. Mutation studies have been conducted on this variety at Bangalore in India to make it suitable for the rainfall pattern of the ragi tract of the Karnataka state. A mutant induced by gamma radiation at 30 K rad has been stabilized. This mutant is early by 30-35 days with comparatively better straw quality. Various morphological characters concerning the yield and yield attributes and the possibility of exploiting it as a genotype for future breeding work and as a promising variety is discussed. Another promising mutant with earhead measuring 15 cm as against the earhead length of 5 to 10 cm of the cultivated varieties is illustrated. (K.B.)

Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

Science.gov (United States)

Cusick, John K; Hager, Elizabeth; Gill, Ronald E

2015-01-01

The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Succinate overproduction: A case study of computational strain design using a comprehensive Escherichia coli kinetic model

Directory of Open Access Journals (Sweden)

Ali eKhodayari

2015-01-01

Full Text Available Computational strain design prediction accuracy has been the focus for many recent efforts through the selective integration of kinetic information into metabolic models. In general, kinetic model prediction quality is determined by the range and scope of genetic and/or environmental perturbations used during parameterization. In this effort, we apply the k-OptForce procedure on a kinetic model of E. coli core metabolism constructed using the Ensemble Modeling (EM method and parameterized using multiple mutant strains data under aerobic respiration with glucose as the carbon source. Minimal interventions are identified that improve succinate yield under both aerobic and anaerobic conditions to test the fidelity of model predictions under both genetic and environmental perturbations. Under aerobic condition, k-OptForce identifies interventions that match existing experimental strategies pointing at a number of unexplored flux redirections such as routing glyoxylate flux through the glycerate metabolism to improve succinate yield. Many of the identified interventions rely on the kinetic descriptions and would not be discoverable by a purely stoichiometric description. In contrast, under fermentative (anaerobic conditions, k-OptForce fails to identify key interventions including up-regulation of anaplerotic reactions and elimination of competitive fermentative products. This is due to the fact that the pathways activated under anaerobic conditions were not properly parameterized as only aerobic flux data were used in the model construction. This study shed light on the importance of condition-specific model parameterization and provides insight onto how to augment kinetic models so as to correctly respond to multiple environmental perturbations.

Carbon and energy metabolism of atp mutants of Escherichia coli

DEFF Research Database (Denmark)

Jensen, Peter Ruhdal; Michelsen, Ole

1992-01-01

strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming......The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...... rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion...

Potency of carbapenems for the prevention of carbapenem-resistant mutants of Pseudomonas aeruginosa: the high potency of a new carbapenem doripenem.

Science.gov (United States)

Sakyo, Shihomi; Tomita, Haruyoshi; Tanimoto, Koichi; Fujimoto, Shuhei; Ike, Yasuyoshi

2006-04-01

The potencies of the carbapenems; doripenem (DRPM), meropenem (MEPM) and imipenem (IPM) in preventing the emergence of carbapenem-resistant mutants were examined in Pseudomonas aeruginosa strains. The carbapenems predominantly selected carbapenem-resistant mutants or carbapenem mutants with reduced susceptibilities that were specifically resistant to carbapenems and had arisen as a result of the reduced level of expression of the outer membrane protein with a molecular weight of about 48,000 (OprD). The potency of carbapenems in preventing the growth of the mutants differed for DRPM, MEPM and IPM. The isolation frequency of the mutant was examined on agar plates containing each of the carbapenems at a concentration of 1/2 or 1/4 MIC of each carbapenem for that mutant. Mutants were not selected on agar containing DRPM at a frequency of greater than 10(-9) per cell per generation, whereas mutants of each strain were selected on agar containing MEPM or IPM at frequencies of 10(-7) to 10(-9) per cell per generation. The drug concentrations and the drug concentration range for the selective increase of carbapenem resistant mutants in the broth culture containing each carbapenem differed for each carbapenem. DRPM exhibited both the lowest drug concentration and the narrowest range of drug concentration for selection of the carbapenem-resistant mutants. The results shown in this report indicated that DRPM exhibited the greatest ability to prevent the emergence of the mutant.

Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

Directory of Open Access Journals (Sweden)

Matthew Dale Woolard

2013-02-01

Full Text Available Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS induces macrophages to synthesize prostaglandin E2 (PGE2. Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112 two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI. Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used

Molecular characterization, fitness and mycotoxin production of Fusarium graminearum laboratory strains resistant to benzimidazoles.

Science.gov (United States)

Sevastos, A; Markoglou, A; Labrou, N E; Flouri, F; Malandrakis, A

2016-03-01

Six benzimidazole (BMZ)-resistant Fusarium graminearum strains were obtained after UV mutagenesis and selection on carbendazim (MBC)-amended medium. In vitro bioassays resulted in the identification of two resistant phenotypes that were highly HR (Rf: 40-170, based on EC50) and moderately MR (Rf: 10-20) resistant to carbendazim. Cross resistance studies with other fungicides showed that all mutant strains tested were also resistant to other BMZs, such as benomyl and thiabendazole, but retained their parental sensitivity to fungicides belonging to other chemical groups. A point mutation at codon 6 (His6Asn) was found in the β2-tubulin gene of MR isolates while another mutation at codon 200 (Phe200Tyr) was present in one MR and one HR isolates. Interestingly, low temperatures suppressed MBC-resistance in all isolates bearing the H6N mutation. The three-dimensional homology model of the wild-type and mutants of β-tubulins were constructed, and the possible carbendazim binding site was analyzed. Studies on fitness parameters showed that the mutation(s) for resistance to BMZs did not affect the mycelial growth rate whereas adverse effects were found in sporulation and conidial germination in most of the resistant mutants. Pathogenicity tests on corn cobs revealed that mutants were less or equally aggressive to the wild-type strain but expressed their BMZ-resistance after inoculation on maize cobs treated with MBC. Analysis of mycotoxin production by high performance liquid chromatography revealed that only two HR strains produced zearalenone (ZEA) at concentrations similar to that of the wild-type strain, while no ZEA levels were detected in the rest of the mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

Achieving mutations of glycogenic strain Aspergillus awamori using ultraviolet radiation

International Nuclear Information System (INIS)

Rykala-Ziobro, M.; Dluzniewska, J.; Jedrzejowska, A.

1994-01-01

It was attempted to increase glucoamylase productivity of strain Aspergillus awamori, using UV light as a mutagenic agent. To this end, the conidias were twice irradiated, what resulted in obtaining the mutant with about 27% higher glucoamylase activity followed first irradiation of the conidias. Mutant form did not differ morphologically from the initial material (author)

[Mechanism of mutant induction in the ade2 gene of diploid Saccharomyces cerevisiae yeasts by ultraviolet rays].

Science.gov (United States)

Gordenin, D A; Inge-Vechtomov, S G

1981-01-01

Ultraviolet light (UV) at 3000 ergs/mm-2 induces ade2 mutants with a frequency about 10(-4) in wild-type haploid strains of yeast and about 10(-5) in diploid wild-type strains. UV irradiation effectively induced mitotic segregation of ade2 in the heterozygous diploid (the frequency of segregation is 6%). Interallelic complementation and localization spectra are similar for mutations induced both in haploids and diploids. The occurrence of ade2 mutants in diploids correlated with mitotic segregation of the marker his8 which is situated in the same arm of XY chromosome as ade2 is, distal to the centromere. Our data about the frequency of ade2 mutants in diploids and haploids, the frequency of ade2 mitotic segregation, mitotic segregation of other markers and genetic characteristics of ade2 mutations confirm the suggestion that the major mechanism of diploid ade2 mutants appearance is mutation in one of the two ADE2 alleles and consequent mitotic homozygotisation of mutation as a result of mitotic crossingover between ade2 and the centromere.

