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Sample records for subtilis rna helicase

  1. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

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    Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

    2007-01-01

    RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

  2. Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

    International Nuclear Information System (INIS)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Brisbarre, Nadège; Lamballerie, Xavier de; Coutard, Bruno; Canard, Bruno; Khromykh, Alexander; Bolognesi, Martino

    2006-01-01

    The C-terminal 440 amino acids of the NS3 protein from Kunjin virus (Flaviviridae) code for a helicase. The protein has been overexpressed and crystallized. Characterization of the isolated monoclinic crystal form and diffraction data (at 3.0 Å resolution) are presented, together with a preliminary molecular-replacement solution. Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3′ nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds

  3. Inhibition of RNA Helicases of ssRNA+ Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families

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    Irene Briguglio

    2011-01-01

    Full Text Available Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

  4. Comparative structural analysis of human DEAD-box RNA helicases.

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    Patrick Schütz

    2010-09-01

    Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  5. Comparative structural analysis of human DEAD-box RNA helicases.

    Science.gov (United States)

    Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

    2010-09-30

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  6. Molecular determinants of nucleolar translocation of RNA helicase A

    International Nuclear Information System (INIS)

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  7. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

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    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  8. RNA helicase A is not required for RISC activity.

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    Liang, Xue-Hai; Crooke, Stanley T

    2013-10-01

    It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects. © 2013. Published by Elsevier B.V. All rights reserved.

  9. Genome-Wide Analysis of the RNA Helicase Gene Family in Gossypium raimondii

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    Jie Chen

    2014-03-01

    Full Text Available The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes, DEAH-box (52 genes, or DExD/H-box (58 genes in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5% are within the identified syntenic blocks. Sixty-six (40.99% helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

  10. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

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    Ana Lúcia Leitão

    2015-01-01

    Full Text Available The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

  11. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans

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    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V.

    2015-01-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  12. Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2.

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    Absmeier, Eva; Becke, Christian; Wollenhaupt, Jan; Santos, Karine F; Wahl, Markus C

    2017-01-02

    RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.

  13. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

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    John L Goodier

    Full Text Available MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC, inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1, Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.

  14. A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

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    Nikolay Kovalev

    2012-02-01

    Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV. To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-RNA, rendering the RNA compatible for initiation of (+-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which is another host factor for TBSV, play non-overlapping functions to enhance (+-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV, a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

  15. The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5

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    Abdullah Shah

    2017-01-01

    Full Text Available DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m6A RNA demethylase. The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.

  16. Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

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    Bhushan, Lokesh; Abraham, Ambily; Choudhury, Nirupam Roy; Rana, Vipin Singh; Mukherjee, Sunil Kumar; Savithri, Handanahal Subbarao

    2015-04-01

    The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

  17. DEAD-Box RNA Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies

    Czech Academy of Sciences Publication Activity Database

    Hafidh, Said; Potěšil, D.; Zdráhal, Z.; Honys, David

    2013-01-01

    Roč. 1, č. 3 (2013) ISSN 2329-9029 R&D Projects: GA ČR GPP501/11/P321; GA ČR(CZ) GAP501/11/1462; GA MŠk(CZ) ED1.1.00/02.0068; GA MŠk(CZ) LD13049 Institutional support: RVO:61389030 Keywords : Translation * mRNA storage * RNA helicase Subject RIV: EB - Genetics ; Molecular Biology

  18. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    International Nuclear Information System (INIS)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

    2016-01-01

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  19. DEAD-box RNA helicase is dispensable for mitochondrial translation in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Richterová, Lenka; Vávrová, Zuzana; Lukeš, Julius

    2011-01-01

    Roč. 127, č. 1 (2011), 300-303 ISSN 0014-4894 R&D Projects: GA ČR GA204/09/1667; GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * Mitochondrial translation * RNA helicase * Cytochrome c oxidase * Mitochondrion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.122, year: 2011

  20. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J., E-mail: nmoorman@med.unc.edu

    2016-02-15

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  1. Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2.

    Science.gov (United States)

    Moreira, Maria-Céu; Klur, Sandra; Watanabe, Mitsunori; Németh, Andrea H; Le Ber, Isabelle; Moniz, José-Carlos; Tranchant, Christine; Aubourg, Patrick; Tazir, Meriem; Schöls, Lüdger; Pandolfo, Massimo; Schulz, Jörg B; Pouget, Jean; Calvas, Patrick; Shizuka-Ikeda, Masami; Shoji, Mikio; Tanaka, Makoto; Izatt, Louise; Shaw, Christopher E; M'Zahem, Abderrahim; Dunne, Eimear; Bomont, Pascale; Benhassine, Traki; Bouslam, Naïma; Stevanin, Giovanni; Brice, Alexis; Guimarães, João; Mendonça, Pedro; Barbot, Clara; Coutinho, Paula; Sequeiros, Jorge; Dürr, Alexandra; Warter, Jean-Marie; Koenig, Michel

    2004-03-01

    Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia. We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP). Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.

  2. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    Science.gov (United States)

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  3. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  4. Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

    Science.gov (United States)

    Theuser, Matthias; Höbartner, Claudia; Wahl, Markus C.; Santos, Karine F.

    2016-01-01

    The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing. PMID:27354531

  5. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

    Science.gov (United States)

    Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  6. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    International Nuclear Information System (INIS)

    Warner, Dennis R.; Bhattacherjee, Vasker; Yin, Xiaolong; Singh, Saurabh; Mukhopadhyay, Partha; Pisano, M. Michele; Greene, Robert M.

    2004-01-01

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  7. The nuclear import of RNA helicase A is mediated by importin-α3

    International Nuclear Information System (INIS)

    Aratani, Satoko; Oishi, Takayuki; Fujita, Hidetoshi; Nakazawa, Minako; Fujii, Ryouji; Imamoto, Naoko; Yoneda, Yoshihiro; Fukamizu, Akiyoshi; Nakajima, Toshihiro

    2006-01-01

    RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-α/β-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-α. Furthermore, the nuclear import of RHA-NLS was supported by importin-α1 and preferentially importin-α3. Our results indicate that the nuclear import of RHA is mediated by the importin-α3/importin-β-dependent pathway and suggest that the specificity for importin may regulate the functions of cargo proteins

  8. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

    NARCIS (Netherlands)

    Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.

    2013-01-01

    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where

  9. Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

    Directory of Open Access Journals (Sweden)

    Robert J Ihry

    Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

  10. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Science.gov (United States)

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

    Directory of Open Access Journals (Sweden)

    Cecilia Zampedri

    2016-10-01

    Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

  12. Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins

    Directory of Open Access Journals (Sweden)

    Rebecca Bish

    2015-07-01

    Full Text Available DDX6 (p54/RCK is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58 of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2 and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2. We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6’s multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6’s interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions

  13. Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems

    Science.gov (United States)

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M.; Hoshika, Shuichi; Frye, Carole; Benner, Steven A.

    2015-01-01

    Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3′-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research. PMID:25953623

  14. The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

    DEFF Research Database (Denmark)

    Lafuente-Barquero, Juan; Luke-Glaser, Sarah; Graf, Marco

    2017-01-01

    of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids...

  15. RNA Helicase DDX5 Regulates MicroRNA Expression and Contributes to Cytoskeletal Reorganization in Basal Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Huang, Jing; Hu, Zhi

    2011-11-15

    RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was upregulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by upregulation of tumor suppressor PDCD4 (a known miR-21 target); as well as upregulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 downregulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.

  16. Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

    Science.gov (United States)

    Ordonez, Heather; Shuman, Stewart

    2013-05-17

    We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

  17. The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

    Science.gov (United States)

    Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Science.gov (United States)

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

    2015-03-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

  19. Structural view of the helicase reveals that Zika virus uses a conserved mechanism for unwinding RNA.

    Science.gov (United States)

    Li, Lei; Wang, Jin; Jia, Zhihui; Shaw, Neil

    2018-04-01

    Recent studies suggest a link between infection by Zika virus (ZIKV) and the development of neurological complications. The lack of ZIKV-specific therapeutics has alarmed healthcare professionals worldwide. Here, crystal structures of apo and AMPPNP- and Mn 2+ -bound forms of the essential helicase of ZIKV refined to 1.78 and 1.3 Å resolution, respectively, are reported. The structures reveal a conserved trimodular topology of the helicase. ATP and Mn 2+ are tethered between two RecA-like domains by conserved hydrogen-bonding interactions. The binding of ligands induces the movement of backbone Cα and side-chain atoms. Numerous solvent molecules are observed in the vicinity of the AMPPNP, suggesting a role in catalysis. These high-resolution structures could be useful for the design of inhibitors targeting the helicase of ZIKV for the treatment of infections caused by ZIKV.

  20. An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

    Science.gov (United States)

    Hodroj, Dana; Recolin, Bénédicte; Serhal, Kamar; Martinez, Susan; Tsanov, Nikolay; Abou Merhi, Raghida; Maiorano, Domenico

    2017-05-02

    Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. © 2017 The Authors.

  1. Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

    Science.gov (United States)

    Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

    2017-12-05

    CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

    Science.gov (United States)

    Hwang, Kyo Seon; Lee, Sang-Myung; Eom, Kilho; Lee, Jeong Hoon; Lee, Yoon-Sik; Park, Jung Ho; Yoon, Dae Sung; Kim, Tae Song

    2007-11-30

    We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

  3. HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

    Science.gov (United States)

    Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent

    2018-01-30

    Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

  4. Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

    Science.gov (United States)

    Mars, Ruben A. T.; Nicolas, Pierre; Denham, Emma L.

    2016-01-01

    SUMMARY Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5′ untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such as Escherichia coli and Salmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacterium Bacillus subtilis. A recent study identified 1,583 putative regulatory RNAs in B. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation in B. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation in B. subtilis mostly involves elements at the 5′ ends of mRNA molecules. These can include 5′ secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs in B. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions in B. subtilis. PMID:27784798

  5. Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression.

    Science.gov (United States)

    Mars, Ruben A T; Nicolas, Pierre; Denham, Emma L; van Dijl, Jan Maarten

    2016-12-01

    Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5' untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such as Escherichia coli and Salmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacterium Bacillus subtilis. A recent study identified 1,583 putative regulatory RNAs in B. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation in B. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation in B. subtilis mostly involves elements at the 5' ends of mRNA molecules. These can include 5' secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs in B. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions in B. subtilis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    Science.gov (United States)

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

  7. 6S-1 RNA function leads to a delay in sporulation in Bacillus subtilis.

    Science.gov (United States)

    Cavanagh, Amy T; Wassarman, Karen M

    2013-05-01

    We have discovered that 6S-1 RNA (encoded by bsrA) is important for appropriate timing of sporulation in Bacillus subtilis in that cells lacking 6S-1 RNA sporulate earlier than wild-type cells. The time to generate a mature spore once the decision to sporulate has been made is unaffected by 6S-1 RNA, and, therefore, we propose that it is the timing of onset of sporulation that is altered. Interestingly, the presence of cells lacking 6S-1 RNA in coculture leads to all cell types exhibiting an early-sporulation phenotype. We propose that cells lacking 6S-1 RNA modify their environment in a manner that promotes early sporulation. In support of this model, resuspension of wild-type cells in conditioned medium from ΔbsrA cultures also resulted in early sporulation. Use of Escherichia coli growth as a reporter of the nutritional status of conditioned media suggested that B. subtilis cells lacking 6S-1 RNA reduce the nutrient content of their environment earlier than wild-type cells. Several pathways known to impact the timing of sporulation, such as the skf- and sdp-dependent cannibalism pathways, were eliminated as potential targets of 6S-1 RNA-mediated changes, suggesting that 6S-1 RNA activity defines a novel mechanism for altering the timing of onset of sporulation. In addition, 6S-2 RNA does not influence the timing of sporulation, providing further evidence of the independent influences of these two related RNAs on cell physiology.

  8. The RNA helicase Rm62 cooperates with SU(VAR3-9 to re-silence active transcription in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Joern Boeke

    Full Text Available Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range.

  9. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

    Science.gov (United States)

    Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

    2002-06-28

    Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

  10. A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation.

    Science.gov (United States)

    Rothstein, David M; Lazinski, David; Osburne, Marcia S; Sonenshein, Abraham L

    2017-07-15

    Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32 P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU , a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes. Copyright © 2017 American Society for Microbiology.

  11. Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

    Science.gov (United States)

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M; Hoshika, Shuichi; Frye, Carole B; Benner, Steven A

    2015-06-15

    Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    Science.gov (United States)

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  13. A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

    Science.gov (United States)

    Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

    2012-08-01

    Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

  14. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Directory of Open Access Journals (Sweden)

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  15. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang

    2012-04-03

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher Kcat and lower Km values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates. © 2012 American Society of Plant Biologists.

  16. Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation

    Energy Technology Data Exchange (ETDEWEB)

    Nelersa, Claudiu M.; Schmier, Brad J.; Malhotra, Arun (Miami-MED)

    2012-05-08

    The final step in RNA degradation is the hydrolysis of RNA fragments five nucleotides or less in length (nanoRNA) to mononucleotides. In Escherichia coli this step is carried out by oligoribonuclease (Orn), a DEDD-family exoribonuclease that is conserved throughout eukaryotes. However, many bacteria lack Orn homologs, and an unrelated DHH-family phosphoesterase, NrnA, has recently been identified as one of the enzymes responsible for nanoRNA degradation in Bacillus subtilis. To understand its mechanism of action, B. subtilis NrnA was purified and crystallized at room temperature using the hanging-drop vapor-diffusion method with PEG 4000, PEG 3350 or PEG MME 2000 as precipitant. The crystals belonged to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 50.62, b = 121.3, c = 123.4 {angstrom}, {alpha} = 90, {beta} = 91.31, {gamma} = 90{sup o}.

  17. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer

    International Nuclear Information System (INIS)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-01-01

    The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms. The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis

  18. RecQL5 promotes genome stabilization through two parallel mechanisms--interacting with RNA polymerase II and acting as a helicase.

    Science.gov (United States)

    Islam, M Nurul; Fox, David; Guo, Rong; Enomoto, Takemi; Wang, Weidong

    2010-05-01

    The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

  19. Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

    Science.gov (United States)

    Plötner, Björn; Nurmi, Markus; Fischer, Axel; Watanabe, Mutsumi; Schneeberger, Korbinian; Holm, Svante; Vaid, Neha; Schöttler, Mark Aurel; Walther, Dirk; Hoefgen, Rainer; Weigel, Detlef; Laitinen, Roosa A E

    2017-07-01

    Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F 2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

    International Nuclear Information System (INIS)

    Fang Jianhua; Acheampong, Edward; Dave, Rajnish; Wang Fengxiang; Mukhtar, Muhammad; Pomerantz, Roger J.

    2005-01-01

    Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to

  1. The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities

    Directory of Open Access Journals (Sweden)

    Mandy Jeske

    2015-07-01

    Full Text Available In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

  2. Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis

    NARCIS (Netherlands)

    Lezzerini, M.; van de Ven, K.; Veerman, M.; Brul, S.; Budovskaya, Y.V.

    2015-01-01

    In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded

  3. NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Ting; Ren, Xiaoming; Adams, Rebecca L.; Pyle, Anna Marie; Ou, J. -H. James

    2017-10-25

    Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.

    IMPORTANCEGenotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important

  4. The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

    Science.gov (United States)

    Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

    2014-08-01

    Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  6. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Science.gov (United States)

    Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-04-29

    Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  7. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  8. Purification and DNA-binding properties of RNA polymerase from Bacillus subtilis.

    Science.gov (United States)

    Giacomoni, P U

    1980-05-01

    Four RNA-polymerizing activities having different subunit composition can be purified from uninfected and from SPO1-infected Bacillus subtilis. Lysozyme and sodium deoxycholate are used for lysing the cells. Polymin P is used for precipitating nucleic acids and DEAE-cellulose chromatography allows separation of enzymatic activity from the residual Polymin P. After these common steps, one can purify core + sigma + delta by chromatography on single-stranded DNA-agarose followed by gel filtration while pure core + sigma can be obtained by chromatography on double-stranded DNA cellulose. Core + delta is obtained by high-salt sucrose/glycerol gradient centrifugation. The host enzyme modified by the product of gene 28 of phage SPO1 can be purified from SPO1 infected cells by chromatography on DNA cellulose (or CNA agarose) followed by chromatography on phosphocellulose. The pH and salt dependance of the initial rate of RNA synthesis of core + sigma has been investigated using SPO1 and SPP1 DNA as templates. The optimum pH for the initial rate of transcription is 8.2 at 30 degrees C in 50 mM N,N-bis(2-hydroxyethyl)glycine buffer, and the optimum Na+ concentration is between 0.1 and 0.15 M. The kinetics of formation and of dissociation of non-filterable complexes between SPP1 DNA and core + sigma have been analyzed at different cationic concentrations. The value of the rate constant of dissociation in 0.1 M NaCl at 30 degrees C is kd = 2.16 x 10(-4) S-1. The value of the rate constant of association, under the same conditions, is ka = 5.5 x 10(8) M-1 S-1; this value is compatible with a diffusion-controlled reaction for promoter selection.

  9. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    Science.gov (United States)

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Zinder, John C. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Tri-Institutional Training Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, United States; Zattas, Dimitrios [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Das, Mom [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Lima, Christopher D. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, United States

    2017-07-25

    Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model.

  11. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

    Science.gov (United States)

    Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

    2015-02-18

    Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. RNases and Helicases in Gram-Positive Bacteria.

    Science.gov (United States)

    Durand, Sylvain; Condon, Ciaran

    2018-04-01

    RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis , the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis , the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.

  13. Elucidating the 16S rRNA 3' boundaries and defining optimal SD/aSD pairing in Escherichia coli and Bacillus subtilis using RNA-Seq data.

    Science.gov (United States)

    Wei, Yulong; Silke, Jordan R; Xia, Xuhua

    2017-12-15

    Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5' UTR of mRNA and the anti-SD (aSD) sequence at the free 3' end of the 16S rRNA (3' TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3' TAIL and 2) extent of the core aSD sequence within the 3' TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3' boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3' TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3' TAIL in B. subtilis and recovered the established 3' TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

  14. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  15. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer.

    Science.gov (United States)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-05-01

    The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq-RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq-RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 A, while the type 2 Hfq-RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 A. Diffraction data were collected to a resolution of 2.20 A from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis.

  16. The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1

    International Nuclear Information System (INIS)

    Nagahama, Masami; Yamazoe, Takeshi; Hara, Yoshimitsu; Tani, Katsuko; Tsuji, Akihiko; Tagaya, Mitsuo

    2006-01-01

    Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase, whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone

  17. Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

    Science.gov (United States)

    Bonner, Eric R.; D’Elia, John N.; Billips, Benjamin K.; Switzer, Robert L.

    2001-01-01

    The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5′-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4–5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure. PMID:11726695

  18. Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

    Science.gov (United States)

    Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

    2017-02-14

    Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

  19. Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

    Science.gov (United States)

    Götze, Michael; Dufourt, Jérémy; Ihling, Christian; Rammelt, Christiane; Pierson, Stephanie; Sambrani, Nagraj; Temme, Claudia; Sinz, Andrea; Simonelig, Martine; Wahle, Elmar

    2017-10-01

    Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression. © 2017 Götze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. Posttranscriptional regulation of the karyogamy gene by Kem1p/Xrn1p exoribonuclease and Rok1p RNA helicase of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kim, Jaehee; Jeon, Soonmee; Yang, Yun-Seok; Kim, Jinmi

    2004-01-01

    The major biochemical activities ascribed to Kem1p/Xrn1p of Saccharomyces cerevisiae are 5'-3' exoribonuclease functioning in RNA turnover and a microtubule-binding protein. Mutational analysis has shown that Kem1p/Xrn1p participates in microtubule-related functions such as nuclear fusion (karyogamy) during mating, chromosome transmission, and spindle pole body duplication. Here, evidence is presented that Kem1p plays a specific role in nuclear fusion by affecting, at the posttranscriptional level, the pheromone induction of the karyogamy-specific transcription factor Kar4p and the expression of Rok1p, a putative RNA helicase. We found that Rok1p itself also affects the pheromone induction of Kar4p and thereby participates in nuclear fusion. Analysis of the active-site mutations, xrn1-D206A or D208A, shows that nuclear fusion as well as the Rok1p synthesis do not require the exoribonuclease activity of Kem1p. Our data provide an important insight into the gene-specific regulatory function mediated by the general RNA-modulating enzymes

  1. Structural basis of Zika virus helicase in recognizing its substrates

    Directory of Open Access Journals (Sweden)

    Hongliang Tian

    2016-07-01

    Full Text Available Abstract The recent explosive outbreak of Zika virus (ZIKV infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

  2. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    International Nuclear Information System (INIS)

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-01-01

    Highlights: ► DP97 interacts with nuclear receptor CAR. ► DP97 enhances CAR-mediated transcriptional activation. ► DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1α. ► DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1α, therefore it might act as mediator between hCAR and appropriate co-activators.