Morphological mutants of Neurospora crassa: possible evidence of abnormal morphology due to changes in DNA composition

Energy Technology Data Exchange (ETDEWEB)

Chaudhuri, R K; Dutta, S.K. Ojha, M.

1973-01-01

DNA from seven experimentally induced morphological mutants and the wild type strain 74A of Neurospora crassa showed typical bimodal denaturation profiles in a Gilford 2400 spectrophotometer. The ''slime'' and ''ropy'' mutants showed a comparatively high proportion of A + T rich DNA sequences. Studies on thermal denaturation, percent hybridization, and thermal stability indicate the DNA sequences of the slime mutant were distinctly different from the normal genomes of parental DNA as well as other wild type DNAs. No such difference was noticed in any other mutant and natural isolate of the species N. crassa tested. These studies indicate possible correlation between a change in DNA nucleotide sequences and abnormal morphogenesis.

Structure and expression of cytochrome f in an Oenothera plastome mutant.

Science.gov (United States)

Johnson, E M; Sears, B B

1990-06-01

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 2

International Nuclear Information System (INIS)

Serres, F.J. de

1980-01-01

UV-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 7 different UV-sensitive strains and a standard wild-type strain. The 7 strains show varying degrees of sensitivity to UV-induced inactivation, with the relative sensitivity being: uvs-2 > uvs-3 > uvs-4 > uvs-6 > upr-1 > uvs-5 > uvs-1. Studies on the induction of ad-3 mutants by UV show that the 2 excision-repair deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, while uvs-4 and uvs-5 exhibit reduced ad-3 mutant frequencies, and uvs-3 completely eliminates UV mutagenesis. The ad-3 mutation-induction curves obtained with uvs-1 or uvs-6 are not significantly different from that found with the wild-type strain. (orig.)

Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

Science.gov (United States)

Mackenzie, Donald A; Jeffers, Faye; Parker, Mary L; Vibert-Vallet, Amandine; Bongaerts, Roy J; Roos, Stefan; Walter, Jens; Juge, Nathalie

2010-11-01

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere.

Science.gov (United States)

Kristensen, K E; Jacobsen, C S; Hansen, L H; Aamand, J; Morgan, J A W; Sternberg, C; Sørensen, S R

2006-09-01

To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.

Malic acid production by chemically induced Aspergillus niger MTCC 281 mutant from crude glycerol.

Science.gov (United States)

Iyyappan, J; Bharathiraja, B; Baskar, G; Jayamuthunagai, J; Barathkumar, S; Anna Shiny, R

2018-03-01

In the present investigation, crude glycerol derived from transesterification process was utilized to produce the commercially-valuable malic acid. A combined resistant on methanol and malic acid strain of Aspergillus niger MTCC 281 mutant was generated in solid medium containing methanol (1-5%) and malic acid (40-80 g/L) by the adaptation process for 22 weeks. The ability of induced Aspergillus niger MTCC 281 mutant to utilize crude glycerol and pure glycerol to produce malic acid was studied. The yield of malic acid was increased with 4.45 folds compared with that of parent strain from crude glycerol. The highest concentration of malic acid from crude glycerol by using beneficial mutant was found to be 77.38 ± 0.51 g/L after 192 h at 25 °C. This present study specified that crude glycerol by-product from biodiesel production could be used for producing high amount of malic acid without any pretreatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Construction and symbiotic competence of a luxA-deletion mutant of Vibrio fischeri.

Science.gov (United States)

Visick, K G; Ruby, E G

1996-10-10

Bioluminescence by the squid Euprymna scolopes requires colonization of its light organ by the symbiotic luminous bacterium Vibrio fischeri. Investigation of the genetic determinants underlying bacterial symbiotic competence in this system has necessitated the continuing establishment and application of molecular genetic techniques in V. fischeri. We developed a procedure for the introduction of plasmid DNA into V. fischeri by electroporation, and isolated a mutant strain that overcame the apparent restriction barrier between V. fischeri and Escherichia coli. Using the technique of electroporation in combination with that of gene replacement, we constructed a non-luminous strain of V. fischeri (delta luxA::erm). In addition, we used the transducing phage rp-1 for the first time to transfer a chromosomal antibiotic resistance marker to another strain of V. fischeri. The luxA mutant was able to colonize E. scolopes as quickly and to the same extent as wild type. This result suggested that, at least during the initial stages of colonization, luminescence per se is not an essential factor for the symbiotic infection.

CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain.

Science.gov (United States)

Yu, Xiaoqin; Zhang, Chao; Yang, Liping; Zhao, Lamei; Lin, Chun; Liu, Zhengjie; Mao, Zichao

2015-02-10

Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis. Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred

Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation

International Nuclear Information System (INIS)

Verbenko, V.N.; Akhmedov, A.T.; Kalinin, V.L.

1986-01-01

By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gam 444 ad Gam 445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 deg C and are induced more intensively during heat shock (in comparison to the parental) wild-tupe strain AB parallel 57). When the missense htp R15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into genome of the Gam 444 mutant, its enhanced radiation-resistance disappeared but could not be restored upon introduction of pKV3 plasmid bearing the htpR, gene. These data show that heat shock Protens are participating in the enhanced radioresistance of Gam mutants

WNIN/GR-Ob - an insulin-resistant obese rat model from inbred WNIN strain.

Science.gov (United States)

Harishankar, N; Vajreswari, A; Giridharan, N V

2011-09-01

WNIN/GR-Ob is a mutant obese rat strain with impaired glucose tolerance (IGT) developed at the National Institute of Nutrition (NIN), Hyderabad, India, from the existing 80 year old Wistar rat (WNIN) stock colony. The data presented here pertain to its obese nature along with IGT trait as evidenced by physical, physiological and biochemical parameters. The study also explains its existence, in three phenotypes: homozygous lean (+/+), heterozygous carrier (+/-) and homozygous obese (-/-). Thirty animals (15 males and 15 females) from each phenotype (+/+, +/-, -/-) and 24 lean and obese (6 males and 6 females) rats were taken for growth and food intake studies respectively. Twelve adult rats from each phenotype were taken for body composition measurement by total body electrical conductivity (TOBEC); 12 rats of both genders from each phenotype at different ages were taken for clinical chemistry parameters. Physiological indices of insulin resistance were calculated according to the homeostasis model assessment for insulin resistance (HOMA-IR) and also by studying U¹⁴C 2-deoxy glucose uptake (2DG). WNINGR-Ob mutants had high growth, hyperphagia, polydipsia, polyurea, glycosuria, and significantly lower lean body mass, higher fat mass as compared with carrier and lean rats. These mutants, at 50 days of age displayed abnormal response to glucose load (IGT), hyperinsulinaemia, hypertriglyceridaemia, hypercholesterolaemia and hyperleptinaemia. Basal and insulin-stimulated glucose uptakes by diaphragm were significantly decreased in obese rats as compared with lean rats. Obese rats of the designated WNIN/GR-Ob strain showed obesity with IGT, as adjudged by physical, physiological and biochemical indices. These indices varied among the three phenotypes, being lowest in lean, highest in obese and intermediate in carrier phenotypes thereby suggesting that obesity is inherited as autosomal incomplete dominant trait in this strain. This mutant obese rat model is easy to

Strain improvement in dye decolourising mutants of Mucor mucedo ...

African Journals Online (AJOL)

The fusant MMFu3 showed very good increase in the production of three enzymes protease (1.90 U/ml), peroxidase (1100 U/ml) and laccase (200 U/ml) when compared to the two parent strains proving that the higher enzymatic secretions are responsible for the decolourisation activity. In protease isozyme analysis, fusants ...

Different action of MMS and EMS in UV-sensitive strains of Aspergillus nidulans.

Science.gov (United States)

Babudri, N; Politi, M G

1989-05-01

The repair of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) damages has been investigated in the fungus Aspergillus nidulans. 4 UV-sensitive mutants, namely uvsB, uvsD, uvsF and uvsH have been tested for their sensitivity and mutability to the above-mentioned agents. The results obtained show that: (1) uvsB and uvsD mutants are no more sensitive than the wild-type strain to the lethal action of EMS. In contrast, they are more sensitive to MMS; (2) uvsF and uvsH mutants are more sensitive than the wild type to EMS at 37 degrees C but not at 20 degrees C. However, they are more sensitive than the wild type to MMS at 37 degrees C as well as at 20 degrees C; (3) the mutation frequencies after treatment with either MMS or EMS plotted against survival are not altered in the UV-sensitive strains compared to the wild-type strain. From these data it may be concluded that the repair of lethal lesions induced by ethylating and methylating agents is under the control of different pathways. Furthermore the mutants tested are not involved in the mutagenic process.

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies.

Science.gov (United States)

Noguchi, Hiroki; Mylemans, Bram; De Zitter, Elke; Van Meervelt, Luc; Tame, Jeremy R H; Voet, Arnout

2018-03-18

β-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Spirogyra varians mutant generated by high dose gamma-irradiation shows increased antioxidant properties

International Nuclear Information System (INIS)

Lee, Hak-Jyung; Yoon, Minchul; Sung, Nak-Yun; Choi, Jong-il

2012-01-01

The aim of this study was to evaluate the antioxidant properties of a Spirogyra varians mutant (Mut) produced by gamma irradiation. Methanol extracts were prepared from Spirogyra varians wild-type and Mut plants, and their antioxidant activities and total phenolic content (TPC) were determined. Antioxidant parameters, including the 2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferric-reducing/antioxidant power, were higher in the Mut extract. Moreover, the TPC level was higher (P<0.05) in the Mut methanol extract. Therefore, these results suggest that gamma irradiation-induced S. varians Mut has superior antioxidant properties. - Highlights: ► The antioxidative properties of a Spirogyra varians mutant produced by gamma-irradiation was investiated. ► The antioxidant activities and total phenolic content levels were higher in mutant strain. ► These results suggest that gamma-irradiation induced algae mutant with superior antioxidant properties.

Isolation of a high malic and low acetic acid-producing sake yeast Saccharomyces cerevisiae strain screened from respiratory inhibitor 2,4-dinitrophenol (DNP)-resistant strains.

Science.gov (United States)

Kosugi, Shingo; Kiyoshi, Keiji; Oba, Takahiro; Kusumoto, Kenichi; Kadokura, Toshimori; Nakazato, Atsumi; Nakayama, Shunichi

2014-01-01

We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+). Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The use of flagella and motility for plant colonization and fitness by different strains of the foodborne pathogen Listeria monocytogenes.

Directory of Open Access Journals (Sweden)

Lisa Gorski

Full Text Available The role of flagella and motility in the attachment of the foodborne pathogen Listeria monocytogenes to various surfaces is mixed with some systems requiring flagella for an interaction and others needing only motility for cells to get to the surface. In nature this bacterium is a saprophyte and contaminated produce is an avenue for infection. Previous studies have documented the ability of this organism to attach to and colonize plant tissue. Motility mutants were generated in three wild type strains of L. monocytogenes by deleting either flaA, the gene encoding flagellin, or motAB, genes encoding part of the flagellar motor, and tested for both the ability to colonize sprouts and for the fitness of that colonization. The motAB mutants were not affected in the colonization of alfalfa, radish, and broccoli sprouts; however, some of the flaA mutants showed reduced colonization ability. The best colonizing wild type strain was reduced in colonization on all three sprout types as a result of a flaA deletion. A mutant in another background was only affected on alfalfa. The third, a poor alfalfa colonizer was not affected in colonization ability by any of the deletions. Fitness of colonization was measured in experiments of competition between mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants of the three strain backgrounds were impaired in fitness of colonization of alfalfa and radish sprouts, and one strain background showed reduced fitness of both mutant types on broccoli sprouts. Together these data indicate a role for flagella for some strains to physically colonize some plants, while the fitness of that colonization is positively affected by motility in almost all cases.

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

OpenAIRE

Raychaudhuri, D; Chatterjee, A N

1985-01-01

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin...

Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

Science.gov (United States)

McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

2016-02-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

Science.gov (United States)

Ghezelbash, Gholam Reza; Nahvi, Iraj; Emamzadeh, Rahman

2014-08-01

The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

Design of cross-sensitive temperature and strain sensor based on sampled fiber grating

Directory of Open Access Journals (Sweden)

Zhang Xiaohang

2017-02-01

Full Text Available In this paper,a cross-sensitive temperature and strain sensor based on sampled fiber grating is designed.Its temperature measurement range is -50-200℃,and the strain measurement rangeis 0-2 000 με.The characteristics of the sensor are obtained using simulation method.Utilizing SPSS software,we found the dual-parameter matrix equations of measurement of temperature and strain,and calibrated the four sensing coefficients of the matrix equations.

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata.

Science.gov (United States)

Fan, Lili; Fu, Kehe; Yu, Chuanjin; Ma, Jia; Li, Yaqian; Chen, Jie

2014-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.

Novel strategy to improve vanillin tolerance and ethanol fermentation performances of Saccharomycere cerevisiae strains.

Science.gov (United States)

Zheng, Dao-Qiong; Jin, Xin-Na; Zhang, Ke; Fang, Ya-Hong; Wu, Xue-Chang

2017-05-01

The aim of this work was to develop a novel strategy for improving the vanillin tolerance and ethanol fermentation performances of Saccharomyces cerevisiae strains. Isogeneic diploid, triploid, and tetraploid S. cerevisiae strains were generated by genome duplication of haploid strain CEN.PK2-1C. Ploidy increments improved vanillin tolerance and diminished proliferation capability. Antimitotic drug methyl benzimidazol-2-ylcarbamate (MBC) was used to introduce chromosomal aberrations into the tetraploid S. cerevisiae strain. Interestingly, aneuploid mutants with DNA contents between triploid and tetraploid were more resistant to vanillin and showed faster ethanol fermentation rates than all euploid strains. The physiological characteristics of these mutants suggest that higher bioconversion capacities of vanillin and ergosterol contents might contribute to improved vanillin tolerance. This study demonstrates that genome duplication and MBC treatment is a powerful strategy to improve the vanillin tolerance of yeast strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bio-succinic acid production: Escherichia coli strains design from genome-scale perspectives

Directory of Open Access Journals (Sweden)

Bashir Sajo Mienda

2017-10-01

Full Text Available Escherichia coli (E. coli has been established to be a native producer of succinic acid (a platform chemical with different applications via mixed acid fermentation reactions. Genome-scale metabolic models (GEMs of E. coli have been published with capabilities of predicting strain design strategies for the production of bio-based succinic acid. Proof-of-principle strains are fundamentally constructed as a starting point for systems strategies for industrial strains development. Here, we review for the first time, the use of E. coli GEMs for construction of proof-of-principles strains for increasing succinic acid production. Specific case studies, where E. coli proof-of-principle strains were constructed for increasing bio-based succinic acid production from glucose and glycerol carbon sources have been highlighted. In addition, a propose systems strategies for industrial strain development that could be applicable for future microbial succinic acid production guided by GEMs have been presented.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

Science.gov (United States)

Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

2016-07-01

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

A novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12.