  3. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp [Faculty of Pharmaceutical Sciences, Toho University, Chiba (Japan); Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio [Faculty of Pharmaceutical Sciences, Toho University, Chiba (Japan)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  4. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    International Nuclear Information System (INIS)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth

    2014-01-01

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers

  5. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  6. Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

    DEFF Research Database (Denmark)

    Nishimura, Kenji; Johansen, Shanna K; Inaoka, Takashi

    2007-01-01

    The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness...... between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants...

  7. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA

    OpenAIRE

    Schertzer , Michael; Jouravleva , Karina; Perderiset , Mylène; Dingli , Florent; Loew , Damarys; Le Guen , Tangui; Bardoni , Barbara; De Villartay , Jean-Pierre; Revy , Patrick; Londono-Vallejo , Arturo

    2015-01-01

    International audience; Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and im-munodeficiency and has been associated with telom-ere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is require...

  8. Structural basis for the function of DEAH helicases

    DEFF Research Database (Denmark)

    He, Yangzi; Andersen, Gregers Rom; Nielsen, Klaus Hvid

    2010-01-01

    DEAH helicases participate in pre‐messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p‐ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide‐binding motif. A β‐hairpin from the second RecA domain is wedged between two carboxy......‐terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C‐terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides...

  9. Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.

    Science.gov (United States)

    Cai, Yanfei; Chandrangsu, Pete; Gaballa, Ahmed; Helmann, John D

    2017-02-01

    Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.

  10. Purification and crystallization of Kokobera virus helicase

    International Nuclear Information System (INIS)

    De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno; Forrester, Naomi L.; Gould, Ernest; Canard, Bruno; Mattevi, Andrea

    2007-01-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å

  11. Purification and crystallization of Kokobera virus helicase

    Energy Technology Data Exchange (ETDEWEB)

    De Colibus, Luigi; Speroni, Silvia [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy); Coutard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Forrester, Naomi L.; Gould, Ernest [Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR (United Kingdom); Canard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Mattevi, Andrea, E-mail: mattevi@ipvgen.unipv.it [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy)

    2007-03-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.

  12. RNA-seq analysis of antibiotic-producing Bacillus subtilis SC-8 in response to signal peptide PapR of Bacillus cereus.

    Science.gov (United States)

    Yeo, In-Cheol; Lee, Nam Keun; Yang, Byung Wook; Hahm, Young Tae

    2014-01-01

    Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

  13. Helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

    2013-10-11

    Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

  14. RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

    Czech Academy of Sciences Publication Activity Database

    Kanagaraj, R.; Huehn, D.; Mackellar, A.; Menigatti, M.; Zheng, L.; Urban, Václav; Shevelev, Igor; Greenleaf, A.L.; Janščák, Pavel

    2010-01-01

    Roč. 38, č. 22 (2010), s. 8131-8140 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Grant - others:SNSF(CH) 3100A0-116008; NIH(US) GM040505 Institutional research plan: CEZ:AV0Z50520514 Keywords : RECQ5 DNA helicase * transcription * genome stability Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.836, year: 2010

  15. AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

    Science.gov (United States)

    Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

    2014-01-01

    The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  16. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    Science.gov (United States)

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-05

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

  17. The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores

    OpenAIRE

    Kodama, Takeko; Takamatsu, Hiromu; Asai, Kei; Kobayashi, Kazuo; Ogasawara, Naotake; Watabe, Kazuhito

    1999-01-01

    The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T2) of sporulation in wild-type cells and in spoIIIG (SigG−) and spoIVCB (SigK−) mutants but not in spoIIAC (SigF−) and spoIIGAB (SigE−) mutants. The transcript...

  18. A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

    DEFF Research Database (Denmark)

    Desmolaize, Benoit; Fabret, Céline; Brégeon, Damien

    2011-01-01

    Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications....... However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme...

  19. RecQ Helicases

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Hickson, Ian D

    2013-01-01

    The RecQ family of DNA helicases is highly conserved throughout -evolution, and is important for the maintenance of genome stability. In humans, five RecQ family members have been identified: BLM, WRN, RECQ4, RECQ1 and RECQ5. Defects in three of these give rise to Bloom's syndrome (BLM), Werner...

  20. Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism.

    Science.gov (United States)

    Mondal, Smarajit; Yakhnin, Alexander V; Babitzke, Paul

    2017-07-15

    The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding. IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory

  1. The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores

    Science.gov (United States)

    Kodama, Takeko; Takamatsu, Hiromu; Asai, Kei; Kobayashi, Kazuo; Ogasawara, Naotake; Watabe, Kazuhito

    1999-01-01

    The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T2) of sporulation in wild-type cells and in spoIIIG (SigG−) and spoIVCB (SigK−) mutants but not in spoIIAC (SigF−) and spoIIGAB (SigE−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T2 of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in l-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T2 of sporulation and that it is required for the l-alanine-stimulated germination pathway. PMID:10419957

  2. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang; Axtell, Michael J.; Fedoroff, Nina V.

    2012-01-01

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display

  3. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  4. PBDE: Structure-Activity Studies for the Inhibition of Hepatitis C Virus NS3 Helicase

    Directory of Open Access Journals (Sweden)

    Kazi Abdus Salam

    2014-04-01

    Full Text Available The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3 is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1 on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1 against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

  5. Regulatory RNAs in Bacillus subtilis : a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

    NARCIS (Netherlands)

    Mars, Ruben A. T.; Nicolas, Pierre; Denham, Emma L.; van Dijl, Jan Maarten

    2016-01-01

    Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5= untranslated region. Thus far, most regulatory RNA research has focused on

  6. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  7. The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

    Directory of Open Access Journals (Sweden)

    Simona Cugusi

    2016-01-01

    Full Text Available RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi and microRNA (miRNA pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

  8. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    International Nuclear Information System (INIS)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  9. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  10. Mitochondrial helicases and mitochondrial genome maintenance

    DEFF Research Database (Denmark)

    de Souza-Pinto, Nadja C; Aamann, Maria Diget; Kulikowicz, Tomasz

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...

  11. Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

    Science.gov (United States)

    Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

    2001-01-01

    The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

  12. Filtration and transport of Bacillus subtilis spores and the F-RNA phage MS2 in a coarse alluvial gravel aquifer: implications in the estimation of setback distances.

    Science.gov (United States)

    Pang, Liping; Close, Murray; Goltz, Mark; Noonan, Mike; Sinton, Lester

    2005-04-01

    Filtration of Bacillus subtilis spores and the F-RNA phage MS2 (MS2) on a field scale in a coarse alluvial gravel aquifer was evaluated from the authors' previously published data. An advection-dispersion model that is coupled with first-order attachment kinetics was used in this study to interpret microbial concentration vs. time breakthrough curves (BTC) at sampling wells. Based on attachment rates (katt) that were determined by applying the model to the breakthrough data, filter factors (f) were calculated and compared with f values estimated from the slopes of log (cmax/co) vs. distance plots. These two independent approaches resulted in nearly identical filter factors, suggesting that both approaches are useful in determining reductions in microbial concentrations over transport distance. Applying the graphic approach to analyse spatial data, we have also estimated the f values for different aquifers using information provided by some other published field studies. The results show that values of f, in units of log (cmax/co) m(-1), are consistently in the order of 10(-2) for clean coarse gravel aquifers, 10(-3) for contaminated coarse gravel aquifers, and generally 10(-1) for sandy fine gravel aquifers and river and coastal sand aquifers. For each aquifer category, the f values for bacteriophages and bacteria are in the same order-of-magnitude. The f values estimated in this study indicate that for every one-log reduction in microbial concentration in groundwater, it requires a few tens of meters of travel in clean coarse gravel aquifers, but a few hundreds of meters in contaminated coarse gravel aquifers. In contrast, a one-log reduction generally only requires a few meters of travel in sandy fine gravel aquifers and sand aquifers. Considering the highest concentration in human effluent is in the order of 10(4) pfu/l for enteroviruses and 10(6) cfu/100 ml for faecal coliform bacteria, a 7-log reduction in microbial concentration would comply with the drinking

  13. Carbohydrate metabolism in Bacillus subtilis

    International Nuclear Information System (INIS)

    Riedel, K.

    1980-01-01

    The glucose metabolism via the glycolytic pathway as well as via the oxidative and inoxidative hexose monophosphate pathways in Bacillus subtilis was studied applying 1- 14 C- and 6- 14 C-glucose, respectively, and determining labelled CO 2 and RNA. A method for calculating the catabolic pathways was developed. In nonproliferating cultures glucose is catabolized to 62% via the glycolytic pathway, to 20% via the oxidative, and to 18% via the inoxidative pathway

  14. Targeting Dengue Virus NS-3 Helicase by Ligand based Pharmacophore Modeling and Structure based Virtual Screening

    Science.gov (United States)

    Halim, Sobia A.; Khan, Shanza; Khan, Ajmal; Wadood, Abdul; Mabood, Fazal; Hussain, Javid; Al-Harrasi, Ahmed

    2017-10-01

    Dengue fever is an emerging public health concern, with several million viral infections occur annually, for which no effective therapy currently exist. Non-structural protein 3 (NS-3) Helicase encoded by the dengue virus (DENV) is considered as a potential drug target to design new and effective drugs against dengue. Helicase is involved in unwinding of dengue RNA. This study was conducted to design new NS-3 Helicase inhibitor by in silico ligand- and structure based approaches. Initially ligand-based pharmacophore model was generated that was used to screen a set of 1201474 compounds collected from ZINC Database. The compounds matched with the pharmacophore model were docked into the active site of NS-3 helicase. Based on docking scores and binding interactions, twenty five compounds are suggested to be potential inhibitors of NS3 Helicase. The pharmacokinetic properties of these hits were predicted. The selected hits revealed acceptable ADMET properties. This study identified potential inhibitors of NS-3 Helicase in silico, and can be helpful in the treatment of Dengue.

  15. Association of RNAs with Bacillus subtilis Hfq.

    Directory of Open Access Journals (Sweden)

    Michael Dambach

    Full Text Available The prevalence and characteristics of small regulatory RNAs (sRNAs have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.

  16. The Regulatory RNAs of Bacillus subtilis

    NARCIS (Netherlands)

    Mars, Ruben

    2014-01-01

    In vrijwel alle organismen wordt RNA aangemaakt dat niet codeert voor eiwit, maar een regulerende functie heeft. Dit proefschrift beschrijft de identificatie van ~1600 nieuwe potentiële regulatie-RNAs in de bodembacterie Bacillus subtilis die veel voor biotechnologische toepassingen ingezet wordt.

  17. TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

    NARCIS (Netherlands)

    G.S. Winkler (Sebastiaan); U. Fiedler; W. Vermeulen (Wim); F. Coin (Frédéric); R.D. Wood (Richard); H.T.M. Timmers (Marc); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S.J. Araú jo; J-M. Egly (Jean-Marc)

    2000-01-01

    textabstractTFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and

  18. A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

    Science.gov (United States)

    Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

    2017-08-01

    Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

  19. Host organisms: Bacillus subtilis

    NARCIS (Netherlands)

    Hohman, Hans-Peter; van Dijl, Jan; Krishnappa, Laxmi; Pragai, Zoltan

    2016-01-01

    Bacillus subtilis and its close Bacillus relatives are important bacterial platforms for industrial production of enzymes and fine chemicals such as vitamin B2 and nucleotides. B. subtilis is an attractive bacterial organism for industrial use mainly because of its straightforward genetic

  20. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    Science.gov (United States)

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  1. A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase

    Directory of Open Access Journals (Sweden)

    Dimitrios Vlachakis

    2013-05-01

    Full Text Available Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12 was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost “U” shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.

  2. Molecular Dynamics of the ZIKA Virus NS3 Helicase

    Science.gov (United States)

    Raubenolt, Bryan; Rick, Steven; The Rick Group Team

    The recent outbreaks of the ZIKA virus (ZIKV) and its connection to microcephaly in newborns has raised its awareness as a global threat and many scientific research efforts are currently underway in attempt to create a vaccine. Molecular Dynamics is a powerful method of investigating the physical behavior of protein complexes. ZIKV is comprised of 3 structural and 7 nonstructural proteins. The NS3 helicase protein appears to play a significant role in the replication complex and its inhibition could be a crucial source of antiviral drug design. This research primarily focuses on studying the structural dynamics, over the course of few hundred nanoseconds, of NS3 helicase in the free state, as well as in complex form with human ssRNA, ATP, and an analogue of GTP. RMSD and RMSF plots of each simulation will provide details on the forces involved in the overall stability of the active and inactive states. Furthermore, free energy calculations on a per residue level will reveal the most interactive residues between states and ultimately the primary driving force behind these interactions. Together these analyses will provide highly relevant information on the binding surface chemistry and thus serve as the basis for potential drug design.

  3. A new role for FBP21 as regulator of Brr2 helicase activity.

    Science.gov (United States)

    Henning, Lisa M; Santos, Karine F; Sticht, Jana; Jehle, Stefanie; Lee, Chung-Tien; Wittwer, Malte; Urlaub, Henning; Stelzl, Ulrich; Wahl, Markus C; Freund, Christian

    2017-07-27

    Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

    Science.gov (United States)

    Balasingham, Seetha V; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L; Laerdahl, Jon K; Bohr, Vilhelm A; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+)/Mn(2+). Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

  5. Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

    Directory of Open Access Journals (Sweden)

    Seetha V Balasingham

    Full Text Available XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB, a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+/Mn(2+. Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

  6. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  7. Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

    Directory of Open Access Journals (Sweden)

    Atsushi Furuta

    2014-01-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3 helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3 and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

  8. Overcoming natural replication barriers: differential helicase requirements.

    Science.gov (United States)

    Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

    2012-02-01

    DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

  9. The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

    Science.gov (United States)

    Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

    2014-04-14

    RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [New antibiotics produced by Bacillus subtilis strains].

    Science.gov (United States)

    Malanicheva, I A; Kozlov, D G; Efimenko, T A; Zenkova, V A; Kastrukha, G S; Reznikova, M I; Korolev, A M; Borshchevskaia, L N; Tarasova, O D; Sineokiĭ, S P; Efremenkova, O V

    2014-01-01

    Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mes6nteroides VKPM B-4177 resistant to glycopep-> tide antibiotics, as well as antifungal activity. The strains were identified as belonging to the "B. subtilis" com- plex based on their morphological and physiological characteristics, as well as by sequencing of the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaen and pentaen, respectively). Strain INA 01086 produced also a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.

  11. The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

    Science.gov (United States)

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

    2014-01-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

  12. Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

    Directory of Open Access Journals (Sweden)

    Fred E Indig

    Full Text Available The Werner protein (WRNp, a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL, an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA.These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

  13. Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

    Science.gov (United States)

    Szewczyk, Maciej; Fedoryszak-Kuśka, Natalia; Tkaczuk, Katarzyna; Dobrucki, Jurek; Waligórska, Agnieszka; Stępień, Piotr P

    2017-01-01

    The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

  14. [RTEL1 (regulator of telomere elongation helicase 1), a DNA helicase essential for genome stability].

    Science.gov (United States)

    Le Guen, Tangui; Jullien, Laurent; Schertzer, Mike; Lefebvre, Axelle; Kermasson, Laetitia; de Villartay, Jean-Pierre; Londoño-Vallejo, Arturo; Revy, Patrick

    2013-12-01

    RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play. © 2013 médecine/sciences – Inserm.

  15. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

    Directory of Open Access Journals (Sweden)

    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  16. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

    Science.gov (United States)

    Tortorici, M Alejandra; Duquerroy, Stéphane; Kwok, Jane; Vonrhein, Clemens; Perez, Javier; Lamp, Benjamin; Bricogne, Gerard; Rümenapf, Till; Vachette, Patrice; Rey, Félix A

    2015-04-01

    Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the

  18. Sigma A recognition sites in the Bacillus subtilis genome

    DEFF Research Database (Denmark)

    Jarmer, Hanne Østergaard; Larsen, Thomas Schou; Krogh, Anders Stærmose

    2001-01-01

    A hidden Markov model of sigma (A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma (A) recognition sites. This work suggests that more information exists...... at the initiation site of transcription in both types of promoters than previously thought. When tested on the entire B. subtilis genome, the model predicts that approximately half of the sigma (A) recognition sites are of the extended type. Some of the response-regulator aspartate phosphatases were among...

  19. Crystal structure of Middle East respiratory syndrome coronavirus helicase.

    Directory of Open Access Journals (Sweden)

    Wei Hao

    2017-06-01

    Full Text Available Middle East respiratory syndrome coronavirus (MERS-CoV remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

  20. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    Science.gov (United States)

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  1. Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

    Directory of Open Access Journals (Sweden)

    Yoomi Choi

    Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

  2. Bacillus subtilis genome diversity.

    Science.gov (United States)

    Earl, Ashlee M; Losick, Richard; Kolter, Roberto

    2007-02-01

    Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.

  3. SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

    Science.gov (United States)

    Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

    2011-01-01

    In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

  4. A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

    Directory of Open Access Journals (Sweden)

    Jacob Thomas

    Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

  5. Essential Bacillus subtilis genes

    DEFF Research Database (Denmark)

    Kobayashi, K.; Ehrlich, S.D.; Albertini, A.

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were...... predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related...... to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from...

  6. Bacillus subtilis and yeast cell wall improve the intestinal health of broilers challenged by Clostridium perfringens.

    Science.gov (United States)

    Li, Z; Wang, W; Lv, Z; Liu, D; Guo, Y

    2017-12-01

    1. The objective was to investigate the effects of Bacillus subtilis, yeast cell wall (YCW) and their combination on intestinal health of broilers challenged by Clostridium perfringens over a 21-d period. 2. Using a 5 × 2 factorial arrangement of treatments, 800 1-d-old male Cobb 500 broilers were used to study the effects of feed additives (without additive or with zinc bacitracin, B. subtilis, YCW, and the combination of B. subtilis and YCW), pathogen challenge (without or with Clostridium perfringens challenge), and their interactive effects. 3. C. perfringens infection increased intestinal lesions scores, damaged intestinal histomorphology, increased serum endotoxin concentration, cytokine mRNA expression and intestinal population of C. perfringens and Escherichia coli and decreased ileal bifidobacteria numbers. The 4 additives decreased serum endotoxin. Zinc bacitracin tended to decrease cytokine mRNA expression and the intestinal number of C. perfringens and E. coli. B. subtilis, YCW and their combination increased cytokine mRNA expression. B. subtilis and YCW decreased the number of C. perfringens and E. coli in the ileum, and their combination decreased pathogens numbers in the ileum and caecum. 4. In conclusion, B. subtilis, YCW and their combination improved the intestinal health of NE-infected broilers, and could be potential alternatives to antibiotics.

  7. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

    Directory of Open Access Journals (Sweden)

    Mehrnoush Dehghani

    2015-03-01

    Full Text Available The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells, posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

  8. Membrane recognition and dynamics of the RNA degradosome

    NARCIS (Netherlands)

    Strahl, H.; Turlan, C.; Khalid, S.; Bond, P.J.; Kebalo, J.M.; Peyron, P.; Poljak, L.; Bouvier, M.; Hamoen, L.; Luisi, B.F.; Carpousis, A.J.

    2015-01-01

    RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane

  9. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Science.gov (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  10. Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track.

    Science.gov (United States)

    Beran, Rudolf K F; Bruno, Michael M; Bowers, Heath A; Jankowsky, Eckhard; Pyle, Anna Marie

    2006-05-12

    The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.

  11. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    Energy Technology Data Exchange (ETDEWEB)

    Möhlmann, Sina [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Mathew, Rebecca [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Neumann, Piotr; Schmitt, Andreas [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Lührmann, Reinhard [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@uni-goettingen.de [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  12. Effects of Bacillus subtilis natto and Different Components in Culture on Rumen Fermentation and Rumen Functional Bacteria In Vitro.