Science.gov (United States)

Miki, Takeyoshi; Yamamoto, Yoshihiro; Matsuda, Hideo

2008-01-01

We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then transfer the disrupted genes from the lambda vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Km(r) cis-diploid mutants harboring a mini-Tn10 insertion mutation and the corresponding wild-type gene on a chromosome, as well as a set of 6954 haploid mutants derived from the cis-diploid mutants. The major advantage of the strategy used is that the indispensable genes or sites for growth can be identified. Preliminary results suggest that 415 open reading frames are indispensable for growth in E. coli cells. A total of 6404 haploid mutants were deposited to Genetic Strains Research Center, National Institute of Genetics, Japan (Chapter 26) and are available for public distribution upon request (http://shigen.lab.nig.ac.jp/ecoli/strain/nbrp/resource.jsp).

Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

Energy Technology Data Exchange (ETDEWEB)

Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

1990-01-01

Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant derivative.

Science.gov (United States)

Lewis, R J; Krulwich, T A; Reynafarje, B; Lehninger, A L

1983-02-25

Obligately alkalophilic Bacillus firmus RAB had a higher molar growth yield on L-malate (Ymal = 38 mg, dry weight/mmol of L-malate) than its non-alkalophilic mutant derivative, strain RABN (Ymal = 12 mg, dry weight/mmol of L-malate). Measurements of respiration dependent proton translocation by the two strains in the presence of K+ and valinomycin showed that the alkalophile also has much higher H+/O stoichiometries (at pH 9.0) than does the mutant (at pH 7.0). H+/O ratios for B. firmus RAB at pH 9.0 were as high as 13, with a frequently observed value of 9. These high values were observed in the first phase of a set of biphasic curves for both oxygen consumption and proton ejection. At pH 7.0, both the wild type and the mutant exhibited H+/O ratios near 4 in a single phase of oxygen consumption and proton ejection. The results are consistent with suggestions that the alkalophilic respiratory chain is especially well adapted for effective energy transduction at alkaline but not neutral pH.

Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Ikai, K.; Tano, K.; Ohnishi, T.; Nozu, K.

1985-01-01

The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

Science.gov (United States)

Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

2014-09-01

Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions.

Science.gov (United States)

Costa, Suelen B; Campos, Ana Carolina C; Pereira, Ana Claudia M; de Mattos-Guaraldi, Ana Luiza; Júnior, Raphael Hirata; Rosa, Ana Cláudia P; Asad, Lídia M B O

2014-09-01

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the

Wireless implantable passive strain sensor: design, fabrication and characterization

International Nuclear Information System (INIS)

Umbrecht, F; Wägli, P; Dechand, S; Hierold, Ch; Gattiker, F; Neuenschwander, J; Sennhauser, U

2010-01-01

This work presents a new passive sensor concept for monitoring the deformation of orthopedic implants. The novel sensing principle of the WIPSS (wireless implantable passive strain sensor) is based on a hydro-mechanical amplification effect. The WIPSS is entirely made from biocompatible PMMA and consists of a microchannel attached to a reservoir, which is filled with an incompressible fluid. As the reservoir is exposed to strain, its volume changes and consequently the fill level inside the microchannel varies. The wireless detection of the microchannel's strain-dependent fill level is based on ultrasound. The WIPSS' sensing principle is proved by finite-element simulations and the reservoir's design is optimized toward maximum volume change, in order to achieve high sensitivity. A fabrication process for WIPSS sensor devices entirely made from PMMA is presented. The obtained measurement results confirmed the sensor's functionality and showed very good agreement with the obtained results of the conducted FE simulations regarding the sensor's sensitivity. A strain resolution of 1.7 ± 0.2 × 10 −5 was achieved. Further, the determination of the cross-sensitivity to temperature and strains applied out of the sensing direction is presented. The response to dynamic inputs (0.1–5 Hz) has been measured and showed decreasing sensor output with increasing frequency. Test structures of the sensor device allow the application of a signal bandwidth up to 1 Hz. Therefore, the proposed sensor concept of the WIPSS presents a promising new sensor system for static in vivo strain monitoring of orthopedic implants. In combination with the developed ultrasound-based read-out method, this new sensor system offers the potential of wireless sensor read-out with medical ultrasound scanners, which are commercially available.

Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

Energy Technology Data Exchange (ETDEWEB)

Ely, Roger L.; Chaplen, Frank W.R.

2014-03-11

enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production.

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants.

Science.gov (United States)

Käfer, E; Mayor, O

1986-07-01

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV

Furfural and hydroxymethylfurfural tolerance in Escherichia coli ΔacrR regulatory mutants.

Science.gov (United States)

Luhe, Annette Lin; Lim, Chan Yuen; Gerken, Henri; Wu, Jinchuan; Zhao, Hua

2015-01-01

The presence of the highly toxic furfural and hydroxymethylfurfural (HMF) in the hydrolysate of lignocellulosic biomass prompted the investigation of the Escherichia coli ΔacrR regulatory mutant for higher tolerance to these compounds, to facilitate the production of biofuels and biochemicals, and further biocatalytic conversions. In comparison with the parental strain, the regulatory mutant with the upregulated efflux pump AcrAB-TolC produced moderately better growth and higher tolerance to concentrations of furfural and HMF between 1 and 2 g L(-1) . © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002.

Science.gov (United States)

Therien, Jesse B; Zadvornyy, Oleg A; Posewitz, Matthew C; Bryant, Donald A; Peters, John W

2014-01-01

The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002. Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability. The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

Science.gov (United States)

Ruiz, Lorena; Motherway, Mary O'Connell; Lanigan, Noreen; van Sinderen, Douwe

2013-01-01

Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

Directory of Open Access Journals (Sweden)

Lorena Ruiz

Full Text Available Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Role of ArlRS in autolysis in methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains.

Science.gov (United States)

Memmi, Guido; Nair, Dhanalakshmi R; Cheung, Ambrose

2012-02-01

Autolysis plays an essential role in bacterial cell division and lysis with β-lactam antibiotics. Accordingly, the expression of autolysins is tightly regulated by several endogenous regulators, including ArlRS, a two component regulatory system that has been shown to negatively regulate autolysis in methicillin-sensitive Staphylococcus aureus (MSSA) strains. In this study, we found that inactivation of arlRS does not play a role in autolysis of methicillin-resistant S. aureus (MRSA) strains, such as community-acquired (CA)-MRSA strains USA300 and MW2 or the hospital-acquired (HA)-MRSA strain COL. This contrasts with MSSA strains, including Newman, SH1000, RN6390, and 8325-4, where autolysis is affected by ArlRS. We further demonstrated that the striking difference in the roles of arlRS between MSSA and MRSA strains is not due to the methicillin resistance determinant mecA. Among known autolysins and their regulators, we found that arlRS represses lytN, while no effect was seen on atl, lytM, and lytH expression in both CA- and HA-MRSA strains. Transcriptional-fusion assays showed that the agr transcripts, RNAII and RNAIII, were significantly more downregulated in the arlRS mutant of MW2 than the MSSA strain Newman. Importantly, provision of agr RNAIII in trans to the MW2 arlRS mutant via a multicopy plasmid induced autolysis in this MRSA strain. Also, the autolytic phenotype in the arlRS mutant of MSSA strain Newman could be rescued by a mutation in either atl or lytM. Together, these data showed that ArlRS impacts autolysis differently in MSSA and MRSA strains.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

Science.gov (United States)

Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

2017-03-01

Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017

Yersinia enterocolitica YopH-Deficient Strain Activates Neutrophil Recruitment to Peyer's Patches and Promotes Clearance of the Virulent Strain.

Science.gov (United States)

Dave, Mabel N; Silva, Juan E; Eliçabe, Ricardo J; Jeréz, María B; Filippa, Verónica P; Gorlino, Carolina V; Autenrieth, Stella; Autenrieth, Ingo B; Di Genaro, María S

2016-11-01

Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effect of Bacillus subtilis mutants on growth performance of piglets fed tryptophan- and valine-deficient diets

DEFF Research Database (Denmark)

Nørgaard, Jan Værum; Canibe, Nuria; Assadi Soumeh, Elham

2016-01-01

The objective was to determine the concentration of l-Trp and l-Val to be substituted by feeding piglets Bacillus subtilis strains developed to overproduce Trp (B. subtilis Trp mutant [BsTrp]) and Val (B. subtilis Val mutant [BsVal]) and by using equations obtained in 3 dose–response studies......-Val per kilogram feed using curvilinear plateau and broken-line equations obtained by modeling the 6 AA levels. Bacillus subtilis Val mutant increased animal performance corresponding to 0.88 and 0.39 g l-Leu and 0.17 and 0.44 g l-Val per kilogram feed for 10x and 100x doses, respectively. Bacillus...... subtilis Trp mutant was equivalent to 0.02 and 0.11 g l-Trp/kg feed for 10x and 100x doses, respectively. Bacillus subtilis Val mutant (10x dose) increased (P Bacillus subtilis Trp mutant tended (P = 0.06) to increase Trp plasma concentrations...

Mutant breeding of ornamental trees for creating variations with high value using Proton Beam

International Nuclear Information System (INIS)

Kwon, H. J.; Lim, J. H.; Woo, S. M.; Hwang, M. J.; Pyo, S. H.; Woo, J. S.

2009-04-01

It is necessary to induce the improved strains of ornamental plants with more disease-resistant and useful for landscape or phytoremediation. Mutation breeding has played an important role in crop improvement, and more than 2,000 mutant cultivars have been released. For the induction of mutation, gamma rays and X-rays are widely used as a mutagen. Proton beam had higher energy than -ray and worked with localized strength, so that proton-beam radiation could be valuable tool to induce useful strains of ornamental plants. Proton ion beam irradiation was used to induce a useful mutant in rice, chrysanthemum, carnation, and so on in Japan. Also, proton ion beam was used to select a useful host strain, in polyhydroxybutyrate (PHB), a member of biodegradable plastic, could be overproduced in Korea. Therefore, we surmise that the effects of proton beam is different from those of gamma rays and X-rays, and we expect proton beam to be a new mutagen. This research was conducted to investigate the proton-beam radiation sensitivity and seed germination rate of the various ornamental plants like as Albizia julibrissin, Ficus religiosa, Rhus chinensis, Sorbaria sorbilfolia and Spiraea chinensis, to survey the quantitative characteristics of proton beam induced strains. To induce the variants of ornamental plants, seeds were irradiated at the dose of 0∼2kGy of proton beam at room temperature. Proton beam energy level was 45 MeV and was irradiated at dose of 0∼2kGy by MC-50 Cyclotron. After irradiation, to assess the effects of proton beam on radiation sensitivity and morphological changes of the plants and the seed germination rate were analysed. By the proton beam radiation, the germination rate decreased at the higher dose. The other hand, the germination rate of Rhus chinensis increased the dose higher, so that it need to investigate the germination rate over 2kGy radiation. The effects of mutation induction by proton beam irradiation on seeds in Lagerstroemia indica were

Mutant breeding of ornamental trees for creating variations with high value using Proton Beam

Energy Technology Data Exchange (ETDEWEB)

Kwon, H. J.; Lim, J. H.; Woo, S. M.; Hwang, M. J.; Pyo, S. H.; Woo, J. S. [Phygen Co., Daejeon (Korea, Republic of)

2009-04-15

It is necessary to induce the improved strains of ornamental plants with more disease-resistant and useful for landscape or phytoremediation. Mutation breeding has played an important role in crop improvement, and more than 2,000 mutant cultivars have been released. For the induction of mutation, gamma rays and X-rays are widely used as a mutagen. Proton beam had higher energy than -ray and worked with localized strength, so that proton-beam radiation could be valuable tool to induce useful strains of ornamental plants. Proton ion beam irradiation was used to induce a useful mutant in rice, chrysanthemum, carnation, and so on in Japan. Also, proton ion beam was used to select a useful host strain, in polyhydroxybutyrate (PHB), a member of biodegradable plastic, could be overproduced in Korea. Therefore, we surmise that the effects of proton beam is different from those of gamma rays and X-rays, and we expect proton beam to be a new mutagen. This research was conducted to investigate the proton-beam radiation sensitivity and seed germination rate of the various ornamental plants like as Albizia julibrissin, Ficus religiosa, Rhus chinensis, Sorbaria sorbilfolia and Spiraea chinensis, to survey the quantitative characteristics of proton beam induced strains. To induce the variants of ornamental plants, seeds were irradiated at the dose of 0{approx}2kGy of proton beam at room temperature. Proton beam energy level was 45 MeV and was irradiated at dose of 0{approx}2kGy by MC-50 Cyclotron. After irradiation, to assess the effects of proton beam on radiation sensitivity and morphological changes of the plants and the seed germination rate were analysed. By the proton beam radiation, the germination rate decreased at the higher dose. The other hand, the germination rate of Rhus chinensis increased the dose higher, so that it need to investigate the germination rate over 2kGy radiation. The effects of mutation induction by proton beam irradiation on seeds in Lagerstroemia

A Francisella tularensis live vaccine strain that improves stimulation of antigen-presenting cells does not enhance vaccine efficacy.

Directory of Open Access Journals (Sweden)

Deanna M Schmitt

Full Text Available Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS, does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

Directory of Open Access Journals (Sweden)

Roland Züst

Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Development and selection of fungal and bacterial mutants using ionizing radiation and radioisotopes for improved enzyme production (cellulase and coagulase)

International Nuclear Information System (INIS)

Markov, K.I.

1975-01-01

Ultraviolet and gamma radiations, chemical mutagens, and combinations of chemical and physical mutagens were used in order to obtain mutants of Bacillus mesentericus and Trichoderma viridae with a higher production of coagulase and cellulase, respectively. It was possible to isolate mutant strains, with enzyme activity increased by a factor of 2 and 3

A comparative study concerning the pigments from two Monascus strains obtained by radio-mutagenesis

International Nuclear Information System (INIS)

Ferdes, M.; Mencinicopschi, G.; Ferdes, O.

1995-01-01

Monascus ruber ICA 3.250 spores suspension was gamma-irradiated at a Co-60 gamma-ray source at doses D=1 to 10 kGy. After dilution it has drown the survival curve and there were isolated two mutant strains, M 1 and M 2 . These were investigated with regard to food red pigment production on solid and liquid submerged (with aeration and stirring) culture media. In comparison with the parental (control strain, the two mutant strains M 1 and M 2 show morphological and physiological modified characteristics which led both to the increase in pigment concentration in culture media and in the reduction of biosynthesis duration. (Author) 3 Figs., 2 Tabs., 11 Refs

Genetic dependence of recombination in recD mutants of Escherichia coli

International Nuclear Information System (INIS)

Lovett, S.T.; Luisi-DeLuca, C.; Kolodner, R.D.