    Science.gov (United States)

    Sun, Peng; Li, Jinan; Bu, Dengpan; Nan, Xuemei; Du, Hong

    2016-05-01

    This study was to investigate the effects of live or autoclaved Bacillus subtilis natto, their fermented products and media on rumen fermentation and rumen functional bacteria in vitro. Rumen fluid from three multiparous lactating Holstein cows was combined and transferred into serum bottles after diluted. Fifteen serum bottles were divided into five treatments, which were designed as following: CTR (the fermentation of 0.5 g TMR and ruminal fluids from dairy cows), LBS (CTR plus a minimum of 10(11) cfu live Bacillus subtilis natto), ABS (CTR plus a minimum of 10(11) cfu autoclaved Bacillus subtilis natto), BSC (CTR plus 1 ml Bacillus subtilis natto fermentation products without bacteria), and BSM (CTR plus 1 ml liquid fermentation medium). When separated from the culture, live Bacillus subtilis natto individually increased the concentrations of ammonia-N (P Bacillus subtilis natto has the similar function with the live bacteria except for the ratio of acetate and propionate. Except B. fibrisolvens, live or autoclaved Bacillus subtilis natto did not influence or decreased the 16S rRNA gene quantification of the detected bacteria. BSC and BSM altered the relative expression of certain functional bacteria in the rumen. These results indicated that it was Bacillus subtilis natto thalli that played the important role in promoting rumen fermentation when applied as a probiotic in dairy ration.

  13. Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

    Energy Technology Data Exchange (ETDEWEB)

    Hood, Iris V.; Berger, James M.

    2016-05-31

    Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

  14. RecQ helicases and cellular responses to DNA damage

    International Nuclear Information System (INIS)

    Wu, Leonard; Hickson, Ian D.

    2002-01-01

    The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process

  15. Enterovirus Exposure Uniquely Discriminates Type 1 Diabetes Patients with a Homozygous from a Heterozygous Melanoma Differentiation-Associated Protein 5/Interferon Induced with Helicase C Domain 1 A946T Genotype

    NARCIS (Netherlands)

    Schulte, B.M.; Gielen, P.R.; Kers-Rebel, E.D.; Prosser, A.C.; Lind, K.; Flodstrom-Tullberg, M.; Tack, C.J.J.; Elving, L.D.; Adema, G.J.

    2016-01-01

    In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma

  16. MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

    Science.gov (United States)

    Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

    2014-01-01

    As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

  17. Ecology and genomics of Bacillus subtilis.

    Science.gov (United States)

    Earl, Ashlee M; Losick, Richard; Kolter, Roberto

    2008-06-01

    Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.

  18. How Changes in Anti-SD Sequences Would Affect SD Sequences in Escherichia coli and Bacillus subtilis.

    Science.gov (United States)

    Abolbaghaei, Akram; Silke, Jordan R; Xia, Xuhua

    2017-05-05

    The 3' end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3' end of the ssu rRNA. Consequently, the 3' end of ssu rRNA (3'TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3' ends of ssu rRNA, being GAUC ACCUCCUUA 3' in E. coli and GAUC ACCUCCUU UCU3' or GAUC ACCUCCUU UCUA3' in B. subtilis Such differences in 3'TAIL lead to species-specific SDs (designated SD Ec for E. coli and SD Bs for B. subtilis ) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3'TAIL is expected to favor SD Bs against SD Ec in B. subtilis , but favor SD Ec against SD Bs in E. coli Among well-positioned SDs, SD Ec is used more in E. coli than in B. subtilis , and SD Bs more in B. subtilis than in E. coli Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli Both species overuse SDs matching the bolded part of the 3'TAIL shown above. The 3'TAIL difference contributes to the host specificity of phages. Copyright © 2017 Abolbaghaei et al.

  19. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  20. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    OpenAIRE

    Leventhal, J M; Chambliss, G H

    1982-01-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phos...

  1. Velocity and processivity of helicase unwinding of double-stranded nucleic acids

    International Nuclear Information System (INIS)

    Betterton, M D; Juelicher, F

    2005-01-01

    Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed

  2. Type I signal peptidases of Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, Harold; Bolhuis, Albert; Bron, Sierd; Jongbloed, Jan; Meijer, Wilfried J.J.; Noback, Michiel; van Roosmalen, Maarten; Venema, Gerhardus; van Dijl, Jan Maarten; Hopsu Havu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from

  3. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    International Nuclear Information System (INIS)

    Zhang, Yuan; Palla, Mirkó; Liao, Jung-Chi; Sun, Andrew

    2013-01-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116. (paper)

  4. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    Science.gov (United States)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  5. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

    Directory of Open Access Journals (Sweden)

    Glenn Hauk

    Full Text Available The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.

  6. TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

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    Beiyu Liu

    2009-09-01

    Full Text Available Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA, is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb, are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

  7. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

    Science.gov (United States)

    Levin, Mikhail K; Gurjar, Madhura; Patel, Smita S

    2005-05-01

    Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

  8. GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

    Science.gov (United States)

    Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

    2016-12-01

    In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

    Science.gov (United States)

    Kubo, Yuji; Rooney, Alejandro P; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

    2011-09-01

    Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

  10. Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production ▿

    Science.gov (United States)

    Kubo, Yuji; Rooney, Alejandro P.; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

    2011-01-01

    Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group. PMID:21764950

  11. Saccharomyces cerevisiae Hrq1 requires a long 3′-tailed DNA substrate for helicase activity

    International Nuclear Information System (INIS)

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-01-01

    Highlights: ► Hrq1 has intrinsic 3′–5′ helicase and DNA strand annealing activities. ► Hrq1 requires a long 3′-tail for efficient DNA unwinding. ► Helicase activity of Hrq1 is stimulated by a fork structure. ► Hrq1 is a moderately processive helicase. -- Abstract: RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3′–5′ helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3′-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases.

  12. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

    Science.gov (United States)

    Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

    2015-01-01

    Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

  13. microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Macovei Anca

    2012-10-01

    Full Text Available Abstract Background Rice (Oryza sativa L., one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. Results In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein, OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase and OsDBH (DEAD-Box Helicase genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5′RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in miRNA

  14. Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

    International Nuclear Information System (INIS)

    Tuteja, N.

    2005-01-01

    Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

  15. The MCM Helicase Motor of the Eukaryotic Replisome.

    Science.gov (United States)

    Abid Ali, Ferdos; Costa, Alessandro

    2016-05-08

    The MCM motor of the CMG helicase powers ahead of the eukaryotic replication machinery to unwind DNA, in a process that requires ATP hydrolysis. The reconstitution of DNA replication in vitro has established the succession of events that lead to replication origin activation by the MCM and recent studies have started to elucidate the structural basis of duplex DNA unwinding. Despite the exciting progress, how the MCM translocates on DNA remains a matter of debate. Copyright © 2016. Published by Elsevier Ltd.

  16. Saccharomyces cerevisiae Hrq1 requires a long 3'-tailed DNA substrate for helicase activity.

    Science.gov (United States)

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-10-26

    RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3'-5' helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3'-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. In TFIIH, XPD helicase is exclusively devoted to DNA repair.

    Directory of Open Access Journals (Sweden)

    Jochen Kuper

    2014-09-01

    Full Text Available The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER. Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

  18. Pirated Siderophores Promote Sporulation in Bacillus subtilis.

    Science.gov (United States)

    Grandchamp, Gabrielle M; Caro, Lews; Shank, Elizabeth A

    2017-05-15

    In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis- produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA , for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis The interaction is mediated by the E

  19. Genome-wide identification of SF1 and SF2 helicases from archaea.

    Science.gov (United States)

    Chamieh, Hala; Ibrahim, Hiba; Kozah, Juliana

    2016-01-15

    Archaea microorganisms have long been used as model organisms for the study of protein molecular machines. Archaeal proteins are particularly appealing to study since archaea, even though prokaryotic, possess eukaryotic-like cellular processes. Super Family I (SF1) and Super Family II (SF2) helicase families have been studied in many model organisms, little is known about their presence and distribution in archaea. We performed an exhaustive search of homologs of SF1 and SF2 helicase proteins in 95 complete archaeal genomes. In the present study, we identified the complete sets of SF1 and SF2 helicases in archaea. Comparative analysis between archaea, human and the bacteria E. coli SF1 and SF2 helicases, resulted in the identification of seven helicase families conserved among representatives of the domains of life. This analysis suggests that these helicase families are highly conserved throughout evolution. We highlight the conserved motifs of each family and characteristic domains of the detected families. Distribution of SF1/SF2 families show that Ski2-like, Lhr, Sfth and Rad3-like helicases are ubiquitous among archaeal genomes while the other families are specific to certain archaeal groups. We also report the presence of a novel SF2 helicase specific to archaea domain named Archaea Specific Helicase (ASH). Phylogenetic analysis indicated that ASH has evolved in Euryarchaeota and is evolutionary related to the Ski2-like family with specific characteristic domains. Our study provides the first exhaustive analysis of SF1 and SF2 helicases from archaea. It expands the variety of SF1 and SF2 archaeal helicases known to exist to date and provides a starting point for new biochemical and genetic studies needed to validate their biological functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Science.gov (United States)

    Furuta, Atsushi; Tsubuki, Masayoshi; Endoh, Miduki; Miyamoto, Tatsuki; Tanaka, Junichi; Abdus Salam, Kazi; Akimitsu, Nobuyoshi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2015-01-01

    Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition. PMID:26262613

  1. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Directory of Open Access Journals (Sweden)

    Atsushi Furuta

    2015-08-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3 helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

  2. A role for the fission yeast Rqh1 helicase in chromosome segregation

    DEFF Research Database (Denmark)

    Win, Thein Z; Mankouri, Hocine W; Hickson, Ian D

    2005-01-01

    Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled...

  3. Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

    Directory of Open Access Journals (Sweden)

    Mahrou Sadri

    2015-02-01

    Full Text Available Objective(s: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3 of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3 using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3. The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

  4. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

    OpenAIRE

    Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including pl...

  5. Emerging importance of helicases in plant stress tolerance: characterization of Oryza sativa repair helicase XPB2 promoter and its functional validation in tobacco under multiple stresses

    OpenAIRE

    Shailendra eRaikwar; Vineet Kumar Shrivastava; Sarvajeet Singh Gill; Renu eTuteja; Narendra eTuteja; Narendra eTuteja

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. DNA hHelicases, the unique molecular motors, are emerged as potentialprospective molecules to engineer stress tolerance in plants and are involved in a variety of DNA nucleic acid metabolismc processes including DNA repair. The DNA repair helicase, OsXPB2 is an evolutionary conserved pr...

  6. DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins.

    Science.gov (United States)

    Sevim, Elif; Gaballa, Ahmed; Beldüz, A Osman; Helmann, John D

    2011-01-01

    σ(D) proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the -10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.

  7. DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σD Proteins▿

    Science.gov (United States)

    Sevim, Elif; Gaballa, Ahmed; Beldüz, A. Osman; Helmann, John D.

    2011-01-01

    σD proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the −10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity. PMID:21097624

  8. DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σD Proteins▿

    OpenAIRE

    Sevim, Elif; Gaballa, Ahmed; Beldüz, A. Osman; Helmann, John D.

    2010-01-01

    σD proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the −10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.

  9. Analysis of Host-Takeover During SPO1 Infection of Bacillus subtilis.

    Science.gov (United States)

    Stewart, Charles R

    2018-01-01

    When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

  10. Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, Apostichopus japonicus Selenka

    Science.gov (United States)

    Zhao, Yancui; Yuan, Lei; Wan, Junli; Sun, Hushan; Wang, Yiyan; Zhang, Qin

    2018-04-01

    The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber ( Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 105, 107, and 109 CFU g-1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 109 CFU g-1 ( P subtilis 2-1 had no significant influence on the lipase activity in sea cucumber ( P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g-1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 109 CFU g-1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.

  11. Characterization of Lipase from Bacillus subtilisI-4 and Its Potential Use in Oil Contaminated Wastewater

    OpenAIRE

    Iqbal, Syeda Abeer; Rehman, Abdul

    2015-01-01

    ABSTRACTA lipase producing bacterium was isolated from oil contaminated effluents of various industries from Sheikhupura Road, Pakistan, and, on the basis of biochemical and 16S rRNA ribotyping, was identified asBacillus subtilis. The optimum temperature and pH for the growth of the culture were 37ºC and 7.0, respectively.B. subtilis I-4 had a lag phase of 4 h in LB medium while this phase prolonged to 6 h in oil containing medium. The optimum temperature and pH for the enzyme activity were 5...

  12. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    Science.gov (United States)

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-06

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

  13. The essential function of B. subtilis RNase III is to silence foreign toxin genes.

    Directory of Open Access Journals (Sweden)

    Sylvain Durand

    Full Text Available RNase III-related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi-RNAs and small interfering (si-RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III-mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.

  14. Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

    Science.gov (United States)

    Radou, Gaël; Dreyer, Frauke N.; Tuma, Roman; Paci, Emanuele

    2014-01-01

    The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring. PMID:25140434

  15. Dicer uses distinct modules for recognizing dsRNA termini.

    Science.gov (United States)

    Sinha, Niladri K; Iwasa, Janet; Shen, Peter S; Bass, Brenda L

    2018-01-19

    Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  16. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

    Science.gov (United States)

    Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

    2000-06-01

    Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

  17. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

    Science.gov (United States)

    Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

    2016-11-01

    A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

  19. Extracellular signaling and multicellularity in Bacillus subtilis.

    Science.gov (United States)

    Shank, Elizabeth Anne; Kolter, Roberto

    2011-12-01

    Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  1. A mechanical mechanism for translocation of ring-shaped helicases on DNA and its demonstration in a macroscopic simulation system

    Science.gov (United States)

    Chou, Y. C.

    2018-04-01

    The asymmetry in the two-layered ring structure of helicases and the random thermal fluctuations of the helicase and DNA molecules are considered as the bases for the generation of the force required for translocation of the ring-shaped helicase on DNA. The helicase comprises a channel at its center with two unequal ends, through which strands of DNA can pass. The random collisions between the portion of the DNA strand in the central channel and the wall of the channel generate an impulsive force toward the small end. This impulsive force is the starting point for the helicase to translocate along the DNA with the small end in front. Such a physical mechanism may serve as a complementary for the chemomechanical mechanism of the translocation of helicase on DNA. When the helicase arrives at the junction of ssDNA and dsDNA (a fork), the collision between the helicase and the closest base pair may produce a sufficient impulsive force to break the weak hydrogen bond of the base pair. Thus, the helicase may advance and repeat the process of unwinding the dsDNA strand. This mechanism was tested in a macroscopic simulation system where the helicase was simulated using a truncated-cone structure and DNA was simulated with bead chains. Many features of translocation and unwinding such as translocation on ssDNA and dsDNA, unwinding of dsDNA, rewinding, strand switching, and Holliday junction resolution were reproduced.

  2. RTEL1: functions of a disease-associated helicase.

    Science.gov (United States)

    Vannier, Jean-Baptiste; Sarek, Grzegorz; Boulton, Simon J

    2014-07-01

    DNA secondary structures that arise during DNA replication, repair, and recombination (3R) must be processed correctly to prevent genetic instability. Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles a variety of DNA secondary structures to facilitate 3R processes and to maintain telomere integrity. The past few years have witnessed the emergence of RTEL1 variants that confer increased susceptibility to high-grade glioma, astrocytomas, and glioblastomas. Mutations in RTEL1 have also been implicated in Hoyeraal-Hreidarsson syndrome, a severe form of the bone-marrow failure and cancer predisposition disorder, dyskeratosis congenita. We review these recent findings and highlight its crucial link between DNA secondary-structure metabolism and human disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  4. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    OpenAIRE

    Mukherjee, Sourav; Weiner, Warren S.; Schroeder, Chad E.; Simpson, Denise S.; Hanson, Alicia M.; Sweeney, Noreena L.; Marvin, Rachel K.; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J.; Frick, David N.

    2014-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previousl...

  5. Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

    Science.gov (United States)

    Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

    2014-10-17

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

  6. Bacillus subtilis biofilm induction by plant polysaccharides.

    Science.gov (United States)

    Beauregard, Pascale B; Chai, Yunrong; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2013-04-23

    Bacillus subtilis is a plant-beneficial Gram-positive bacterium widely used as a biofertilizer. However, relatively little is known regarding the molecular processes underlying this bacterium's ability to colonize roots. In contrast, much is known about how this bacterium forms matrix-enclosed multicellular communities (biofilms) in vitro. Here, we show that, when B. subtilis colonizes Arabidopsis thaliana roots it forms biofilms that depend on the same matrix genes required in vitro. B. subtilis biofilm formation was triggered by certain plant polysaccharides. These polysaccharides served as a signal for biofilm formation transduced via the kinases controlling the phosphorylation state of the master regulator Spo0A. In addition, plant polysaccharides are used as a source of sugars for the synthesis of the matrix exopolysaccharide. The bacterium's response to plant polysaccharides was observed across several different strains of the species, some of which are known to have beneficial effects on plants. These observations provide evidence that biofilm genes are crucial for Arabidopsis root colonization by B. subtilis and provide insights into how matrix synthesis may be triggered by this plant.

  7. The methionine salvage pathway in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Danchin Antoine

    2002-04-01

    Full Text Available Abstract Background Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR. Very little was known about MTR recycling for methionine salvage in Bacillus subtilis. Results Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere as a major step in MTR recycling. Conclusions A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane.

  8. The Cell Wall of Bacillus subtilis

    NARCIS (Netherlands)

    Scheffers, Dirk-Jan; Graumann, Peter

    2012-01-01

    The cell wall of Bacillus subtilis is a rigid structure on the outside of the cell that forms the first barrier between the bacterium and the environment, and at the same time maintains cell shape and withstands the pressure generated by the cell’s turgor. In this chapter, the chemical composition

  9. Close encounters for the first time: Helicase interactions with DNA damage.

    Science.gov (United States)

    Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

    2015-09-01

    DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

  10. ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

    Science.gov (United States)

    Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

    2017-01-01

    Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  12. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  13. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

    Directory of Open Access Journals (Sweden)

    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  14. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    Science.gov (United States)

    Leventhal, J M; Chambliss, G H

    1982-12-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

  15. Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

    Science.gov (United States)

    Hyun, Moonjung; Park, Sojin; Kim, Eunsun; Kim, Do-Hyung; Lee, Se-Jin; Koo, Hyeon-Sook; Seo, Yeon-Soo; Ahn, Byungchan

    2012-02-21

    The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

  16. Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

    Science.gov (United States)

    Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

    2014-06-01

    Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Development of Bacillus subtilis mutants to produce tryptophan in pigs

    DEFF Research Database (Denmark)

    Bjerre, Karin; Cantor, Mette D.; Nørgaard, Jan Værum

    2017-01-01

    Objectives To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. Results A novel concept has been investigated—to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis......-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications....

  18. Logarithmic sensing in Bacillus subtilis aerotaxis.

    Science.gov (United States)

    Menolascina, Filippo; Rusconi, Roberto; Fernandez, Vicente I; Smriga, Steven; Aminzare, Zahra; Sontag, Eduardo D; Stocker, Roman

    2017-01-01

    Aerotaxis, the directed migration along oxygen gradients, allows many microorganisms to locate favorable oxygen concentrations. Despite oxygen's fundamental role for life, even key aspects of aerotaxis remain poorly understood. In Bacillus subtilis, for example, there is conflicting evidence of whether migration occurs to the maximal oxygen concentration available or to an optimal intermediate one, and how aerotaxis can be maintained over a broad range of conditions. Using precisely controlled oxygen gradients in a microfluidic device, spanning the full spectrum of conditions from quasi-anoxic to oxic (60 n mol/l-1 m mol/l), we resolved B. subtilis' 'oxygen preference conundrum' by demonstrating consistent migration towards maximum oxygen concentrations ('monotonic aerotaxis'). Surprisingly, the strength of aerotaxis was largely unchanged over three decades in oxygen concentration (131 n mol/l-196 μ mol/l). We discovered that in this range B. subtilis responds to the logarithm of the oxygen concentration gradient, a rescaling strategy called 'log-sensing' that affords organisms high sensitivity over a wide range of conditions. In these experiments, high-throughput single-cell imaging yielded the best signal-to-noise ratio of any microbial taxis study to date, enabling the robust identification of the first mathematical model for aerotaxis among a broad class of alternative models. The model passed the stringent test of predicting the transient aerotactic response despite being developed on steady-state data, and quantitatively captures both monotonic aerotaxis and log-sensing. Taken together, these results shed new light on the oxygen-seeking capabilities of B. subtilis and provide a blueprint for the quantitative investigation of the many other forms of microbial taxis.