1988-01-01

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations

PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

Science.gov (United States)

Foulquier, Elodie; Pompeo, Frédérique; Freton, Céline; Cordier, Baptiste; Grangeasse, Christophe; Galinier, Anne

2014-08-22

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

In Vivo Differences in the Virulence, Pathogenicity, and Induced Protective Immunity of wboA Mutants from Genetically Different Parent Brucella spp.

Science.gov (United States)

Wang, Zhen; Niu, Jianrui; Wang, Shuangshan

2013-01-01

To explore the effects of the genetic background on the characteristics of wboA gene deletion rough mutants generated from different parent Brucella sp. strains, we constructed the rough-mutant strains Brucella melitensis 16 M-MB6, B. abortus 2308-SB6, B. abortus S19-RB6, and B. melitensis NI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks for B. abortus S19-RB6 to 11 weeks for B. abortus 2308-SB6 and B. melitensis NI-NB6. However, B. abortus S19-RB6 and B. melitensis 16 M-MB6, with a shorter survival time in mice, offered better protection against challenges with B. abortus 2308 in protection tests than B. abortus 2308-SB6 and B. melitensis NI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival time in vivo. In the cross-protection assay, both B. melitensis 16 M-MB6 and B. abortus S19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated with B. abortus S19-RB6 and B. melitensis 16 M-MB6, B. abortus S19-RB6 did not induce abortion, whereas B. melitensis 16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunity in vivo in the wboA deletion mutants from genetically different parent Brucella spp. and also indicated that future rough vaccine strain development could be promising if suitable parent Brucella strains and/or genes were selected. PMID:23239800

Survival and mutant production induced by mutagenic agents in Metarhizium anisopliae Sobrevivência e obtenção de mutantes induzidos por agentes mutagênicos em Metarhizium anisopliae

Directory of Open Access Journals (Sweden)

V. Kava - Cordeiro

1995-12-01

Full Text Available A wild strain of Metarhizium anisopliae, an entomopathogenic fungus, was submitted to three mutagenic agents: gamma radiation, ultraviolet light and nitrous acid. Survival curves were obtained and mutants were selected using different mutagenic doses which gave 1 to 5% survival. Morphological and auxotrophic mutants were isolated. Morphological mutants were grouped in a class with yellow conidia and other with pale vinaceous conidia as opposed to the green wild type conidia. Auxotrophic mutants had requirements for vitamin and aminoacid biosynthesis. More than 58% of the total auxotrophk mutants required proline/aipnine. Gamma radiation showed to be the most efficient mutagenic agent giving 0.2% of auxotrophk mutants followed by ultraviolet light (0.12% and nitrous acid (0.06%.The conidial colour and auxotrophk mutants isolated until now from M. anisopliae were reviewed.Uma linhagem selvagem do fungo entomopatogênico Metarhizium anisopliae foi submetida à ação de três agentes mutagênicos: radiação gama, luz ultravioleta e ácido nitroso. Curvas de sobrevivência foram obtidas para cada mutagênicos utilizado e mutantes foram selecionados a partir de doses dos mutagênicos que proporcionassem de 1 a 5% de sobrevivência. Mutantes morfológicos para a coloração de conídios e mutantes auxotróficos foram isolados. Mutantes para coloração de conidios foram agrupados em duas classes, uma com conídios amarelos e outra com conídios vinho pálido. Os mutantes auxotróficos obtidos foram deficientes para aminoácidos e vitaminas e mais de 58% deles eram auxotróficos para prolina/argmina. Radiação gama foi o mutagênico mais eficiente com uma porcentagem de obtenção de mulantes auxotróficos de aproximadamente 0,2%, seguido pela luz ultravioleta (0.12% e pelo ácido nitroso (0.06%.Os mulantes morfológicos e auxotróficos obtidos até o momento em Metarhizium anisopliae foram revistos.

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis.

Science.gov (United States)

Bozue, Joel; Mou, Sherry; Moody, Krishna L; Cote, Christopher K; Trevino, Sylvia; Fritz, David; Worsham, Patricia

2011-06-01

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis. Published by Elsevier India Pvt Ltd.

Rational design of botulinum neurotoxin A1 mutants with improved oxidative stability.

Science.gov (United States)

López de la Paz, Manuela; Scheps, Daniel; Jurk, Marcel; Hofmann, Fred; Frevert, Jürgen

2018-06-01

Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT-containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell. Stabilization of the LC by reducing vulnerability towards denaturants would thus be advantageous for the development of BoNT-based therapeutics. In this work, we focused on increasing the stability of LC of BoNT/A1 (LC/A1) towards oxidative stress. We tackled this task by rational design of mutations at cysteine and methionine LC/A1 sites. Designed mutants showed improved oxidative stability in vitro and equipotency to wildtype toxin in vivo. Our results suggest that suitable modification of the catalytic domain can lead to more stable BoNTs without impairing their therapeutic efficacy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

Energy Technology Data Exchange (ETDEWEB)

Xinle, Liang; Qian, Shou; Hong, Zhang; Min, Chen [Department of Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang (China); Xuan, Liu [Beihai Institute of Environmental Science, Beihai, Guangxi (China)

2009-08-15

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with {sup 60}Co {gamma}-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6 (nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

International Nuclear Information System (INIS)

Liang Xinle; Shou Qian; Zhang Hong; Chen Min; Liu Xuan

2009-01-01

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with 60 Co γ-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6( nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

Science.gov (United States)

Navon, G; Shulman, R G; Yamane, T; Eccleshall, T R; Lam, K B; Baronofsky, J J; Marmur, J

1979-10-16

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.

The Multiple Localized Glyceraldehyde-3-Phosphate Dehydrogenase Contributes to the Attenuation of the Francisella tularensis dsbA Deletion Mutant

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Ivona Pavkova

2017-12-01

Full Text Available The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

Science.gov (United States)

Anisimov, Andrey P; Bakhteeva, Irina V; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Kravchenko, Tat'yana B; Platonov, Mikhail E; Titareva, Galina M; Kombarova, Tat'yana I; Ivanov, Sergey A; Rakin, Alexander V; Amoako, Kingsley K; Dentovskaya, Svetlana V

2009-01-01

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

Incomplete excision repair process after UV-irradiation in MUT-mutants of Proteus mirabillis

International Nuclear Information System (INIS)

Stoerl, K.

1977-01-01

MUT-mutants of P. mirabilis seem to be able to perform the incision step in the course of excision repair. In contrast to the corresponding wildtype strains with MUT-mutants the number of single-strand breaks formed after UV-irradiation is independent of the UV-dose up to about 720 erg/mm 2 . Incubation in minimal medium over a longer time does not result in completion of excision repair; about 3-6 single-strand breaks in the DNA of these mutants remain open. Likewise, the low molecular weight of the newly synthesized daughter DNA confirms an incompletely proceeding or delayed repair process. As a possible reason for the mutator phenotype an alteration of the DNA-polymerase playing a role in excision and resynthesis steps of excision repair is discussed. (author)

Design and analysis of MEMS MWCNT / epoxy strain sensor using ...

Indian Academy of Sciences (India)

Gaurav Sapra

2017-06-20

Jun 20, 2017 ... In this paper, highly sensitive MEMS-based multi- walled (MWCNT)/epoxy strain sensor has been designed using ... This paper also discusses the process flow for fabricating MWCNT/epoxy thin film ... stone bridge, i.e., connected to the gold metal pad of the sensor. The change in resistance with respect to.

Influence of the protein kinase C activator phorbol myristate acetate on the intracellular activity of antibiotics against hemin- and menadione-auxotrophic small-colony variant mutants of Staphylococcus aureus and their wild-type parental strain in human THP-1 cells.

Science.gov (United States)

Garcia, Laetitia G; Lemaire, Sandrine; Kahl, Barbara C; Becker, Karsten; Proctor, Richard A; Tulkens, Paul M; Van Bambeke, Françoise

2012-12-01

In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700-3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H(2)O(2). In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H(2)O(2). Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

Science.gov (United States)

Hama, S; Kimura, G

1980-01-01

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A

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Jianli Wang

2014-03-01

Full Text Available 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo2-lipid A is the conserved structure domain of lipopolysaccharide found in most Gram-negative bacteria, and it is believed to stimulate the innate immune system through the TLR4/MD2 complex. Therefore, Kdo2-lipid A is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants. Kdo2-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain, WBB06. WBB06 cells grow slowly and have to grow in the presence of tetracycline. In this study, a novel E. coli mutant strain, WJW00, that could synthesize Kdo2-lipid A was constructed by deleting the rfaD gene from the genome of E. coli W3110. The rfaD gene encodes ADP-l-glycero-d-manno-heptose-6-epimerase RfaD. Based on the analysis by SDS-PAGE, thin layer chromatography (TLC and electrospray ionization mass spectrometry (ESI/MS, WJW00 could produce similar levels of Kdo2-lipid A to WBB06. WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth. Compared with the wild-type strain, W3110, WJW00 showed increased hydrophobicity, higher cell permeability, greater autoaggregation and decreased biofilm-forming ability. Therefore, WJW00 could be a more suitable strain than WBB06 for producing Kdo2-lipid A and a good base strain for developing lipid A adjuvants.

Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

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Christopher M Brennan

Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

OpenAIRE

Dri, A M; Rouviere-Yaniv, J; Moreau, P L

1991-01-01

Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The...

Improvement of xylanase production by a parasexual cross between Aspergillus niger strains

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Octavio Loera

2003-03-01

Full Text Available A diploid strain (D4 isolated via parasexual recombination between two Aspergillus niger xylanase overproducing mutants was characterised in terms of enzyme production and catabolite repression by glucose. This strain increased xylanase production (607 nkat/ml, which was nearly 100% higher than titers achieved by the wild type strain (305 nkat/ml and 28% higher than the best mutant used to induce parasexual cycle. Diploid D4 was also less sensitive to carbon catabolite repression by glucose, since xylanolytic activity was detected under conditions normally repressing production by the wild type strain. No decrease in maximal xylanase levels was observed in the presence of glucose for diploid D4.Um cepa diplóide (D4 isolada por combinação parasexual entre dois Aspergillus niger, mutantes superprodutores de xylanase foi caracterizado através da produção de (607 nkat/ml e repressão catabólica por glicose. Essa cepa aumenta a produção de xylanase em mais de 100% em comparação com uma cepa selvagem (305 nkat/ml e 28% superior do que o melhor mutante usado para induzir o ciclo parasexual. A cepa diplóide D4 foi também menos sensível a repressão catabólica pela glicose, sendo que a atividade xylanolitica foi detectada sob condições normalmente de produção repressiva pela cepa selvagem. Não foi observado um decréscimo na produção máxima de xylanase em presença de glicose para o diplóide D4.

Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice.

Science.gov (United States)

Lalsiamthara, Jonathan; Gogia, Neha; Goswami, Tapas K; Singh, R K; Chaudhuri, Pallab

2015-05-21

Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Energy Technology Data Exchange (ETDEWEB)

Brown, Steven D [ORNL; Guss, Adam M [ORNL; Karpinets, Tatiana V [ORNL; Parks, Jerry M [ORNL; Smolin, Nikolai [ORNL; Yang, Shihui [ORNL; Land, Miriam L [ORNL; Klingeman, Dawn Marie [ORNL; Bhandiwad, Ashwini [Thayer School of Engineering at Dartmouth; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Shao, Xiongjun [Thayer School of Engineering at Dartmouth; Mielenz, Jonathan R [ORNL; Smith, Jeremy C [ORNL; Keller, Martin [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

2011-01-01

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

Branched-chain fatty acid biosynthesis in a branched-chain amino acid aminotransferase mutant of Staphylococcus carnosus

DEFF Research Database (Denmark)

Beck, Hans Christian

2005-01-01

Fatty acid biosynthesis by a mutant strain of Staphylococcus carnosus deficient in branched-chain amino acid aminotransferase (IlvE) activity was analysed. This mutant was unable to produce the appropriate branched-chain alpha-ketoacid precursors for branched-chain fatty acid biosynthesis from...... in rich medium and growth in defined medium supplemented with 2-methylpropanoic acid lead to extensive alteration of the fatty acid composition in the cell membrane. In rich medium, a change from 51.7% to 17.1% anteiso-C15:0, and from 3.6% to 33.9% iso-C14:0 fatty acids as compared to the wild-type strain...... for 2-methylpropanoic acid production, revealing that the IlvE protein plays an important, but not essential role in the biosynthesis of branched-chain fatty acids and secondary metabolites in S. carnosus....

Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

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Bogumil Jacek Karas

2014-07-01

Full Text Available With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in

Design of homo-organic acid producing strains using multi-objective optimization

DEFF Research Database (Denmark)

Kim, Tae Yong; Park, Jong Myoung; Kim, Hyun Uk

2015-01-01

Production of homo-organic acids without byproducts is an important challenge in bioprocess engineering to minimize operation cost for separation processes. In this study, we used multi-objective optimization to design Escherichia coli strains with the goals of maximally producing target organic ...

The mutation studies of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts

International Nuclear Information System (INIS)

Schultz, R.A.; Chang, C.C.; Trosko, J.E.

1981-01-01

We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to the parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis

Improved Biosurfactant Production by Bacillus subtilis SPB1 Mutant Obtained by Random Mutagenesis and Its Application in Enhanced Oil Recovery in a Sand System.

Science.gov (United States)

Bouassida, Mouna; Ghazala, Imen; Ellouze-Chaabouni, Semia; Ghribi, Dhouha

2018-01-28

Biosurfactants or microbial surfactants are surface-active biomolecules that are produced by a variety of microorganisms. Biodegradability and low toxicity have led to the intensification of scientific studies on a wide range of industrial applications for biosurfactants in the field of environmental bioremediation as well as the petroleum industry and enhanced oil recovery. However, the major issues in biosurfactant production are high production cost and low yield. Improving the bioindustrial production processes relies on many strategies, such as the use of cheap raw materials, the optimization of medium-culture conditions, and selecting hyperproducing strains. The present work aims to obtain a mutant with higher biosurfactant production through applying mutagenesis on Bacillus subtilis SPB1 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on blood agar and subsequent formation of halos, the mutated strains were examined for emulsifying activity of their culture broth. A mutant designated B. subtilis M2 was selected as it produced biosurfactant at twice higher concentration than the parent strain. The potential of this biosurfactant for industrial uses was shown by studying its stability to environmental stresses such as pH and temperature and its applicability in the oil recovery process. It was practically stable at high temperature and at a wide range of pH, and it recovered above 90% of motor oil adsorbed to a sand sample.