  19. Cdt1 stabilizes an open MCM ring for helicase loading.

    Science.gov (United States)

    Frigola, Jordi; He, Jun; Kinkelin, Kerstin; Pye, Valerie E; Renault, Ludovic; Douglas, Max E; Remus, Dirk; Cherepanov, Peter; Costa, Alessandro; Diffley, John F X

    2017-06-23

    ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.

  20. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    Directory of Open Access Journals (Sweden)

    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  1. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-01-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

  2. Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis.

    Science.gov (United States)

    Stickel, Felix; Droz, Sara; Patsenker, Eleonora; Bögli-Stuber, Katja; Aebi, Beat; Leib, Stephen L

    2009-01-01

    Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

  3. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  4. Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production ▿

    OpenAIRE

    Kubo, Yuji; Rooney, Alejandro P.; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

    2011-01-01

    Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analy...

  5. Distinct functions of human RecQ helicases during DNA replication.

    Science.gov (United States)

    Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

    2017-06-01

    DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Science.gov (United States)

    Tuteja, Narendra; Banu, Mst Sufara Akhter; Huda, Kazi Md Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  7. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Directory of Open Access Journals (Sweden)

    Narendra Tuteja

    Full Text Available The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum and its novel function in salinity stress tolerance in plant.The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities.To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  8. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Acquired Dependence of Acute Myeloid Leukemia on the DEAD-Box RNA Helicase DDX5

    Directory of Open Access Journals (Sweden)

    Anthony Mazurek

    2014-06-01

    Full Text Available Acute myeloid leukemia (AML therapy involves compounds that are cytotoxic to both normal and cancer cells, and relapsed AML is resistant to subsequent chemotherapy. Thus, agents are needed that selectively kill AML cells with minimal toxicity. Here, we report that AML is dependent on DDX5 and that inhibiting DDX5 expression slows AML cell proliferation in vitro and AML progression in vivo but is not toxic to cells from normal bone marrow. Inhibition of DDX5 expression in AML cells induces apoptosis via induction of reactive oxygen species (ROS. This apoptotic response can be blocked either by BCL2 overexpression or treatment with the ROS scavenger N-acetyl-L-cysteine. Combining DDX5 knockdown with a BCL2 family inhibitor cooperates to induce cell death in AML cells. By inhibiting DDX5 expression in vivo, we show that DDX5 is dispensable for normal hematopoiesis and tissue homeostasis. These results validate DDX5 as a potential target for blocking AML.

  10. Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6

    NARCIS (Netherlands)

    Reuß, Daniel R; Thürmer, Andrea; Daniel, Rolf; Quax, Wim J; Stülke, Jörg

    2016-01-01

    Bacillus subtilis ∆6 is a genome-reduced strain that was cured from six prophages and AT-rich islands. This strain is of great interest for biotechnological applications. Here, we announce the full-genome sequence of this strain. Interestingly, the conjugative element ICEBs1 has most likely

  11. Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

    DEFF Research Database (Denmark)

    Kusumoto-Matsuo, Rika; Opresko, Patricia L; Ramsden, Dale

    2010-01-01

    Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere...... D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo....

  12. Biodegradation of naphthalene and phenanthren by Bacillus subtilis 3KP

    Science.gov (United States)

    Ni'matuzahroh, Trikurniadewi, N.; Pramadita, A. R. A.; Pratiwi, I. A.; Salamun, Fatimah, Sumarsih, Sri

    2017-06-01

    The purposes of this research were to know growth response, degradation ability, and uptake mechanism of naphthalene and phenanthrene by Bacillus subtilis 3KP. Bacillus subtilis 3KP was grown on Mineral Synthetic (MS) medium with addition of 1% yeast extract and naphthalene and phenanthrene respectively 200 ppm in different cultures. Bacillus subtilis 3KP growth response was monitored by Total Plate Count (TPC) method, the degradation ability was monitored by UV-Vis spectrophotometer, and the uptake mechanism of hydrocarbon was monitored by emulsification activity, decrease of surface tension, and activity of Bacterial Adherence to Hydrocarbon (BATH). Bacillus subtilis 3KP was able to grow and show biphasic growth pattern on both of substrates. Naphthalene and phenanthrene were used as a carbon source for Bacillus subtilis 3KP growth that indicated by the reduction of substrate concomitant with the growth. At room temperature conditions (± 30°C) and 90 rpm of agitation for 7 days, Bacillus subtilis 3KP could degrade naphthalene in the amount of 70.5% and phenanthrene in the amount of 24.8%. Based on the analysis of UV-Vis spectrophotometer, three metabolites, 1-hydroxy-2-naphthoic acid, salicylic acid, and pyrocatechol were found in both cultures. The metabolite identification became basis of propose degradation pathway of naphthalene and phenanthrene by Bacillus subtilis 3KP. The results of hydrocarbon uptake mechanism test show that Bacillus subtilis 3KP used all of the mechanism to degrade naphthalene and phenanthrene.

  13. Bacillus subtilis Hfq: A role in chemotaxis and motility

    Indian Academy of Sciences (India)

    2016-07-16

    Jul 16, 2016 ... motility, thus assigning a new function for Hfq in B. subtilis. 1. Introduction. Hfq in ... to play a role in pathogenecity in mice, tolerance to osmotic and ethanol stress ...... in B. subtilis is characterized by events like surfactin pro- duction .... SM Cutting (New York: John Wiley and Sons Inc) pp 442–444. Nicolas P ...

  14. Effect of Bacillus subtilis on Granite Weathering: A Laboratory Experiment

    Science.gov (United States)

    Song, W.; Ogawa, N.; Oguchi, C. T.; Hatta, T.; Matsukura, Y.

    2006-12-01

    We performed a comparative experiment to investigate how the ubiquitous soil bacterium Bacillus subtilis weathers granite and which granite-forming minerals weather more rapidly via biological processes. Batch type experiments (granite specimen in a 500 ml solution including NaCl, glucose, yeast extract and bacteria Bacillus subtilis at 27°E C) were carried out for 30 days. Granite surfaces were observed by SEM before and after the experiment. Bacillus subtilis had a strong influence on granite weathering by forming pits. There were 2.4 times as many pits and micropores were 2.3 times wider in granite exposed to Bacillus subtilis when compared with bacteria-free samples. Bacillus subtilis appear to preferentially select an optimum place to adhere to the mineral and dissolve essential elements from the mineral to live. Plagioclase was more vulnerable to bacterial weathering than biotite among the granite composing minerals.

  15. Engineering of Bacillus subtilis 168 for increased nisin resistance

    DEFF Research Database (Denmark)

    Hansen, Mette; Wangari, Romilda; Hansen, Egon Bech

    2009-01-01

    . Bacillus subtilis had been suggested as a potential host for the biosynthesis of nisin but was discarded due to its sensitivity to the lethal action of nisin. In this study, we have reevaluated the potential of B. subtilis as a host organism for the heterologous production of nisin. We applied...... transcriptome and proteome analyses of B. subtilis and identified eight genes upregulated in the presence of nisin. We demonstrated that the overexpression of some of these genes boosts the natural defenses of B. subtilis, which allows it to sustain higher levels of nisin in the medium. We also attempted...... to overcome the nisin sensitivity of B. subtilis by introducing the nisin resistance genes nisFEG and nisI from L. lactis under the control of a synthetic promoter library....

  16. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

    Science.gov (United States)

    Munoz, L; Sadaie, Y; Doi, R H

    1978-10-10

    The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

  17. Antibacterial activity of antagonistic bacterium Bacillus subtilis DJM-51 against phytopathogenic Clavibacter michiganense subsp. michiganense ATCC 7429 in vitro.

    Science.gov (United States)

    Jung, W J; Mabood, F; Souleimanov, A; Whyte, L G; Niederberger, T D; Smith, D L

    2014-12-01

    To investigate antibacterial activity against the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429 (Cmm ATCC 7429), Bacillus subtilis DJM-51 was isolated from rhizosphere soil. For isolation of bacteria, samples were taken from rhizosphere soil. The isolate, DJA-51, had strong antagonistic ability against Tomato pathogen Cmm ATCC 7429 on nutrient-broth yeast extract agar (NBYA) as indicated by inhibition zones around colonies. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium has been identified as B. subtilis DJM-51. The growth of Cmm ATCC 7429 on NBYA plates was inhibited by culture broth of B. subtilis DJM-51 including cells, by the supernatant of culture broth of B. subtilis DJM-51, and by the liquid material resulting from butanol extract of bacterial cultures. The OD value in co-culture mixture was lower than the control throughout the entire incubation period. Antibiotics obtained from B. subtilis DJM-51 inhibited the growth of Tomato pathogen Cmm ATCC 7429. These results provide potentially information about the protection of tomato from pathogen Cmm ATCC 7429 under greenhouse conditions in Quebec. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

    Directory of Open Access Journals (Sweden)

    Manjula Pandey

    2014-03-01

    Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

  19. Potassium sensing histidine kinase in Bacillus subtilis.

    Science.gov (United States)

    López, Daniel; Gontang, Erin A; Kolter, Roberto

    2010-01-01

    The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Cannibalism enhances biofilm development in Bacillus subtilis.

    Science.gov (United States)

    López, Daniel; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2009-11-01

    Cannibalism is a mechanism to delay sporulation in Bacillus subtilis. Cannibal cells express the skf and sdp toxin systems to lyse a fraction of their sensitive siblings. The lysed cells release nutrients that serve to feed the community, effectively delaying spore formation. Here we provide evidence that the subpopulation of cells that differentiates into cannibals is the same subpopulation that produces the extracellular matrix that holds cells together in biofilms. Cannibalism and matrix formation are both triggered in response to the signalling molecule surfactin. Nutrients released by the cannibalized cells are preferentially used by matrix-producing cells, as they are the only cells expressing resistance to the Skf and Sdp toxins. As a result this subpopulation increases in number and matrix production is enhanced when cannibalism toxins are produced. The cannibal/matrix-producing subpopulation is also generated in response to antimicrobials produced by other microorganisms and may thus constitute a defense mechanism to protect B. subtilis from the action of antibiotics in natural settings.

  1. Fast Neutron Radiation Effects on Bacillus Subtili

    International Nuclear Information System (INIS)

    Chen Xiaoming; Zhang Jianguo; Chu Shijin; Ren Zhenglong; Zheng Chun; Yang Chengde; Tan Bisheng

    2009-01-01

    To examine the sterilizing effect and mechanism of neutron radiation, Bacillus subtilis var. niger. strain (ATCC 9372) spores were irradiated with the fast neutron from the Chinese fast burst reactor II(CFBR-II). The plate-count results indicated that the D 10 value was 384.6 Gy with a neutron radiation dose rate of 7.4 Gy/min. The rudimental catalase activity of the spores declined obviously with the increase in the radiation dose. Meanwhile, under the scanning electron microscope, no visible influence of the neutron radiation on the spore configuration was detected even if the dose was increased to 4 kGy. The content and distribution of DNA double-strand breaks induced by neutron radiation at different doses were measured and quantified by pulsed-field gel electrophoresis (PFGE). Further analysis of the DNA release percentage (PR), the DNA breakage level (L), and the average molecular weight, indicated that DNA fragments were obviously distributed around the 5 kb regions at different radiation doses, which suggests that some points in the DNA molecule were sensitive to neutron radiation. Both PR and L varied regularly to some extent with the increase in radiation dose. Thus neutron radiation has a high sterilization power, and can induce falling enzyme activity and DNA breakage in Bacillus subtilis spores

  2. A Small Molecule Inhibitor of the BLM Helicase Modulates Chromosome Stability in Human Cells

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Dexheimer, Thomas S; Rosenthal, Andrew S

    2013-01-01

    The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM f...

  3. BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation

    Czech Academy of Sciences Publication Activity Database

    Yodh, J.G.; Stevens, B.C.; Kanagaraj, R.; Janščák, Pavel; Ha, T.

    2009-01-01

    Roč. 28, č. 4 (2009), s. 405-416 ISSN 0261-4189 Institutional research plan: CEZ:AV0Z50520514 Keywords : Bloom syndrome * FRET * helicase * hRPA * single molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.993, year: 2009

  4. BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes

    NARCIS (Netherlands)

    van Wietmarschen, Niek; Merzouk, Sarra; Halsema, Nancy; Spierings, Diana C J; Guryev, Victor; Lansdorp, Peter M

    2018-01-01

    Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing

  5. Human RecQL4 helicase plays critical roles in prostate carcinogenesis

    DEFF Research Database (Denmark)

    Su, Yanrong; Meador, Jarah A; Calaf, Gloria M

    2010-01-01

    Prostate cancer is the second leading cause of cancer-associated deaths among men in the western countries. Here, we report that human RecQL4 helicase, which is implicated in the pathogenesis of a subset of cancer-prone Rothmund-Thomson syndrome, is highly elevated in metastatic prostate cancer c...

  6. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

    Science.gov (United States)

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

  7. MRE11 complex links RECQ5 helicase to sites of DNA damage

    Czech Academy of Sciences Publication Activity Database

    Zheng, L.; Kanagaraj, R.; Mihaljevic, B.; Schwendener, S.; Sartori, A.A.; Gerrits, B.; Shevelev, Igor; Janščák, Pavel

    2009-01-01

    Roč. 37, č. 8 (2009), s. 2645-2657 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Institutional research plan: CEZ:AV0Z50520514 Keywords : homologous recombination, * RECQ5 helicase * MRE11 * DNA repair Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.479, year: 2009

  8. FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

    Czech Academy of Sciences Publication Activity Database

    Šimandlová, Jitka; Zagelbaum, J.; Payne, M.J.; Chu, W.K.; Shevelev, Igor; Hanada, K.; Chatterjee, S.; Reid, D.A.; Liu, Y.; Janščák, Pavel; Rothenberg, E.; Hickson, I.D.

    2013-01-01

    Roč. 288, č. 47 (2013), s. 34168-34180 ISSN 0021-9258 R&D Projects: GA ČR GAP305/10/0281 Institutional support: RVO:68378050 Keywords : DNA damage * DNA helicase * DNA recombination * DNA repair * DNA replication Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.600, year: 2013

  9. Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

    DEFF Research Database (Denmark)

    Suhasini, Avvaru N; Rawtani, Nina A; Wu, Yuliang

    2011-01-01

    Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact...

  10. DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

    Science.gov (United States)

    Costa, Alessandro; Renault, Ludovic; Swuec, Paolo; Petojevic, Tatjana; Pesavento, James J; Ilves, Ivar; MacLellan-Gibson, Kirsty; Fleck, Roland A; Botchan, Michael R; Berger, James M

    2014-01-01

    The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. DOI: http://dx.doi.org/10.7554/eLife.03273.001 PMID:25117490

  11. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

    Directory of Open Access Journals (Sweden)

    Stefanie C Wolski

    2008-06-01

    Full Text Available DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

  12. Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

    Energy Technology Data Exchange (ETDEWEB)

    Matsuo, Eiko [Microbiology and Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-City 657-8501 (Japan); Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom); Leon, Esther; Matthews, Steve J. [Division of Molecular Biosciences, Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ (United Kingdom); Roy, Polly, E-mail: polly.roy@lshtm.ac.uk [Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom)

    2014-09-05

    Highlights: • NMR analysis on BTV VP6 reveals two large loop regions. • The loss of a loop (aa 34–130) does not affect the overall fold of the protein. • A region of VP6 (aa 34–92) is not required for BTV replication. • A region of VP6 (aa 93–130) plays an essential role in the virus replication. - Abstract: Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

  13. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    Science.gov (United States)

    Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

    2002-01-01

    Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

  14. DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

    Science.gov (United States)

    Jain, Aklank; Bacolla, Albino; Del Mundo, Imee M; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M

    2013-12-01

    Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

  15. Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM.

    Science.gov (United States)

    Xu, Yuli; Gristwood, Tamzin; Hodgson, Ben; Trinidad, Jonathan C; Albers, Sonja-Verena; Bell, Stephen D

    2016-11-22

    The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS•Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.

  16. Solution structure of GSP13 from Bacillus subtilis exhibits an S1 domain related to cold shock proteins

    International Nuclear Information System (INIS)

    Yu Wenyu; Hu Jicheng; Yu Bingke; Xia Wei; Jin Changwen; Xia Bin

    2009-01-01

    GSP13 encoded by gene yugI is a σ B -dependent general stress protein in Bacillus subtilis, which can be induced by heat shock, salt stress, ethanol stress, glucose starvation, oxidative stress and cold shock. Here we report the solution structure of GSP13 and it is the first structure of S1 domain containing protein in Bacillus subtilis. The structure of GSP13 mainly consists of a typical S1 domain along with a C-terminal 50-residue flexible tail, different from the other known S1 domain containing proteins. Comparison with other S1 domain structures reveals that GSP13 has a conserved RNA binding surface, and it may function similarly to cold shock proteins in response to cold stress

  17. The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

    Science.gov (United States)

    Busa, Veronica F; Rector, Maxwell J; Russell, Rick

    2017-07-18

    DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (k cat /K M ) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

  18. Sticking together: building a biofilm the Bacillus subtilis way.

    Science.gov (United States)

    Vlamakis, Hera; Chai, Yunrong; Beauregard, Pascale; Losick, Richard; Kolter, Roberto

    2013-03-01

    Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

  19. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

    Science.gov (United States)

    Cruz-Reyes, Jorge; Mooers, Blaine H.M.; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    2016-01-01

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. PMID:27540585

  20. Role of the ATPase/helicase maleless (MLE in the assembly, targeting, spreading and function of the male-specific lethal (MSL complex of Drosophila

    Directory of Open Access Journals (Sweden)

    Morra Rosa

    2011-04-01

    Full Text Available Abstract Background The male-specific lethal (MSL complex of Drosophila remodels the chromatin of the X chromosome in males to enhance the level of transcription of most X-linked genes, and thereby achieve dosage compensation. The core complex consists of five proteins and one of two non-coding RNAs. One of the proteins, MOF (males absent on the first, is a histone acetyltransferase that specifically acetylates histone H4 at lysine 16. Another protein, maleless (MLE, is an ATP-dependent helicase with the ability to unwind DNA/RNA or RNA/RNA substrates in vitro. Recently, we showed that the ATPase activity of MLE is sufficient for the hypertranscription of genes adjacent to a high-affinity site by MSL complexes located at that site. The helicase activity is required for the spreading of the complex to the hundreds of positions along the X chromosome, where it is normally found. In this study, to further understand the role of MLE in the function of the MSL complex, we analyzed its relationship to the other complex components by creating a series of deletions or mutations in its putative functional domains, and testing their effect on the distribution and function of the complex in vivo. Results The presence of the RB2 RNA-binding domain is necessary for the association of the MSL3 protein with the other complex subunits. In its absence, the activity of the MOF subunit was compromised, and the complex failed to acetylate histone H4 at lysine 16. Deletion of the RB1 RNA-binding domain resulted in complexes that maintained substantial acetylation activity but failed to spread beyond the high-affinity sites. Flies bearing this mutation exhibited low levels of roX RNAs, indicating that these RNAs failed to associate with the proteins of the complex and were degraded, or that MLE contributes to their synthesis. Deletion of the glycine-rich C-terminal region, which contains a nuclear localization sequence, caused a substantial level of retention of the

  1. A singular enzymatic megacomplex from Bacillus subtilis.

    Science.gov (United States)

    Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

    2007-01-02

    Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

  2. Studies on DNA repair in Bacillus subtilis

    International Nuclear Information System (INIS)

    Inoue, Tadashi; Kada, Tsuneo

    1977-01-01

    An enzyme which enhances the priming activity of γ-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells. The enzyme preferentially degraded γ-irradiated DNA into acid-soluble materials. DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme. However, sonication rendered DNA susceptible to the enzyme to some extent. From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by γ-irradiation to serve as effective primer of sites for repair synthesis by the type I DNA polymerase

  3. A love affair with Bacillus subtilis.

    Science.gov (United States)

    Losick, Richard

    2015-01-30

    My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Bacillus subtilis as potential producer for polyhydroxyalkanoates

    Directory of Open Access Journals (Sweden)

    Patel Sanjay KS

    2009-07-01

    Full Text Available Abstract Polyhydroxyalkanoates (PHAs are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB, the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.

  5. Bacillus subtilis as potential producer for polyhydroxyalkanoates.

    Science.gov (United States)

    Singh, Mamtesh; Patel, Sanjay Ks; Kalia, Vipin C

    2009-07-20

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process - for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.

  6. Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism

    DEFF Research Database (Denmark)

    Buescher, Joerg Martin; Liebermeister, Wolfram; Jules, Matthieu

    2012-01-01

    Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical...

  7. Two Genes Encoding Uracil Phosphoribosyltransferase Are Present in Bacillus subtilis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Glaser, Philippe; Andersen, Paal S.

    1995-01-01

    Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative...

  8. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity. ... Generally, alkaline proteases are produced using submerged fermentation .... biopolymer concentrations were reported to have an influence ... adding nitrogenous compounds stimulate microorganism growth and ...

  9. Enhanced biomass production study on probiotic Bacillus subtilis ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... INTRODUCTION. Probiotic organisms find their potential use in food and ..... complex nutrients, temperature and pH on bacteriocin production by. Bacillus subtilis ... B, Gupta R (2004). Application of statistical experimental.

  10. Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis.

    Science.gov (United States)

    Ulvatne, Hilde; Samuelsen, Ørjan; Haukland, Hanne H; Krämer, Manuela; Vorland, Lars H

    2004-08-15

    Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action. Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis. Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target. In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria. In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis. After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly. In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min. After 20 min RNA-synthesis increases. The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

  11. Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis

    OpenAIRE

    Yüksel, Melih; Power, Jeffrey J.; Ribbe, Jan; Volkmann, Thorsten; Maier, Berenike

    2016-01-01

    In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relati...

  12. DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr [Department of Life Sciences, University of Ulsan, Ulsan (Korea, Republic of); Bohr, Vilhelm A. [Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, Baltimore, MD (United States)

    2011-08-12

    Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

  13. Global transcriptional responses of Bacillus subtilis to xenocoumacin 1.

    Science.gov (United States)

    Zhou, T; Zeng, H; Qiu, D; Yang, X; Wang, B; Chen, M; Guo, L; Wang, S

    2011-09-01

    To determine the global transcriptional response of Bacillus subtilis to an antimicrobial agent, xenocoumacin 1 (Xcn1). Subinhibitory concentration of Xcn1 applied to B. subtilis was measured according to Hutter's method for determining optimal concentrations. cDNA microarray technology was used to study the global transcriptional response of B. subtilis to Xcn1. Real-time RT-PCR was employed to verify alterations in the transcript levels of six genes. The subinhibitory concentration was determined to be 1 μg ml(-1). The microarray data demonstrated that Xcn1 treatment of B. subtilis led to more than a 2.0-fold up-regulation of 480 genes and more than a 2.0-fold down-regulation of 479 genes (q ≤ 0.05). The transcriptional responses of B. subtilis to Xcn1 were determined, and several processes were affected by Xcn1. Additionally, cluster analysis of gene expression profiles after treatment with Xcn1 or 37 previously studied antibiotics indicated that Xcn1 has similar mechanisms of action to protein synthesis inhibitors. These microarray data showed alterations of gene expression in B. subtilis after exposure to Xcn1. From the results, we identified various processes affected by Xcn1. This study provides a whole-genome perspective to elucidate the action of Xcn1 as a potential antimicrobial agent. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  14. Bacillus subtilis strain specificity affects performance improvement in broilers.

    Science.gov (United States)

    Rhayat, L; Jacquier, V; Brinch, K S; Nielsen, P; Nelson, A; Geraert, P-A; Devillard, E

    2017-07-01

    The study reports the effects on broiler performance of a newly isolated Bacillus subtilis strain, which is phylogenetically not closely related to already well-described strains of B. subtilis. In the first experiment, birds were reared in battery cages and exposed to C. perfringens. An increase in growth performance was observed with the strain when compared to the challenged animals. Three additional growth trials were conducted to 35 d of age, in different rearing conditions (genetic breeds, corn-soybean meal-based diet with or without animal proteins, in presence or absence of phytase, on fresh or used litter) to investigate the efficacy and the specificity of this new B. subtilis strain on the improvement of BWG and FCR of broilers in comparison with a B. subtilis-based DFM already used in the field. Whatever the rearing conditions tested, the new B. subtilis strain led to an average 3.2% improvement in feed conversion ratio or bodyweight. Comparatively, the commercial Bacillus strain significantly improved broiler performance in only one trial out of 3 with an average improvement reaching 2%. All these results indicate that this new B. subtilis strain consistently improves broiler performances. © 2017 Poultry Science Association Inc.

  15. Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

    Science.gov (United States)

    Nicolas, Pierre; Repoila, Francis; Bardowski, Jacek; Aymerich, Stéphane

    2017-01-01

    In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks. PMID:28723971

  16. Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

    Science.gov (United States)

    Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

    2017-05-01

    Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

  17. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3′ to 5′ translocase and helicase activities

    OpenAIRE

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-01-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and...

  18. hSSB1 associates with and promotes stability of the BLM helicase

    OpenAIRE

    O'BYRNE, KEN

    2017-01-01

    Background Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. Results In this s...

  19. The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability

    Czech Academy of Sciences Publication Activity Database

    Singh, D.K.; Popuri, V.; Kulikowicz, T.; Shevelev, Igor; Ghosh, A.K.; Ramamoorthy, M.; Rossi, M.L.; Janščák, Pavel; Croteau, D.L.; Bohr, V.A.

    2012-01-01

    Roč. 40, č. 14 (2012), s. 6632-6648 ISSN 0305-1048 R&D Projects: GA ČR GAP305/10/0281 Grant - others:NIH(US) Z01-AG000726-17 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : RecQ helicase * genome stability * BLM * RECQL4 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  20. Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity

    Czech Academy of Sciences Publication Activity Database

    Schwendener, S.; Raynard, S.; Paliwal, S.; Cheng, A.; Kanagaraj, R.; Shevelev, Igor; Stark, J.M.; Sung, P.; Janscak, P.

    2010-01-01

    Roč. 285, č. 21 (2010), s. 15739-15745 ISSN 0021-9258 Grant - others:NIH(US) R01CA120954; NIH(US) ES015632; SNSF(CH) 3100A0-116008 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA helicase * double-strand breaks * homologous recombination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.328, year: 2010

  1. Distinct functions of human RecQ helicases during DNA replication

    Czech Academy of Sciences Publication Activity Database

    Urban, Václav; Dobrovolná, Jana; Janščák, Pavel

    2017-01-01

    Roč. 225, červen (2017), s. 20-26 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GA14-05743S; GA MŠk LH14037 Institutional support: RVO:68378050 Keywords : DNA replication * Replication stress * RecQ helicases * Genomic instability * Cancer Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.402, year: 2016

  2. Relocalization of nuclear DNA helicase II during the growth period of bovine oocytes

    Czech Academy of Sciences Publication Activity Database

    Baran, V.; Kovářová, Hana; Klíma, Jiří; Hozák, Pavel; Motlík, Jan

    2006-01-01

    Roč. 125, 1-2 (2006), s. 155-164 ISSN 0948-6143 R&D Projects: GA ČR GA523/03/0857 Grant - others:Slovenská Akademie věd(SK) VEGA 2/3065/23 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50390512 Keywords : DNA helicase II * fibroblasts * oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor : 3.220, year: 2006

  3. Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

    Science.gov (United States)

    Wang, Weiping; Manna, David; Simmons, Daniel T

    2007-05-01

    The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

  4. RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination

    OpenAIRE

    Uringa, Evert-Jan; Youds, Jillian L.; Lisaingo, Kathleen; Lansdorp, Peter M.; Boulton, Simon J.

    2010-01-01

    Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron–sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homolo...

  5. Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function

    Science.gov (United States)

    Miller, Justin M.; Enemark, Eric J.

    2015-01-01

    In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. PMID:26539061

  6. Isolation and characterization of atrazine mineralizing Bacillus subtilis strain HB-6.

    Directory of Open Access Journals (Sweden)

    Jinhua Wang

    Full Text Available Atrazine is a widely used herbicide with great environmental concern due to its high potential to contaminate soil and waters. An atrazine-degrading bacterial strain HB-6 was isolated from industrial wastewater and the 16S rRNA gene sequencing identified HB-6 as a Bacillus subtilis. PCR assays indicated that HB-6 contained atrazine-degrading genes trzN, atzB and atzC. The strain HB-6 was capable of utilizing atrazine and cyanuric acid as a sole nitrogen source for growth and even cleaved the s-triazine ring and mineralized atrazine. The strain demonstrated a very high efficiency of atrazine biodegradation with a broad optimum pH and temperature ranges and could be enhanced by cooperating with other bacteria, suggesting its huge potential for remediation of atrazine-contaminated sites. To our knowledge, there are few Bacillus subtilis strains reported that can mineralize atrazine, therefore, the present work might provide some new insights on atrazine remediation.

  7. Characterization of Lipase from Bacillus subtilisI-4 and Its Potential Use in Oil Contaminated Wastewater

    Directory of Open Access Journals (Sweden)

    Syeda Abeer Iqbal

    2015-10-01

    Full Text Available ABSTRACTA lipase producing bacterium was isolated from oil contaminated effluents of various industries from Sheikhupura Road, Pakistan, and, on the basis of biochemical and 16S rRNA ribotyping, was identified asBacillus subtilis. The optimum temperature and pH for the growth of the culture were 37ºC and 7.0, respectively.B. subtilis I-4 had a lag phase of 4 h in LB medium while this phase prolonged to 6 h in oil containing medium. The optimum temperature and pH for the enzyme activity were 50ºC and 7.0, respectively. Maximum lipase activity was found in the presence of Ca ions. Olive oil and Tween 80 induced lipase gene in the bacterium while concentration of oil greater than 2% retarded the growth of the organism. In addition to lipaseB. subtilis I-4 also produced alkane hydroxylase and biosurfactant which could make this bacterium potential candidate for lipase production as well as bioremediation of oil-contaminated wastewater.

  8. Escherichia coli and Neisseria gonorrhoeae UvrD helicase unwinds G4 DNA structures.

    Science.gov (United States)

    Shukla, Kaustubh; Thakur, Roshan Singh; Ganguli, Debayan; Rao, Desirazu Narasimha; Nagaraju, Ganesh

    2017-10-18

    G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  9. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.

    Science.gov (United States)

    Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K; McDevitt, Shane; Kashkina, Ekaterina; Hoang, Trung M; Pomerantz, Richard T; Sfeir, Agnel

    2017-12-01

    Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

  10. Non-B DNA Secondary Structures and Their Resolution by RecQ Helicases

    Directory of Open Access Journals (Sweden)

    Sudha Sharma

    2011-01-01

    Full Text Available In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics.

  11. Structural mechanisms of human RecQ helicases WRN and BLM

    Directory of Open Access Journals (Sweden)

    Ken eKitano

    2014-10-01

    Full Text Available The RecQ family DNA helicases WRN (Werner syndrome protein and BLM (Bloom syndrome protein play a key role in protecting the genome against deleterious changes. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. WRN and BLM are distinguished from other helicases by possessing signature tandem domains toward the C terminus, referred to as the RecQ C-terminal (RQC and helicase-and-ribonuclease D-C-terminal (HRDC domains. Although the precise function of the HRDC domain remains unclear, the previous crystal structure of a WRN RQC-DNA complex visualized a central role for the RQC domain in recognizing, binding and unwinding DNA at branch points. In particular, a prominent hairpin structure (the β-wing within the RQC winged-helix motif acts as a scalpel to induce the unpairing of a Watson-Crick base pair at the DNA duplex terminus. A similar RQC-DNA interaction was also observed in the recent crystal structure of a BLM-DNA complex. I review the latest structures of WRN and BLM, and then provide a docking simulation of BLM with a Holliday junction. The model offers an explanation for the efficient branch migration activity of the RecQ family toward recombination and repair intermediates.

  12. Helicase properties of the Escherichia coli UvrAb protein complex

    International Nuclear Information System (INIS)

    Oh, E.Y.; Grossman, L.

    1987-01-01

    The Escherichia coli UvrA protein has an associated ATPase activity with a turnover number affected by the presence of UvrB protein as well as by DNA. Specifically, the structure of DNA significantly influences the turnover rate of the UvrAB ATPase activity. Double-stranded DNA maximally activates the turnover rate 10-fold whereas single-stranded DNA maximally activates the turnover rate 20-fold, suggesting that the mode of interaction of UvrAB protein with different DNAs is distinctive. We have previously shown that the UvrAB protein complex, driven by the binding energy of ATP, can locally unwind supercoiled DNA. The nature of the DNA unwinding activity and single-stranded DNA activation of ATPase activity suggest potential helicase activity. In the presence of a number of helicase substrates, the UvrAB complex, indeed, manifests a strand-displacement activity-unwinding short duplexes and D-loop DNA, thereby generating component DNA structures. The energy for the activity is derived from ATP or dATP hydrolysis. Unlike the E. coli DnaB, the UvrAB helicase is sensitive to UV-induced photoproducts

  13. Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

    Energy Technology Data Exchange (ETDEWEB)

    Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka; Pigli, Ying Z.; Boyle-Vavra, Susan; Rice , Phoebe A. (UC)

    2016-08-29

    Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

  14. The Arabidopsis thaliana homolog of the helicase RTEL1 plays multiple roles in preserving genome stability.

    Science.gov (United States)

    Recker, Julia; Knoll, Alexander; Puchta, Holger

    2014-12-01

    In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. © 2014 American Society of Plant Biologists. All rights reserved.

  15. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    Science.gov (United States)

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  16. Bistability and Biofilm Formation in Bacillus subtilis

    Science.gov (United States)

    Chai, Yunrong; Chu, Frances; Kolter, Roberto; Losick, Richard

    2008-01-01

    Summary Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others. PMID:18047568

  17. Homolactic fermentation from glucose and cellobiose using Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Martinez Alfredo

    2009-04-01

    Full Text Available Abstract Backgroung Biodegradable plastics can be made from polylactate, which is a polymer made from lactic acid. This compound can be produced from renewable resources as substrates using microorganisms. Bacillus subtilis is a Gram-positive bacterium recognized as a GRAS microorganism (generally regarded as safe by the FDA. B. subtilis produces and secretes different kind of enzymes, such as proteases, cellulases, xylanases and amylases to utilize carbon sources more complex than the monosaccharides present in the environment. Thus, B. subtilis could be potentially used to hydrolyze carbohydrate polymers contained in lignocellulosic biomass to produce chemical commodities. Enzymatic hydrolysis of the cellulosic fraction of agroindustrial wastes produces cellobiose and a lower amount of glucose. Under aerobic conditions, B. subtilis grows using cellobiose as substrate. Results In this study, we proved that under non-aerated conditions, B. subtilis ferments cellobiose to produce L-lactate with 82% of the theoretical yield, and with a specific rate of L-lactate production similar to that one obtained fermenting glucose. Under fermentative conditions in a complex media supplemented with glucose, B. subtilis produces L-lactate and a low amount of 2,3-butanediol. To increase the L-lactate production of this organism, we generated the B subtilis CH1 alsS- strain that lacks the ability to synthesize 2,3-butanediol. Inactivation of this pathway, that competed for pyruvate availability, let a 15% increase in L-lactate yield from glucose compared with the parental strain. CH1 alsS- fermented 5 and 10% of glucose to completion in mineral medium supplemented with yeast extract in four and nine days, respectively. CH1 alsS- produced 105 g/L of L-lactate in this last medium supplemented with 10% of glucose. The L-lactate yield was up to 95% using mineral media, and the optical purity of L-lactate was of 99.5% since B. subtilis has only one gene (lctE that

  18. Production of Bioactive Compounds by Bacillus subtilis against Sclerotium rolfsii

    Directory of Open Access Journals (Sweden)

    Nalisha, I.

    2006-01-01

    Full Text Available This study aims to investigate the characteristic of bioactive compound produced by Bacillus subtilis against Sclerotium rolfsii and the influence of additive supplements on the antagonistic activity of B. subtilis. The fact that B. subtilis produced an antifungal substance which has inhibitory effect on wide range of fungi, including S. rolfsii, is well known. To learn the effect of pH, temperature and light condition on the production of antifungal compound, B. subtilis was inoculated in Potato Dextrose Broth at various initial pH, temperatures and light conditions, respectively. This antagonist was found to produce antifungal compound that stable at 80C with 58.3 % inhibition on S. rolfsii. The activity was constant within a wide range of pH (3–11. However, treatment with pH11 lead to higher antifungal activity (31.57 % inhibition and it was also found to produce substance that can endure dark condition (46.24 % inhibition with fungicidal effect on S. rolfsii. A series of experiments also been carried out to enhance the antifungal production by supplementing different carbon source preparation into bacterial liquid culture. B. subtilis were grown in minimal medium containing 1 % of oil palm root, Ganoderma lucidum or chitin, respectively prior to bioassay. Crude culture from oil palm root supplemented culture shown significantly reduction in S. rolfsii growth compared to other carbon source crude culture or the antagonism alone, suggesting that this approach may provide improved biocontrol efficiency.

  19. Removing Bacillus subtilis from fermentation broth using alumina nanoparticles.

    Science.gov (United States)

    Mu, Dashuai; Mu, Xin; Xu, Zhenxing; Du, Zongjun; Chen, Guanjun

    2015-12-01

    In this study, an efficient separation technology using Al2O3 nanoparticles (NPs) was developed for removing Bacillus subtilis from fermentation broth. The dosage of alumina nanoparticles used for separating B. subtilis increased during the culture process and remained stable in the stationary phase of the culture process. The pH of the culture-broth was also investigated for its effects on flocculation efficiency, and showed an acidic pH could enhance the flocculation efficiency. The attachment mechanisms of Al2O3 NPs to the B. subtilis surface were investigated, and the zeta potential analysis showed that Al2O3 NPs could attach to B. subtilis via electrostatic attachment. Finally, the metabolite content and the antibacterial effect of the fermentation supernatants were detected and did not significantly differ between alumina nanoparticle separation and centrifugation separation. Together, these results indicate a great potential for a highly efficient and economical method for removing B. subtilis from fermentation broth using alumina nanoparticles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. The ribosome uses two active mechanisms to unwind messenger RNA during translation.

    Science.gov (United States)

    Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

    2011-07-06

    The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

  1. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

    2016-04-22

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  2. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    International Nuclear Information System (INIS)

    Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  3. 40 CFR 180.1111 - Bacillus subtilis GB03; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis GB03; exemption from... FOOD Exemptions From Tolerances § 180.1111 Bacillus subtilis GB03; exemption from the requirement of a tolerance. The biofungicide Bacillus subtilis GB03 is exempted from the requirement of a tolerance in or on...

  4. 40 CFR 180.1128 - Bacillus subtilis MBI 600; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis MBI 600; exemption... FOOD Exemptions From Tolerances § 180.1128 Bacillus subtilis MBI 600; exemption from the requirement of... biofungicide Bacillus subtilis MBI 600 in or on all food commodities, including residues resulting from post...

  5. Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat ...

    African Journals Online (AJOL)

    Three strains, Bacillus subtilis B1, B. subtilis B18 and Bacillus thuringiensis B12, were screened from wheat bran to produce milk-clotting enzyme. Among them, B. subtilis B1 exhibited considerable milkclotting activity with low proteolytic activity. After response surface methodology optimization, milkclotting activity was ...

  6. Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

    Science.gov (United States)

    Awate, Sanket; Brosh, Robert M

    2017-06-08

    Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

  7. Crystallization and preliminary X-ray diffraction analysis of YisP protein from Bacillus subtilis subsp. subtilis strain 168

    International Nuclear Information System (INIS)

    Hu, Yumei; Jia, Shiru; Ren, Feifei; Huang, Chun-Hsiang; Ko, Tzu-Ping; Mitchell, Douglas A.; Guo, Rey-Ting; Zheng, Yingying

    2012-01-01

    A bacteria biofilm formation involved enzyme, BsYisP, from Bacillus subtilis subsp. subtilis strain 168, was crystallized and diffracted to 1.92 Å. YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Å resolution. Structure determination using MAD and MIR methods is in progress

  8. Fast neutron radiation inactivation of Bacillus subtilis: Absorbed dose determination

    International Nuclear Information System (INIS)

    Song Lingli; Zheng Chun; Ai Zihui; Li Junjie; Dai Shaofeng

    2011-01-01

    In this paper, fast neutron inactivation effects of Bacillus subtilis were investigated with fission fast neutrons from CFBR-II reactor of INPC (Institute of Nuclear Physics and Chemistry) and mono-energetic neutrons from the Van de Graaff accelerator at Peking University. The method for determining the absorbed dose in the Bacillus subtilis suspension contained in test tubes is introduced. The absorbed dose, on account of its dependence on the volume and the form of confined state, was determined by combined experiments and Monte Carlo method. Using the calculation results of absorbed dose, the fast neutron inactivation effects on Bacillus subtilis were studied. The survival rates and absorbed dose curve was constructed. (authors)

  9. 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.