Strain response of stretchable micro-electrodes: Controlling sensitivity with serpentine designs and encapsulation

International Nuclear Information System (INIS)

Gutruf, Philipp; Walia, Sumeet; Nur Ali, Md; Sriram, Sharath; Bhaskaran, Madhu

2014-01-01

The functionality of flexible electronics relies on stable performance of thin film micro-electrodes. This letter investigates the behavior of gold thin films on polyimide, a prevalent combination in flexible devices. The dynamic behavior of gold micro-electrodes has been studied by subjecting them to stress while monitoring their resistance in situ. The shape of the electrodes was systematically varied to examine resistive strain sensitivity, while an additional encapsulation was applied to characterize multilayer behavior. The realized designs show remarkable tolerance to repetitive strain, demonstrating that curvature and encapsulation are excellent approaches for minimizing resistive strain sensitivity to enable durable flexible electronics

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

Science.gov (United States)

Costa, José M.; Loper, Joyce E.

1994-01-01

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out- mutants of E. carotovora subsp. carotovora. Out- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out- mutants of Ecb168 were complemented to the Out+ phenotype by introduction of the corresponding cloned HindIII fragment. Out- mutants of Ecb168 were less virulent than the Out+ parental strain on potato tubers. Strain Ecb168 and Out- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora. PMID:16349316

Citric acid fermentation by gamma ray induced mutants of Aspergillus niger in different carbohydrate media

Energy Technology Data Exchange (ETDEWEB)

Anjuman Ara Begum; Naiyyum Choudhury; Mohammad Serajul Islam (Institute of Food and Radiation Biology, Dacca (Bangladesh))

1990-01-01

A natural isolate of Aspergillus niger, CA16, and two of its second step mutants, 136/40 and 277/30, grown on different sugar substrates gave maximum citric acid yields of 34, 70, and 126 mg/ml respectively in sucrose medium. Combination of two sugars in the medium at 50% of each improved the yields of citric acid for the sucrose: glucose, glucose: sorbitol, glucose: xylose, and xylose: sorbitol combinations with the mutant strains. Inclusion of galactose in combinations decreased the citric acid yield. (author).

Citric acid fermentation by gamma ray induced mutants of Aspergillus niger in different carbohydrate media

International Nuclear Information System (INIS)

Anjuman Ara Begum; Naiyyum Choudhury; Mohammad Serajul Islam

1990-01-01

A natural isolate of Aspergillus niger, CA16, and two of its second step mutants, 136/40 and 277/30, grown on different sugar substrates gave maximum citric acid yields of 34, 70, and 126 mg/ml respectively in sucrose medium. Combination of two sugars in the medium at 50% of each improved the yields of citric acid for the sucrose: glucose, glucose: sorbitol, glucose: xylose, and xylose: sorbitol combinations with the mutant strains. Inclusion of galactose in combinations decreased the citric acid yield. (author)

Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP and generation of a mutant library with diverse phenotypes.

Directory of Open Access Journals (Sweden)

Mingyue Fang

Full Text Available In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

Butyric acid fermentation from pre-treated wheat straw by a mutant clostridium tyrobutyricum strain

DEFF Research Database (Denmark)

Baroi, George Nabin; Baumann, Ivan; Westermann, Peter

Only little research on butyric acid fermentation has been carried out in relationship to bio-refinery perspectives involving strain selection, development of adapted strains, physiological analyses for higher yield, productivity and selectivity. However, a major step towards the development...... strain could grow in up to 80% pre-treated wheat straw and can ferment both glucose and xylose. The yield of butyric acid without optimization was 0,37±0,051 g butyric acid/g sugar monomers and the acetate yield was 0,06±0,021 g acetic acid/g sugar monomers. Moreover, the strain could grow without...... addition of yeast extract. Further optimization of yield and productivity is under investigation....

Statistical optimization for enhanced yields of probiotic Bacillus coagulans and its phage resistant mutants followed by kinetic modelling of the process.

Science.gov (United States)

Pandey, Kavita R; Joshi, Chetan; Vakil, Babu V

2016-01-01

Probiotics are microorganisms which when administered in adequate amounts confer health benefits to the host. A leading pharmaceutical company producing Bacillus coagulans as a probiotic was facing the problem of recurring phage attacks. Two mutants viz. B. co PIII and B. co MIII that were isolated as phage resistant mutants after UV irradiation and MMS treatment of phage sensitive B. coagulans parental culture were characterized at functional and molecular level and were noted to have undergone interesting genetic changes. The non-specific genetic alterations induced by mutagenesis can also lead to alterations in cell performance. Hence, in the current study the parental strain and the two mutants were selected for shake flask optimization. Plackett-Burman design was used to select the significant culture variables affecting biomass production. Evolutionary operation method was applied for further optimization. The study showed wide variations in the nutritional requirements of phage resistant mutants, post exposure to mutagens. An increment of 150, 134 and 152 % was observed in the biomass productions of B. coagulans (parental type) and mutants B.co PIII and B.co MIII respectively, compared to the yield from one-factor-at-a-time technique. Using Logistic and modified Leudeking-Piret equations, biomass accumulation and substrate utilization efficiency of the bioprocess were determined. The experimental data was in agreement with the results predicted by statistical analysis and modelling. The developed model may be useful for controlling the growth and substrate consumption kinetics in large scale fermentation using B. coagulans .

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-yl carbamate.

Science.gov (United States)

Li, M; Zhang, H Y; Liang, B

2013-01-01

Twelve-low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe-the molecular lesion likely to be responsible-for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at-amino acid 168, having Phe (TTC) instead of Ser (TCC)', was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.

Senior management leadership, social support, job design and stressor-to-strain relationships in hospital practice.

Science.gov (United States)

Buttigieg, Sandra C; West, Michael A

2013-01-01

The purpose of this paper is to examine the effect of the quality of senior management leadership on social support and job design, whose main effects on strains, and moderating effects on work stressors-to-strains relationships were assessed. A survey involving distribution of questionnaires was carried out on a random sample of health care employees in acute hospital practice in the UK. The sample comprised 65,142 respondents. The work stressors tested were quantitative overload and hostile environment, whereas strains were measured through job satisfaction and turnover intentions. Structural equation modelling and moderated regression analyses were used in the analysis. Quality of senior management leadership explained 75 per cent and 94 per cent of the variance of social support and job design respectively, whereas work stressors explained 51 per cent of the variance of strains. Social support and job design predicted job satisfaction and turnover intentions, as well as moderated significantly the relationships between quantitative workload/hostility and job satisfaction/turnover intentions. The findings are useful to management and to health employees working in acute/specialist hospitals. Further research could be done in other counties to take into account cultural differences and variations in health systems. The limitations included self-reported data and percept-percept bias due to same source data collection. The quality of senior management leaders in hospitals has an impact on the social environment, the support given to health employees, their job design, as well as work stressors and strains perceived. The study argues in favour of effective senior management leadership of hospitals, as well as ensuring adequate support structures and job design. The findings may be useful to health policy makers and human resources managers.

Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.

Science.gov (United States)

Su, Yue; Rhee, Mun Su; Ingram, Lonnie O; Shanmugam, K T

2011-03-01

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.

Physiological Study of Lactobacillus delbrueckii subsp. bulgaricus Strains in a Novel Chemically Defined Medium

Science.gov (United States)

Chervaux, Christian; Ehrlich, S. Dusko; Maguin, Emmanuelle

2000-01-01

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h−1. MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions. PMID:11097906