  10. Ultrasensitivity of the Bacillus subtilis sporulation decision.

    Science.gov (United States)

    Narula, Jatin; Devi, Seram N; Fujita, Masaya; Igoshin, Oleg A

    2012-12-11

    Starving Bacillus subtilis cells execute a gene expression program resulting in the formation of stress-resistant spores. Sporulation master regulator, Spo0A, is activated by a phosphorelay and controls the expression of a multitude of genes, including the forespore-specific sigma factor σ(F) and the mother cell-specific sigma factor σ(E). Identification of the system-level mechanism of the sporulation decision is hindered by a lack of direct control over Spo0A activity. This limitation can be overcome by using a synthetic system in which Spo0A activation is controlled by inducing expression of phosphorelay kinase KinA. This induction results in a switch-like increase in the number of sporulating cells at a threshold of KinA. Using a combination of mathematical modeling and single-cell microscopy, we investigate the origin and physiological significance of this ultrasensitive threshold. The results indicate that the phosphorelay is unable to achieve a sufficiently fast and ultrasensitive response via its positive feedback architecture, suggesting that the sporulation decision is made downstream. In contrast, activation of σ(F) in the forespore and of σ(E) in the mother cell compartments occurs via a cascade of coherent feed-forward loops, and thereby can produce fast and ultrasensitive responses as a result of KinA induction. Unlike σ(F) activation, σ(E) activation in the mother cell compartment only occurs above the KinA threshold, resulting in completion of sporulation. Thus, ultrasensitive σ(E) activation explains the KinA threshold for sporulation induction. We therefore infer that under uncertain conditions, cells initiate sporulation but postpone making the sporulation decision to average stochastic fluctuations and to achieve a robust population response.

  11. 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

  12. Enhancement of Cellulase Production by Cellulomonas Fimi and Bacillus Subtilis

    International Nuclear Information System (INIS)

    Omer, A.M.

    2012-01-01

    Two bacterial strains identified as Cellulomonas fimi and Baciliius subtilus are cosidered as highly active cellulytic bacteria. Trials for maximizing the cellulolytic activites of the two strains were conducted. A maximum cellulase production was achieved at 1 and 1.5%carboxy methyl cellulose as carbon source, sodium nitrate and yeast as nitrogen source for Cellulomonas fimi and Bacillus subtilis, respectively. Incubation temprature at 30 and 45 degree C, ph at 6 and 7 achieved the highest activity of cellulase for Cellulomonas fimi and bacillus subtilis, respectively

  13. Antibiotic Stimulation of a Bacillus subtilis Migratory Response

    Science.gov (United States)

    Liu, Yongjin; Kyle, Steven

    2018-01-01

    ABSTRACT Competitive interactions between bacteria reveal physiological adaptations that benefit fitness. Bacillus subtilis is a Gram-positive species with several adaptive mechanisms for competition and environmental stress. Biofilm formation, sporulation, and motility are the outcomes of widespread changes in a population of B. subtilis. These changes emerge from complex, regulated pathways for adapting to external stresses, including competition from other species. To identify competition-specific functions, we cultured B. subtilis with multiple species of Streptomyces and observed altered patterns of growth for each organism. In particular, when plated on agar medium near Streptomyces venezuelae, B. subtilis initiates a robust and reproducible mobile response. To investigate the mechanistic basis for the interaction, we determined the type of motility used by B. subtilis and isolated inducing metabolites produced by S. venezuelae. Bacillus subtilis has three defined forms of motility: swimming, swarming, and sliding. Streptomyces venezuelae induced sliding motility specifically in our experiments. The inducing agents produced by S. venezuelae were identified as chloramphenicol and a brominated derivative at subinhibitory concentrations. Upon further characterization of the mobile response, our results demonstrated that subinhibitory concentrations of chloramphenicol, erythromycin, tetracycline, and spectinomycin all activate a sliding motility response by B. subtilis. Our data are consistent with sliding motility initiating under conditions of protein translation stress. This report underscores the importance of hormesis as an early warning system for potential bacterial competitors and antibiotic exposure. IMPORTANCE Antibiotic resistance is a major challenge for the effective treatment of infectious diseases. Identifying adaptive mechanisms that bacteria use to survive low levels of antibiotic stress is important for understanding pathways to

  14. Evolution of exploitative interactions during diversification in Bacillus subtilis biofilms

    DEFF Research Database (Denmark)

    Dragoš, Anna; Lakshmanan, Nivedha; Martin, Marivic

    2018-01-01

    variants. These variants can settle in alternative biofilm niches and develop new types of interactions that greatly influence population productivity. Here, we explore the evolutionary diversification of pellicle biofilms of the Gram positive, spore-forming bacterium Bacillus subtilis. We discover that......-similarly to other species-B. subtilis diversifies into distinct colony variants. These variants dramatically differ in biofilm formation abilities and expression of biofilm-related genes. In addition, using a quantitative approach, we reveal striking differences in surface complexity and hydrophobicity...

  15. Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Juan Campos Guillen

    2017-01-01

    Full Text Available Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD, which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3′ terminus of tRNAs may also be important in oxidative stress. The addition of the 3′ terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.

  16. 77 FR 73934 - Bacillus subtilis Strain QST 713 Variant Soil; Amendment to an Exemption From the Requirement of...

    Science.gov (United States)

    2012-12-12

    ... Bacillus subtilis Strain QST 713 To Include Residues of Bacillus subtilis Strain QST 713 Variant Soil... existing exemption from the requirement of a tolerance for residues of the Bacillus subtilis strain QST 713 in or on all food commodities by including residues of Bacillus subtilis strain QST 713 variant soil...

  17. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

    Science.gov (United States)

    Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

  18. Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

    DEFF Research Database (Denmark)

    Rossi, Marie L; Ghosh, Avik K; Kulikowicz, Tomasz

    2010-01-01

    Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer...... provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N...... activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases....

  19. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    Science.gov (United States)

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  20. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  1. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  2. Dissection of the functional domains of an archaeal holliday junction helicase

    DEFF Research Database (Denmark)

    Hong, Ye; Chu, Mingzhu; Li, Yansheng

    2012-01-01

    Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from...... conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting...

  3. Yeast as a model system to study RecQ helicase function

    DEFF Research Database (Denmark)

    Ashton, Thomas M; Hickson, Ian David

    2010-01-01

    Mutations in the highly conserved RecQ helicase, BLM, cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1(+), respectively. Studies in these yeast species have revealed a plethora...... of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast...

  4. New roles of the human Suv3 helicase in genome maintenance

    DEFF Research Database (Denmark)

    Venø, Susanne Trillingsgaard

    During her PhD studies, Susanne Trillingsgaard Venø carried out research into the role of the human Suv3 protein in stabilising the human genome – DNA. Suv3 is a helicase that separates the two strands of the DNA’s double helix. Throughout our lives, the DNA in our cells is constantly exposed...... maintenance. Based on these new research results, the Suv3 protein could be a valuable model for genome stability as an important factor in our understanding of why we get old....

  5. RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination.

    Science.gov (United States)

    Uringa, Evert-Jan; Youds, Jillian L; Lisaingo, Kathleen; Lansdorp, Peter M; Boulton, Simon J

    2011-03-01

    Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron-sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homologous recombination and discuss models linking RTEL1's enzymatic activity to its function in telomere maintenance and DNA repair.

  6. The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients

    DEFF Research Database (Denmark)

    Jensen, Martin Borch; Dunn, Christopher A; Keijzers, Guido

    2012-01-01

    RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations....... These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase...

  7. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2018-03-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  8. Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea

    OpenAIRE

    Bao-Hong Lee; Yi-Syuan Lai; She-Ching Wu

    2015-01-01

    Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained us...

  9. The impact of manganese on biofilm development of Bacillus subtilis

    NARCIS (Netherlands)

    Mhatre, Eisha; Troszok, Agnieszka; Gallegos-Monterrosa, Ramses; Lindstädt, Stefanie; Hölscher, Theresa; Kuipers, Oscar P.; Kovács, Ákos T.

    2016-01-01

    Bacterial biofilms are dynamic and structurally complex communities, involving cell-to-cell interactions. In recent years, various environmental signals were identified that induce the complex biofilm development of the Gram-positive bacterium Bacillus subtilis. These signaling molecules are often

  10. Enhanced biomass production study on probiotic Bacillus subtilis ...

    African Journals Online (AJOL)

    The culture conditions of lactose fermenting, spore forming probiotic Bacillus subtilis SK09 isolated from dairy effluent were optimized by response surface methodology to maximize the biomass production. The student's t-test of the Placket-Burman screening design revealed that the effects of pH, ammonium citrate and ...

  11. Characterization of a thermostable Bacillus subtilis β-amylase

    African Journals Online (AJOL)

    ... 70 0C respectively, and the thermal stability curve gave a maximum activity of 9.75 U at 70oC for 60 min of incubation. Bacillus subtilis â-amylase is valuable for maltose production, which can be hydrolyzed further by other groups of amylase for the production of high cassava glucose syrup used as sweeteners in the food ...

  12. PRODIGIOSIN INDUCES AUTOLYSINS IN ACTIVELY GROWN Bacillus subtilis CELLS

    Directory of Open Access Journals (Sweden)

    Tjasa eDanevcic

    2016-01-01

    Full Text Available Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on B. subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within two hours. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80 % compared to the wild-type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

  13. A part toolbox to tune genetic expression in Bacillus subtilis

    Science.gov (United States)

    Guiziou, Sarah; Sauveplane, Vincent; Chang, Hung-Ju; Clerté, Caroline; Declerck, Nathalie; Jules, Matthieu; Bonnet, Jerome

    2016-01-01

    Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis. PMID:27402159

  14. The transcriptionally active regions in the genome of Bacillus subtilis

    DEFF Research Database (Denmark)

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard

    2009-01-01

    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome...

  15. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

    NARCIS (Netherlands)

    Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P.; de, Vicente A.

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool

  16. Extracellular protease produced by Bacillus subtilis isolated from ...

    African Journals Online (AJOL)

    In a study to evaluate the microbiological safety of some paracetamol oral solutions sold in some Nigerian drug stores, 40.0% of the samples examined was contaminated with protease-producing Bacillus subtilis. The production of extracellular protease was induced by casein in the minimal medium and was found to be the ...

  17. Assembly properties of the Bacillus subtilis actin, MreB.

    Science.gov (United States)

    Mayer, Joshua A; Amann, Kurt J

    2009-02-01

    The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

  18. bmr3, a third multidrug transporter gene of Bacillus subtilis.

    OpenAIRE

    Ohki, R; Murata, M

    1997-01-01

    A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase.

  19. Genome engineering using a synthetic gene circuit in Bacillus subtilis.

    Science.gov (United States)

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-03-31

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis

    NARCIS (Netherlands)

    Krishnappa, Laxmi; Monteferrante, Carmine G; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten

    The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold

  1. Protein export in bacillus subtilis and escherichia coli

    NARCIS (Netherlands)

    Dijl, Jan Maarten van

    1990-01-01

    The export of heterologous proteins in Bacillus subtilis and Escherichia coli is often inefficient. Frequently observed problems are: 1) accumulation of the precursor form of the exported protein in the cytoplasm or in the membrane; 2), inefficient or incorrect processing of the precursor; 3),

  2. Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

    Science.gov (United States)

    Powers, Matthew J.; Sanabria-Valentín, Edgardo; Bowers, Albert A.

    2015-01-01

    ABSTRACT Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. IMPORTANCE Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that

  3. Anaerobic growth of Bacillus subtilis alters the spectrum of spontaneous mutations in the rpoB gene leading to rifampicin resistance.

    Science.gov (United States)

    Nicholson, Wayne L; Park, Roy

    2015-12-01

    Spontaneous rifampicin-resistant (RFM(R)) mutants were isolated from Bacillus subtilis 168 cultivated in the presence or absence of oxygen. By DNA sequencing, the mutations were located within Cluster I of the rpoB gene encoding the β subunit of RNA polymerase. The spectrum of RFM(R) rpoB mutations isolated from B. subtilis cells grown anaerobically differed from aerobically grown cells, not only with respect to the location of mutations within Cluster I but also in the class of mutation observed (transition versus transversion). In the absence of RFM, RFM(R) mutants exhibited poorer growth under anaerobic conditions than did the wild-type strain, indicating their lower fitness in the absence of antibiotic selection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. BIOMASS PRODUCTION AND FORMULATION OF Bacillus subtilis FOR BIOLOGICAL CONTROL

    Directory of Open Access Journals (Sweden)

    Amran Muis

    2016-10-01

    Full Text Available Bacillus subtilis is a widespread bacterium found in soil, water, and air. It controls the growth of certain harmful bacteria and fungi, presumably by competing for nutrients, growth sites on plants, and by directly colonizing and attaching to fungal pathogens. When applied to seeds, it colonizes the developing root system of the plants and continues to live on the root system and provides protection throughout the growing season. The study on biomass production and formulation of B. subtilis for biological control was conducted in the laboratory of Department of Plant Pathology, College of Agriculture, University of the Philippines Los Baños (UPLB-CA, College, Laguna from May to July 2005. The objective of the study was to determine the optimum pH and a good carbon source for biomass production of B. subtilis and to develop a seed treatment formulation of B. subtilis as biological control agent. Results showed that the optimum pH for growth of B. subtilis was pH 6 (1.85 x 109 cfu/ml. In laboratory tests for biomass production using cassava flour, corn flour, rice flour, and brown sugar as carbon sources, it grew best in brown sugar plus yeast extract medium (6.8 x 108 cfu ml-1 in sterile distilled water and 7.8 x 108 cfu ml-1 in coconut water. In test for bacterial biomass carriers, talc proved to be the best in terms of number of bacteria recovered from the seeds (3.98 x 105 cfu seed-1.

  5. Genetic Competence Drives Genome Diversity in Bacillus subtilis

    Science.gov (United States)

    Chevreux, Bastien; Serra, Cláudia R; Schyns, Ghislain; Henriques, Adriano O

    2018-01-01

    Abstract Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. PMID:29272410

  6. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    Science.gov (United States)

    Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

    2016-08-11

    Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

  7. Mechanisms of Action of Probiotics based on Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    A.V. Savustyanenko

    2016-04-01

    Full Text Available The bacterium B.subtilis is one of the most promi­sing probiotics studied in recent decades. Mechanisms of its probiotic action are associated with the synthesis of antimicrobial agents, increasing of non-specific and specific immunity, stimulation of growth of normal microflora of the intestine and the releasing of digestive enzymes. B.subtilis releases ribosomally synthesized peptides, non-ribosomally synthesized peptides and non-peptide substances with a broad spectrum of antimicrobial activity covering Gram-positive, Gram-negative bacteria, viruses and fungi. Resistance to these antimicrobial agents is rare. Enhancement of non-specific immunity is associated with macrophage activation and the release of pro-inflammatory cytokines from them, increasing of barrier function of the intestinal mucosa, releasing of vitamins and amino acids (including essential ones. Enhancement of specific immunity manifests by activation of T- and B-lymphocytes and the release from the latter of immunoglobulins — IgG and IgA. B.subtilis stimulates the growth of normal intestinal flora, in particular, bacteria of the genus Lactobacillus and Bifidobacterium. Furthermore, probiotic increases the diversity of intestinal microflora. Probiotic secretes all major digestive enzymes to the intestinal lumen: amylases, lipases, proteases, pectinases and cellulases. In addition to digestion, these enzymes destroy antinutritional factors and allergenic substances contained in the food. These mechanisms of action make reasonable the use of B.subtilis in the combination therapy to treat intestinal infections; prevention of respiratory infections during the cold season; prevention of antibiotic-associated diarrhea; for the correction of food digestion and movement impairments of various origin (errors in the diet, changes in the diet, diseases of the gastrointestinal tract, disorders of the autonomic nervous system, etc.. B.subtilis does not usually cause side effects. This

  8. Dimethylglycine provides salt and temperature stress protection to Bacillus subtilis.

    Science.gov (United States)

    Bashir, Abdallah; Hoffmann, Tamara; Smits, Sander H J; Bremer, Erhard

    2014-05-01

    Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a Kd value of approximate 172 μM; its Kd for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.

  9. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  10. Characterization of a novel single-stranded RNA mycovirus in pleurotus ostreatus

    International Nuclear Information System (INIS)

    Yu, Hyun Jae; Lim, Dongbin; Lee, Hyun-Sook

    2003-01-01

    A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group

  11. Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

    Science.gov (United States)

    Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

    2015-11-02

    The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

  12. Bloom syndrome helicase in meiosis: Pro-crossover functions of an anti-crossover protein.

    Science.gov (United States)

    Hatkevich, Talia; Sekelsky, Jeff

    2017-09-01

    The functions of the Bloom syndrome helicase (BLM) and its orthologs are well characterized in mitotic DNA damage repair, but their roles within the context of meiotic recombination are less clear. In meiotic recombination, multiple repair pathways are used to repair meiotic DSBs, and current studies suggest that BLM may regulate the use of these pathways. Based on literature from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, we present a unified model for a critical meiotic role of BLM and its orthologs. In this model, BLM and its orthologs utilize helicase activity to regulate the use of various pathways in meiotic recombination by continuously disassembling recombination intermediates. This unwinding activity provides the meiotic program with a steady pool of early recombination substrates, increasing the probability for a DSB to be processed by the appropriate pathway. As a result of BLM activity, crossovers are properly placed throughout the genome, promoting proper chromosomal disjunction at the end of meiosis. This unified model can be used to further refine the complex role of BLM and its orthologs in meiotic recombination. © 2017 WILEY Periodicals, Inc.

  13. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

    2011-09-21

    We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

  14. Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

    Science.gov (United States)

    Lõoke, Marko; Maloney, Michael F; Bell, Stephen P

    2017-02-01

    Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication. © 2017 Lõoke et al.; Published by Cold Spring Harbor Laboratory Press.

  15. G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

    Science.gov (United States)

    Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2015-01-01

    The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

  16. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita.

    Science.gov (United States)

    Ballew, Bari J; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P; Savage, Sharon A

    2013-04-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.

  17. Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

    Science.gov (United States)

    Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

    2014-01-01

    Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

  18. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  19. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  20. Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I

    2014-01-01

    Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathologic...

  1. Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

    DEFF Research Database (Denmark)

    Boskovic, Jasminka; Bragado-Nilsson, Elisabeth; Saligram Prabhakar, Bhargav

    2016-01-01

    DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replicati...

  2. Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

    Science.gov (United States)

    Seol, Yeonee; Strub, Marie-Paule; Neuman, Keir C.

    2016-01-01

    Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism. PMID:27131595

  3. Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

    Directory of Open Access Journals (Sweden)

    Marija Maric

    2017-03-01

    Full Text Available Disassembly of the Cdc45-MCM-GINS (CMG DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated. Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

  4. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  5. Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment

    DEFF Research Database (Denmark)

    Compaore, C. S.; Nielsen, Dennis S.; Ouoba, L. I. I.

    2013-01-01

    inhibited in the agar spot assay while only Gram-positive pathogens were inhibited in the agar well diffusion assay. Cell free supernatants (CFS) of pure cultures of 3 B. subtilis subsp. subtilis (G2, H4 and F1) strains inhibited growth of Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus...

  6. Emerging importance of helicases in plant stress tolerance: characterization of Oryza sativa repair helicase XPB2 promoter and its functional validation in tobacco under multiple stresses

    Directory of Open Access Journals (Sweden)

    Shailendra eRaikwar

    2015-12-01

    Full Text Available Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. DNA hHelicases, the unique molecular motors, are emerged as potentialprospective molecules to engineer stress tolerance in plants and are involved in a variety of DNA nucleic acid metabolismc processes including DNA repair. The DNA repair helicase, OsXPB2 is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress The analysis of promoter sequence from plant genome is important in understanding the gene regulation. Hereconditions. Here, we report the in silico analysis of novel stress inducible promoter of rice Oryza sativa OsXPB2 (OsXPB2. gene is reported. The in vivo validation of functionality/activity of novel stress inducible promoter of rice OsXPB2 gene promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. Our resultsThe present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration or cold and hormone (Auxin, ABA or MeJA induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA or ABA responsive, respectively. Functional analysis was done by Agrobacterium-transient assays using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present

  7. Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

    Science.gov (United States)

    Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

    2016-01-15

    Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Selective RNA targeting and regulated signaling by RIG-I is controlled by coordination of RNA and ATP binding.

    Science.gov (United States)

    Fitzgerald, Megan E; Rawling, David C; Potapova, Olga; Ren, Xiaoming; Kohlway, Andrew; Pyle, Anna Marie

    2017-02-17

    RIG-I is an innate immune receptor that detects and responds to infection by deadly RNA viruses such as influenza, and Hepatitis C. In the cytoplasm, RIG-I is faced with a difficult challenge: it must sensitively detect viral RNA while ignoring the abundance of host RNA. It has been suggested that RIG-I has a ‘proof-reading’ mechanism for rejecting host RNA targets, and that disruptions of this selectivity filter give rise to autoimmune diseases. Here, we directly monitor RNA proof-reading by RIG-I and we show that it is controlled by a set of conserved amino acids that couple RNA and ATP binding to the protein (Motif III). Mutations of this motif directly modulate proof-reading by eliminating or enhancing selectivity for viral RNA, with major implications for autoimmune disease and cancer. More broadly, the results provide a physical explanation for the ATP-gated behavior of SF2 RNA helicases and receptor proteins.

  9. Antimicrobial gageomacrolactins characterized from the fermentation of the marine-derived bacterium Bacillus subtilis under optimum growth conditions.

    Science.gov (United States)

    Tareq, Fakir Shahidullah; Kim, Ji Hye; Lee, Min Ah; Lee, Hyi-Seung; Lee, Jong-Seok; Lee, Yeon-Ju; Shin, Hee Jae

    2013-04-10

    Marine bacteria are a potential source of structurally diversified bioactive secondary metabolites that are not found in terrestrial sources. In our continuous effort to search for new antimicrobial agents from marine-derived bacteria, we isolated bacterial strain 109GGC020 from a marine sediment sample collected from Gageocho, Republic of Korea. The strain was identified as Bacillus subtilis based on a 16s rRNA sequence analysis. After a 7-day fermentation of the B. subtilis strain under optimum growth conditions three new and four known secondary metabolites were discovered using chromatographic procedures, and their biological activities were evaluated against both bacteria and crop-devastating fungi. The discovered metabolites were confirmed by extensive 2D NMR and high-resolution ESI-MS data analyses to have the structures of new macrolactin derivatives gageomacrolactins 1-3 and known macrolactins A (4), B (5), F (6), and W (7). The stereoconfigurations of 1-3 were assigned based on coupling constant values, chemical derivatization studies, and a literature review. The coupling constants were very crucial to determine the relative geometries of olefins in 1-3 because of overlap of the ¹H NMR signals. The NMR data of these compounds were recorded in different solvents to overcome this problem and obtain accurate coupling constant values. The new macrolactin derivatives 1-3 displayed good antibiotic properties against both Gram-positive (S. aureus, B. subtilis, and B. cereus) and Gram-negative (E. coli, S. typhi, and P. aeruginosa) bacteria with minimum inhibitory concentration (MIC) values of 0.02-0.05 μM. Additionally, the antifungal activities of 1-7 were evaluated against pathogenic fungi and found to inhibit mycelial growth of A. niger, B. cinerea, C. acutatum, C. albicans, and R. solani with MIC values of 0.04-0.3 μM, demonstrating that these compounds were good fungicides.

  10. Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications.

    Science.gov (United States)

    Gu, Yang; Xu, Xianhao; Wu, Yaokang; Niu, Tengfei; Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Liu, Long

    2018-05-15

    Bacillus subtilis is the most characterized gram-positive bacterium that has significant attributes, such as growing well on cheap carbon sources, possessing clear inherited backgrounds, having mature genetic manipulation methods, and exhibiting robustness in large-scale fermentations. Till date, B. subtilis has been identified as attractive hosts for the production of recombinant proteins and chemicals. By applying various systems and synthetic biology tools, the productivity features of B. subtilis can be thoroughly analyzed and further optimized via metabolic engineering. In the present review, we discussed why B. subtilis is the primary organisms used for metabolic engineering and industrial applications. Additionally, we summarized the recent advances in systems and synthetic biology, engineering strategies for improving cellular performances, and metabolic engineering applications of B. subtilis. In particular, we proposed emerging opportunities and essential strategies to enable the successful development of B. subtilis as microbial cell factories. Copyright © 2018. Published by Elsevier Inc.

  11. Tryptophan provision by dietary supplementation of a Bacillus subtilis mutant strain in piglets

    DEFF Research Database (Denmark)

    Torres-Pitarch, A; Nielsen, B.; Canibe, Nuria

    2015-01-01

    Supplementing Bacillus (B.) subtilis mutants selected to overproduce a specific amino acid (AA) may be an alternative method to provide essential AA in pig diets. Two experiments on a B. subtilis strain selected to overproduce Trp were conducted using 8-kg pigs fed Trp-deficient diets for 20 d. B....... subtilis were supplied in a low or high dose in Experiments 1 and 2, respectively. The Trp-deficient diet (0.15 SID Trp:Lys) reduced (p subtilis strain was not able...... to counterbalance the Trp deficiency in any of the two experiments. No effect of B. subtilis supplementation to piglet diets was observed on the plasma AA profile. In conclusion, this mutant strain of B. subtilis was not able to compensate a Trp deficiency in the tested doses....

  12. Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response.

    Directory of Open Access Journals (Sweden)

    Nicolas Tremblay

    2016-07-01

    Full Text Available Spliceosomal SNRNP200 is a Ski2-like RNA helicase that is associated with retinitis pigmentosa 33 (RP33. Here we found that SNRNP200 promotes viral RNA sensing and IRF3 activation through the ability of its amino-terminal Sec63 domain (Sec63-1 to bind RNA and to interact with TBK1. We show that SNRNP200 relocalizes into TBK1-containing cytoplasmic structures upon infection, in contrast to the RP33-associated S1087L mutant, which is also unable to rescue antiviral response of SNRNP200 knockdown cells. This functional rescue correlates with the Sec63-1-mediated binding of viral RNA. The hindered IFN-β production of knockdown cells was further confirmed in peripheral blood cells of RP33 patients bearing missense mutation in SNRNP200 upon infection with Sendai virus (SeV. This work identifies a novel immunoregulatory role of the spliceosomal SNRNP200 helicase as an RNA sensor and TBK1 adaptor for the activation of IRF3-mediated antiviral innate response.

  13. Generation of multiple cell types in Bacillus subtilis.

    Science.gov (United States)

    Lopez, Daniel; Vlamakis, Hera; Kolter, Roberto

    2009-01-01

    Bacillus subtilis is a Gram-positive bacterium that is well known for its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In fact, populations of genetically identical B. subtilis comprise numerous distinct cell types. In addition to spores, cells can become genetically competent, motile, produce extracellular matrix or degradative enzymes, or secrete toxins that allow them to cannibalize their neighbors. Many of the cell fates listed above appear to be mutually exclusive. In this review, we discuss how individual cells within a population control their gene expression to ensure that proper regulation of differentiation occurs. These different cell fates are regulated by an intricate network that relies primarily on the activity of three major transcriptional regulators: Spo0A, DegU, and ComK. While individual cells must choose distinct cell fates, the population as a whole exhibits a spectrum of phenotypes whose diversity may increase fitness.

  14. Study of UV-mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Lotareva, O.V.; Filippov, V.D.

    1974-01-01

    The sensitivity of Bac. subtilis to the inactivating and mutagenic effects of UV-mutants has been determined: uvr, which does not extract pyrimidine dimers from damaged DNA; recsub(x), which exhibits a reduced activity of ATP-dependent DNAase; poll, which is devoid of DNA polymerase, and wild strains (DT). The sensitivity of these strains to the inactivating effects of UV rays increases in the order: DT<= recsub(x) << uvr < poll, and UV mutability in the order: DT = rec(sub(x) < poll<< uvr. A comparison of UV mutagenesis in Bac. subtilis and E. coli suggests the hypothesis that the mechanisms of UV mutation formation are similar in these two organisms. (author)

  15. Study of UV-induced mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Filippov, V.D.; Lotareva, O.V.

    1978-01-01

    The mechanism of UV-induced mutagenesis was studied in Bacillus subtilis departing from the assumption that a lower yield of UV-induced mutations should be found in mutants deficient in the recombination if production of mutations is coupled with the recombination process. Three recombination-deficient strains were used: two (recA and recF) with defects in different recombination pathways and the third (recB) has a block at a stage common for both of them. UV light induced reversions to prototrophy in recB cells and did not in recA and recF strains. Direct mutations, which confer to the cell additional growth requirements, were induced by UV light in recA and recF mutants. It is concluded that UV-induced mutagenesis in B subtilis is independent of the two known recombination mechanisms

  16. Sensitivity of thermally treated Bacillus subtilis spores to subsequent irradiation

    International Nuclear Information System (INIS)

    Mostafa, S.A.; El-Zawahry, Y.A.; Awny, N.M.

    1986-01-01

    B. subtilis spores exposed to thermal treatment at 70 or 80 0 C for 1 hr were more sensitive to subsequent radiation exposure than non-heated spores. Deactivation of previously heated spores by increasing dose of 0-radiation followed an exponential function while, for non-heated spores a shoulder followed by exponential deactivation was noticed. Combined heat-radiation treatment exhibited a synergistic effect on spore deactivation at low irradiation doses, while at high irradiation doses, the effect was more or less additive. Added values of spore injury was higher for B. subtilis spores that received heat and radiation separately than the observed injury for spores that received combined treatment (heat followed by radiation). Results of spore deactivation and injury due to heat followed by radiation treatment are discussed in comparison to those of spores that received radiation-heat sequence

  17. Biodegradation of furfural by Bacillus subtilis strain DS3.

    Science.gov (United States)

    Zheng, Dan; Bao, Jianguo; Lu, Jueming; Lv, Quanxi

    2015-07-01

    An aerobic bacterial strain DS3, capable of growing on furfural as sole carbon source, was isolated from actived sludge of wastewater treatment plant in a diosgenin factory after enrichment. Based on morphological physiological tests as well as 16SrDNA sequence and Biolog analyses it was identified as Bacillus subtilis. The study revealed that strain DS3 utilized furfural, as analyzed by high-performance liquid chromatography (HPLC). Under following conditions: pH 8.0, temperature 35 degrees C, 150 rpm and 10% inoculum, strain DS3 showed 31.2% furfural degradation. Furthermore, DS3 strain was found to tolerate furfural concentration as high as 6000 mg(-1). The ability of Bacillus subtilis strain DS3 to degrade furfural has been demonstrated for the first time in the present study.

  18. Production, regulation and transportation of bacillibactin in bacillus subtilis

    International Nuclear Information System (INIS)

    Raza, W.; Hussain, Q.; Shen, Q.

    2012-01-01

    Bacillus subtilis produces a catecholate type siderophore 'Bacillibactin'. This review focuses on the non-ribosomal synthesis, transport and regulation of bacillibactin. Bacillibactin biosynthetic operon contains five genes (dhbACEBF). The uptake of bacillibactin requires the FeuABC transporter, inner-membrane permease, FepDG and YusV ATPase and an esterase encoding gene, besA and while export required YmfE major facilitator super-family (MFS)-type transporter. Fur is the major iron-controlled transcriptional regulator in B. subtilis, which acts as an iron-dependent repressor of the dhb operon in vivo while an iron-independent repressor in vitro. Knowledge of the Fur regulon will be useful in interpreting other global analysis of transcriptional responses. (author)

  19. Effect of Bacillus subtilis microecological probiotics on livestock breeding

    Directory of Open Access Journals (Sweden)

    Xiaohui ZHOU

    2016-10-01

    Full Text Available As a kind of green and healthy microecologics, Bacillus subtilis could balance the intestinal flora, promote the nutrient absorption and enhance immunity. Microecologics is one of the ideal antibiotics alternative, which are effective in preventing and treating animal disease and promoting the growth and development of the animal. Because of its advantages, such as no toxin side effect and no residual or drug-resistant, microecologics has been used in livestock breeding widely. Here, we concluded the characteristics and mechanism of Bacillus subtilis,elaborated application of microecologics on livestock breeding, discussed its problems and suggested its solved methods. In the end, the future of microecologics was expected in order to provide a reference for subsequent livestock breeding.

  20. [Molecular cloning and expression of Nattokinase gene in Bacillus subtilis].

    Science.gov (United States)

    Liu, B Y; Song, H Y

    2002-05-01

    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR. The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene. The supernatant of the culture was collected after 15 h culture. The target proteins were identified by SDS-PAGE. Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture. The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively.

  1. Characterization of high hydrostatic pressure-injured Bacillus subtilis cells.

    Science.gov (United States)

    Inaoka, Takashi; Kimura, Keitarou; Morimatsu, Kazuya; Yamamoto, Kazutaka

    2017-06-01

    High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.

  2. Enhanced hydrocarbon biodegradation by a newly isolated bacillus subtilis strain

    International Nuclear Information System (INIS)

    Christova, N.; Tuleva, B.; Nikolova-Damyanova, B.

    2004-01-01

    The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new bacillus subtilis 22BN strain was investigated. The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l -1 ). Biosurfactant production was detected by surface tension lowering and emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 ± 1% and 75 ± 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first report of bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant. (orig.)

  3. Enhanced secretion of natto phytase by Bacillus subtilis.

    Science.gov (United States)

    Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

    2015-01-01

    Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

  4. Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

    OpenAIRE

    Nicholson, W L; Chambliss, G H

    1987-01-01

    Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strai...

  5. Transcriptional regulation of the Bacillus subtilis menp1 promoter.

    OpenAIRE

    Qin, X; Taber, H W

    1996-01-01

    The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone. The menp1 promoter previously was shown to be the primary cis element for menFD gene expression. In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase. The effect on menp1 activity by a particular end product (such as acetoin or ...

  6. Screen for agents that induce autolysis in Bacillus subtilis.

    Science.gov (United States)

    Lacriola, Christopher J; Falk, Shaun P; Weisblum, Bernard

    2013-01-01

    The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis. SH-536 distinguishes between the two groups of autolysis-inducing agents by synthesizing and then releasing β-galactosidase (β-Gal) in late stationary phase at a time that cells have nearly stopped growing and are therefore tolerant of cell wall synthesis inhibitors. Four hits, named compound 2, compound 3, compound 5, and compound 24, obtained previously as inducers of autolysis by screening a 10,080-compound discovery library with BAU-102, were probed with SH-536 and found to release β-Gal, indicating that their mode of action was to permeabilize the B. subtilis cell membrane. The four primary hits inhibited growth in Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis, and Bacillus anthracis, with MICs in the 12.5- to 25-μg/ml (20 to 60 μM) range. The four primary hits were further used to probe B. subtilis, and their action was partially characterized with respect to the dependence of induced autolysis on specific autolysins.

  7. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    OpenAIRE

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, K?r?ad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor pro...

  8. Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis.

    Science.gov (United States)

    Makroczyová, Jana; Jamroškovič, Ján; Krascsenitsová, Eva; Labajová, Nad'a; Barák, Imrich

    2016-06-01

    In rod-shaped bacteria, the proper placement of the division septum at the midcell relies, at least partially, on the proteins of the Min system as an inhibitor of cell division. The main principle of Min system function involves the formation of an inhibitor gradient along the cell axis; however, the establishment of this gradient differs between two well-studied gram-negative and gram-positive bacteria. While in gram-negative Escherichia coli, the Min system undergoes pole-to-pole oscillation, in gram-positive Bacillus subtilis, proper spatial inhibition is achieved by the preferential attraction of the Min proteins to the cell poles. Nevertheless, when E.coli Min proteins are inserted into B.subtilis cells, they still oscillate, which negatively affects asymmetric septation during sporulation in this organism. Interestingly, homologs of both Min systems were found to be present in various combinations in the genomes of anaerobic and endospore-forming Clostridia, including the pathogenic Clostridium difficile. Here, we have investigated the localization and behavior of C.difficile Min protein homologs and showed that MinDE proteins of C.difficile can oscillate when expressed together in B.subtilis cells. We have also investigated the effects of this oscillation on B.subtilis sporulation, and observed decreased sporulation efficiency in strains harboring the MinDE genes. Additionally, we have evaluated the effects of C.difficile Min protein expression on vegetative division in this heterologous host. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  9. Use of bacillus subtilis strains to inhibit postharvest pathogenic fungi

    International Nuclear Information System (INIS)

    Arras, G.; Gambella, F.; Demontis, S.; Petretto, A.

    1995-01-01

    An isolate (87) of the bacillus subtilis strains isolated from cold stored citrus fruit 13 proved to inhibit the growth in vitro of the penicillium italicum used in the experiment (from 50.6% to 92.2%) and to inhibit botrytis cinerea (from 65.3% to 95.9%). A further test, superimposing on plates containing PDA strains Nos. 13, 173, and 160, totally inhibited the fungi. Tested in vivo on artificially bruised oranges, they significantly inhibited two fungi

  10. Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5.

    Science.gov (United States)

    Louber, Jade; Brunel, Joanna; Uchikawa, Emiko; Cusack, Stephen; Gerlier, Denis

    2015-07-28

    The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5'-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5-2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial. A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged. The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response

  11. Sporulation during growth in a gut isolate of Bacillus subtilis.

    Science.gov (United States)

    Serra, Cláudia R; Earl, Ashlee M; Barbosa, Teresa M; Kolter, Roberto; Henriques, Adriano O

    2014-12-01

    Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis.

    Science.gov (United States)

    Chen, H; Wang, L; Su, C X; Gong, G H; Wang, P; Yu, Z L

    2008-09-01

    Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics. The antagonistic activity of JA was tested in vitro; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l(-1) HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C18 column (5 microm, 250 x 4.6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization-mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments. Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens. This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis.

  13. Foam separation of Pseudomonas fluorescens and Bacillus subtilis var. niger.

    Science.gov (United States)

    Grieves, R B; Wang, S L

    1967-01-01

    An experimental investigation established the effect of the presence of inorganic salts on the foam separation of Pseudomonas fluorescens and of Bacillus subtilis var. niger (B. globigii) from aqueous suspension by use of a cationic surfactant. For P. fluorescens, 5.0 mueq/ml of NaCl, KCl, Na(2)SO(4), K(2)SO(4), CaCl(2), CaSO(4), MgCl(2), or MgSO(4) produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 x 10(5) up to 1.6 x 10(6) to 2.8 x 10(7) cells per milliliter (initial suspensions contain from 3.3 x 10(7) to 4.8 x 10(7) cells per milliliter). The exceptional influence of magnesium was overcome by bringing the cells into contact first with the surfactant and then the salt. For B. subtilis, the presence of 5.0 mueq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 x 10(4) to about 4.0 x 10(5) cells per milliliter. This occurred regardless of the sequence of contact as long as the surfactant contact period was sufficient. The presence of salts increased collapsed foam volumes with P. fluorescens and decreased collapsed foam volumes with B. subtilis.

  14. Bottleneck in secretion of α-amylase in Bacillus subtilis.

    Science.gov (United States)

    Yan, Shaomin; Wu, Guang

    2017-07-19

    Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.

  15. Molecular mechanisms involved in Bacillus subtilis biofilm formation

    Science.gov (United States)

    Mielich-Süss, Benjamin; Lopez, Daniel

    2014-01-01

    Summary Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil-dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programs of cellular specialization and cell-cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis. In particular, a special emphasis is placed on summarizing the most recent discoveries in the field and integrating them into the general view of these truly sophisticated microbial communities. PMID:24909922

  16. Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

    Science.gov (United States)

    Bleich, Rachel; Watrous, Jeramie D; Dorrestein, Pieter C; Bowers, Albert A; Shank, Elizabeth A

    2015-03-10

    Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

  17. RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

    Science.gov (United States)

    Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

    2018-05-08

    R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Inducible error-prone repair in B. subtilis. Progress report, September 1, 1979-February 28, 1981

    International Nuclear Information System (INIS)

    Yasbin, R.E.

    1980-10-01

    The mechanism of activation and the mode of action of the SOS system in Bacillus subtilis are being investigated. Interesting aspects of the SOS system in B. subtilis include: (1) the differences between the SOS functions in this bacterium and in the enteric bacteria; (2) the spontaneous activation of SOS functions in competent cells; and (3) the difficulty in establishing the presence of error-prone repair in this bacterium. In order to characterize the SOS system of B. subtilis, attempts will be made to: (1) isolate bacteria mutated in genes controlling various repair functions; (2) investigate inducible repair; (3) determine the role of endogenous prophages in DNA repair phenomena; and (4) utilize competent B. subtilis as a tester system for the detection of potential carcinogens. Data obtained during the past 18 months demonstrate: (1) the ability of the B. subtilis Comptest to detect potential environmental carcinogens; (2) the importance of DNA polymerase III in W-reactivation in B. subtilis; and (3) the control the bacteriophage SPβ has over the inducible DNA modification system in B. subtilis. Furthermore, the data also suggests the lack of error-prone repair in B. subtilis, and the differences which exist between the Bacilli and the enteric bacteria with regards to SOS phenomena. In order to further characterize inducible repair functions in B. subtilis, results will also be presented on attempts to mobilize error-prone repair systems of other bacterial species

  19. Genetic transformation of Bacillus strains close to bacillus subtilis and isolated from the soil

    International Nuclear Information System (INIS)

    Van, C.K.; Kuzin, Yu.Yu.; Kozlovskii, Yu.E.; Prozorov, A.A.

    1986-01-01

    Chromosomal and plasmid transformation was found in five out of 118 Bacillus strains, close or identical to Bacillus subtilis, and isolated from soil in Moscow or in the Moscow district. The efficiency of transformation in these strains was lower than that in derivatives of Bac. subtilis strain 168. In these strains the ability to undergo transformation was dependent on the rate of sporulation and the presence of restrictases. As in the case of Bac. subtilis 168 the strains isolated may be used as models in genetic transformation studies on Bac. subtilis

  20. Inducible error-prone repair in B. subtilis. Progress report, September 1, 1978-August 31, 1979

    International Nuclear Information System (INIS)

    Yasbin, R.E.

    1979-01-01

    The mechanism of activation and the mode of action of the SOS system in the bacterium Bacillus subtilis is under study. Interesting aspects of the SOS system in B. subtilis are: (1) the differences between SOS functions in this bacterium and in the enteric bacteria; (2) the spontaneous activation of SOS functions in component cells; and (3) the difficulty in obtaining consistent results for mutation studies in this bacterium. In order to characterize the SOS system of B. subtilis, it was proposed to: (1) isolate bacteria mutated in genes controlling various repair function; (2) investigate inducible repair; (3) determine the role of endogeneous Bacillus prophages in SOS functions; and (4) develop a tester system for potential carcinogens from competent Bacillus subtilis cells. Research has been able to: (1) isolate strains of B. subtilis in which the endogeneous prophages have been removed or neutralized; (2) demonstrate the association of one SOS function with prophage SPB; (3) demonstrate that the survival of uv-irradiated B. subtilis is not significantly altered by the removal and neutralization of the endogeneous prophages; (4) develop competant B. subtilis into a tester system; and (5) show that DNA polymerase III is absolutely necessary for W reactivation. In addition, uv and mitomycin C resistant mutants have been isolated and inducible postreplication repair in excision-repair deficient mutants of B. subtilis has been studied. The last two results are somewaht confusing but highly exciting in regards to DNA repair mechanisms in B. subtilis

  1. Inducible error-prone repair in B. subtilis. Progress report, May 1, 1983-April 30, 1984

    International Nuclear Information System (INIS)

    Yasbin, R.E.

    1983-12-01

    DNA repair mechanisms in Bacillus subtilis were investigated following mutagenesis via ultraviolet radiation or by chemical mutagens. A bioassay is described whereby the efficiency of repair mechanisms can be measured. DNA cloning studies to transfer the photoreactivation gene from E. coli to B. subtilis are reported. The mutation, which induces the SOS-like system in B. subtilis when grown at 45 0 C, was characterized in order to begin delineation of the genes controlling this system, efforts directed at isolation and cloning of a DNA Polymerase III gene of B. subtilis are related. (DT)

  2. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells

    DEFF Research Database (Denmark)

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J

    2013-01-01

    Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD......51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences...... filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under...

  3. RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.

    Science.gov (United States)

    Vannier, Jean-Baptiste; Sandhu, Sumit; Petalcorin, Mark I R; Wu, Xiaoli; Nabi, Zinnatun; Ding, Hao; Boulton, Simon J

    2013-10-11

    Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

  4. Association between regulator of telomere elongation helicase1 (RTEL1) gene and HAPE risk

    Science.gov (United States)

    Rong, Hao; He, Xue; Zhu, Linhao; Zhu, Xikai; Kang, Longli; Wang, Li; He, Yongjun; Yuan, Dongya; Jin, Tianbo

    2017-01-01

    Abstract High altitude pulmonary edema (HAPE) is a paradigm of pulmonary edema. Mutations in regulator of telomere elongation helicase1 (RTEL1) represent an important contributor to risk for pulmonary fibrosis. However, little information is found about the association between RTEL1 and HAPE risk. The present study was undertaken to tentatively explore the potential relation between single-nucleotide polymorphisms (SNPs) in RTEL1 and HAPE risk in Chinese Han population. A total of 265 HAPE patients and 303 healthy controls were included in our case-control study. Four SNPs in RTEL1 were selected and genotyped using the Sequenom MassARRAY method. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression with adjustment for gender and age. All P values were Bonferroni corrected, and statistical significance was set at P RTEL1 and a decreased risk HAPE in the Chinese population. The results need further confirmation. PMID:28953687

  5. Crystallization and preliminary X-ray analysis of the stress-response PPM phosphatase RsbX from Bacillus subtilis

    International Nuclear Information System (INIS)

    Suganuma, Masatoshi; Teh, Aik Hong; Makino, Masatomo; Shimizu, Nobutaka; Kaneko, Tomonori; Hirata, Kunio; Yamamoto, Masaki; Kumasaka, Takashi

    2009-01-01

    The bacterial PPM phosphatase RsbX from B. subtilis was expressed in E. coli, purified and crystallized. The crystal belonged to space group P1 and diffracted to 1.06 Å resolution. RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase σ factor SigB. In order to elucidate the structural–functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 Å resolution with an R merge of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 Å, α = 98.8, β = 90.0, γ = 108.4°

  6. Salt-sensitivity of σ(H) and Spo0A prevents sporulation of Bacillus subtilis at high osmolarity avoiding death during cellular differentiation.

    Science.gov (United States)

    Widderich, Nils; Rodrigues, Christopher D A; Commichau, Fabian M; Fischer, Kathleen E; Ramirez-Guadiana, Fernando H; Rudner, David Z; Bremer, Erhard

    2016-04-01

    The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete. © 2015 John Wiley & Sons Ltd.

  7. Salt-sensitivity of σH and Spo0A prevents sporulation of Bacillus subtilis at high osmolarity avoiding death during cellular differentiation

    Science.gov (United States)

    Widderich, Nils; Rodrigues, Christopher D.A.; Commichau, Fabian M.; Fischer, Kathleen E.; Ramirez-Guadiana, Fernando H.; Rudner, David Z.; Bremer, Erhard

    2016-01-01

    Summary The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete. PMID:26712348

  8. Effect of individual or combined treatment by γ-irradiation or temperature (high or low) on bacillus subtilis spores and its application for sterilization of ground beef

    International Nuclear Information System (INIS)

    El-Zawahry, Y.A.; Mostafa, S.A.; Awny, N.M.

    1986-01-01

    The combination of two lethal agents such as irradiation and temperature (high or sub zero) resulted in synergistic death or B. subtilis spores (as indicated by decrease in the thermal D-value). The extent of this synergism in killing a spore population depended mainly on the sequence on application of the two physical agents. Irradiation-temperature (high or sub zero) sequence killed more but injured less B. subtilis spores than temperature irradiation sequence or irradiation and temperature applied separately. Storage at -20 0 C killed more spores than storage at -2 0 C if carried after irradiation, while the reverse was true of storage was prior irradiation. An irradiation dose of 8 KGY followed by thermal exposure to 70 0 C for 1 hr is suggested for the sterilization of ground beef. Irradiation induced certain quantitative changes on the amino-N, protein-N, RNA and DNA of the first subcultures of irradiated spores with stimulatory effect at low irradiation doses and inhibitory effect at the high irradiation doses. This might explain the increased sensitivity of irradiated spores to subsequent exposure to unfavourable temperature (high or sub zero). Exposure of B. subtilis spore to 70 0 C induced a stimulation in the amino- and protein-N of the resulting cells while exposure to 80 0 C resulted in a significant decrease in the amino-N. The protein-N remained more or less the same

  9. Chl1 DNA helicase regulates Scc2 deposition specifically during DNA-replication in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Soumya Rudra

    Full Text Available The conserved family of cohesin proteins that mediate sister chromatid cohesion requires Scc2, Scc4 for chromatin-association and Eco1/Ctf7 for conversion to a tethering competent state. A popular model, based on the notion that cohesins form huge ring-like structures, is that Scc2, Scc4 function is essential only during G1 such that sister chromatid cohesion results simply from DNA replisome passage through pre-loaded cohesin rings. In such a scenario, cohesin deposition during G1 is temporally uncoupled from Eco1-dependent establishment reactions that occur during S-phase. Chl1 DNA helicase (homolog of human ChlR1/DDX11 and BACH1/BRIP1/FANCJ helicases implicated in Fanconi anemia, breast and ovarian cancer and Warsaw Breakage Syndrome plays a critical role in sister chromatid cohesion, however, the mechanism through which Chl1 promotes cohesion remains poorly understood. Here, we report that Chl1 promotes Scc2 loading unto DNA such that both Scc2 and cohesin enrichment to chromatin are defective in chl1 mutant cells. The results further show that both Chl1 expression and chromatin-recruitment are tightly regulated through the cell cycle, peaking during S-phase. Importantly, kinetic ChIP studies reveals that Chl1 is required for Scc2 chromatin-association specifically during S-phase, but not during G1. Despite normal chromatin enrichment of both Scc2 and cohesin during G1, chl1 mutant cells exhibit severe chromosome segregation and cohesion defects--revealing that G1-loaded cohesins is insufficient to promote cohesion. Based on these findings, we propose a new model wherein S-phase cohesin loading occurs during DNA replication and in concert with both cohesion establishment and chromatin assembly reactions--challenging the notion that DNA replication fork navigates through or around pre-loaded cohesin rings.

  10. Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis

    International Nuclear Information System (INIS)

    Marrero, R.; Yasbin, R.E.

    1988-01-01

    By use of the Bacillus subtilis bacteriophage cloning vehicle Phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages Phi 105Rec Phi1 (3.85-kilobase insert) and Phi 105Rec Phi4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE + strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage Phi105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either Phi 105Rec Phi 1 or Phi 105RecPhi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages Phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages Phi 105RecPhi 1 and Phi 105Rec Phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA + gene product antibodies. Collectively, these data demonstrate that the recE + gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination

  11. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2017-01-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424

  12. Transcriptional regulation and adaptation to a high-fiber environment in Bacillus subtilis HH2 isolated from feces of the giant panda.

    Directory of Open Access Journals (Sweden)

    Ziyao Zhou

    Full Text Available In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the panda's vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous study, we determined that several strains of B. subtilis isolated from pandas exhibited good cellulose decomposition ability, and we hypothesized that this bacterial species can survive in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed RNA-Seq technology to analyze the differentially expressed genes of the selected strain B. subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon sources (cellulose and glucose. In addition, we used bioinformatics software and resources to analyze the functions and pathways of differentially expressed genes. Interestingly, comparison of the cellulose and glucose groups revealed that the up-regulated genes were involved in amino acid and lipid metabolism or transmembrane transport, both of which are involved in cellulose utilization. Conversely, the down-regulated genes were involved in non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 triggered a series of adaptive mechanisms at the transcriptional level, which suggests that this bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellulose is not a very suitable carbon source for this bacterial species. In this study, we present a model to understand the dynamic organization of and interactions between various functional and regulatory networks for unicellular organisms in a high-fiber environment.

  13. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    Science.gov (United States)

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  14. Mutations of the RTEL1 Helicase in a Hoyeraal-Hreidarsson Syndrome Patient Highlight the Importance of the ARCH Domain.

    Science.gov (United States)

    Jullien, Laurent; Kannengiesser, Caroline; Kermasson, Laetitia; Cormier-Daire, Valérie; Leblanc, Thierry; Soulier, Jean; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Callebaut, Isabelle; Revy, Patrick

    2016-05-01

    The DNA helicase RTEL1 participates in telomere maintenance and genome stability. Biallelic mutations in the RTEL1 gene account for the severe telomere biology disorder characteristic of the Hoyeraal-Hreidarsson syndrome (HH). Here, we report a HH patient (P4) carrying two novel compound heterozygous mutations in RTEL1: a premature stop codon (c.949A>T, p.Lys317*) and an intronic deletion leading to an exon skipping and an in-frame deletion of 25 amino-acids (p.Ile398_Lys422). P4's cells exhibit short and dysfunctional telomeres similarly to other RTEL1-deficient patients. 3D structure predictions indicated that the p.Ile398_Lys422 deletion affects a part of the helicase ARCH domain, which lines the pore formed with the core HD and the iron-sulfur cluster domains and is highly specific of sequences from the eukaryotic XPD family members. © 2016 WILEY PERIODICALS, INC.

  15. Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

    NARCIS (Netherlands)

    Veening, JW; Hamoen, LW; Kuipers, OP

    Spore formation in the Gram- positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate

  16. Plant methyl salicylate induces defense responses in the rhizobacterium Bacillus subtilis.

    Science.gov (United States)

    Kobayashi, Kazuo

    2015-04-01

    Bacillus subtilis is a rhizobacterium that promotes plant growth and health. Cultivation of B. subtilis with an uprooted weed on solid medium produced pleat-like architectures on colonies near the plant. To test whether plants emit signals that affect B. subtilis colony morphology, we examined the effect of plant-related compounds on colony morphology. Bacillus subtilis formed mucoid colonies specifically in response to methyl salicylate, which is a plant-defense signal released in response to pathogen infection. Methyl salicylate induced mucoid colony formation by stimulating poly-γ-glutamic acid biosynthesis, which formed enclosing capsules that protected the cells from exposure to antimicrobial compounds. Poly-γ-glutamic acid synthesis depended on the DegS-DegU two-component regulatory system, which activated DegSU-dependent gene transcription in response to methyl salicylate. Bacillus subtilis did not induce plant methyl salicylate production, indicating that the most probable source of methyl salicylate in the rhizosphere is pathogen-infected plants. Methyl salicylate induced B. subtilis biosynthesis of the antibiotics bacilysin and fengycin, the latter of which exhibited inhibitory activity against the plant pathogenic fungus Fusarium oxysporum. We propose that B. subtilis may sense plants under pathogen attack via methyl salicylate, and express defense responses that protect both B. subtilis and host plants in the rhizosphere. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  17. 75 FR 862 - Bacillus subtilis; Registration Review Proposed Decision; Notice of Availability

    Science.gov (United States)

    2010-01-06

    ...: Bacillus subtilis strain GB03 is used to prevent, control and suppress plant disease on barley, berries, bulb vegetables, cole crops, cotton, cucurbits, fruiting vegetables, herbs, leafy crops, legumes... subtilis strain MBI 600 is used to suppress disease organisms such as Botrytis, Alternaria, Rhizoctonia...

  18. Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

    Science.gov (United States)

    Mun Su Rhee; Lusha Wei; Neha Sawhney; John D. Rice; Franz J. St. John; Jason C. Hurlbert; James F. Preston

    2014-01-01

    Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and...

  19. Genomic comparisons of two Bacillus subtilis biocontrol strains with different modes of actions

    Science.gov (United States)

    Bacillus subtilis strains AS 43.3 and OH131.1 were isolated from wheat anthers and shown to be efficacious in managing Fusarium head blight in greenhouse and some field trials. Chemical analysis of the cell-free culture supernatant identified B. subtilis strain AS 43.3 to be a potent producer of the...

  20. Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

    NARCIS (Netherlands)

    Westers, Lidia; Westers, Helga; Zanen, Geeske; Antelmann, Haike; Hecker, Michael; Noone, David; Devine, Kevin M.; van Dijl, Jan Maarten; Quax, Wim J.

    Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously

  1. Cell Physiology and Protein Secretion of Bacillus licheniformis Compared to Bacillus subtilis

    NARCIS (Netherlands)

    Voigt, Birgit; Antelmann, Haike; Albrecht, Dirk; Ehrenreich, Armin; Maurer, Karl-Heinz; Evers, Stefan; Gottschalk, Gerhard; van Dijl, Jan Maarten; Schweder, Thomas; Hecker, Michael

    2009-01-01

    The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can

  2. Towards a complete proteome of Bacillus subtilis : cytosolic, cell wall-associated and extracellular proteins

    NARCIS (Netherlands)

    Antelmann, Haike; van Dijl, Jan Maarten; Hecker, Michael

    2003-01-01

    Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable

  3. Effect of Bacillus subtilis natto on growth performance in Muscovy ducks

    Directory of Open Access Journals (Sweden)

    T Sheng-Qiu

    2013-09-01

    Full Text Available The aim of the present study was to determine whether dietary Bacillus subtilis natto could affect growth performance of Muscovy ducks. A total of 120 hundred Muscovy ducks at the age of 1 day were randomly assigned to four groups (30 Muscovy ducks/group, and fed with diets supplemented with 0% (control group, 0.1%, 0.2%, and 0.4% Bacillus subtilis natto, respectively during the 6-week feeding period. Weight gain, feed intake and feed conversion efficiency of Muscovy ducks were significantly improved by the dietary addition of Bacillus subtilis natto, and the results were more significant in 0.4% dietary Bacillus subtilis natto treatment group; Also, Bacillus subtilis natto reduced Escherichia coli and Salmonella colonies, and increased lactobacilli population in the ileum and the cecum. Biochemical parameters, including total protein, GOT (glutamic oxaloacetic transaminase, GPT (glutamic pyruvic transaminase, AKP (alkaline phosphatase, triiodothyronine (T3 and tetraiodothyronine (T4 contents (pBacillus subtilis natto was added to the diets (p0.05. The results of the present study indicate that diets with 0.4% Bacillus subtilis natto improved the growth performance of Muscovy ducks by increasing the absorption of protein, simulating hormone secretion, suppressing harmful microflora, and improving the duodenal structure and immune functions of Muscovy ducks. It is suggested that Bacillus subtilis natto is a potential candidate to be used use as a probiotic to improve the growth performance of Muscovy ducks.

  4. Effect of Bacillus subtilis on the growth and survival rate of shrimp ...

    African Journals Online (AJOL)

    The effect ofBacillus subtilis, isolated from digestive tract of Macrobrachium rosenbergii was investigated on growth and survival rate of Litopenaeus vannamei during 60 days of culture. Sixteen aquaria with four replicates were used for treatments and controls. Treatment groups were consisted of Bacillus subtilis, isolated ...

  5. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens strain...

  6. 40 CFR 180.1209 - Bacillus subtilis strain QST 713; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis strain QST 713... RESIDUES IN FOOD Exemptions From Tolerances § 180.1209 Bacillus subtilis strain QST 713; exemption from the... the microbial pesticide Bacillus subtilis strain QST 713 when used in or on all food commodities. [65...

  7. GLYCOGEN IN BACILLUS-SUBTILIS - MOLECULAR CHARACTERIZATION OF AN OPERON ENCODING ENZYMES INVOLVED IN GLYCOGEN BIOSYNTHESIS AND DEGRADATION

    NARCIS (Netherlands)

    KIEL, JAKW; BOELS, JM; BELDMAN, G; VENEMA, G

    Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 degrees on the B. subtilis chromosome. It

  8. Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

    Directory of Open Access Journals (Sweden)

    Shinagawa Hideo

    2008-03-01

    Full Text Available Abstract Background DNA double-strand breaks (DSBs are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. Results To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

  9. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs and cytokines expression patterns in jejunum and ileum of broilers.

    Directory of Open Access Journals (Sweden)

    Imran Rashid Rajput

    Full Text Available The present study was designed to evaluate the effects of Saccharomyces boulardii (Sb and Bacillus subtilis B10 (Bs on intestinal epithelial Toll like receptors (TLR, and Cytokine expression response to understand the intestinal epithelial innate immune mechanism in broilers. A total of 300 birds (Sanhuang broilers were allotted into three groups (n = 100 and each divided into five replications (n = 20. Control group (Ctr birds were fed basal diet, broilers in experimental groups received (1×108cfu/kg feed Sb and Bs respectively in addition to basal diet for 72 days. The result showed significant increase in mRNA expression level of TLR2, TLR4 and TLR15. Down streaming MyD88, TRAF6, TAB2 and NF-κB mRNA level noted higher, in the jejunum and ileum as compared to control group. Meanwhile, IL-6, TNFα, IL-10, TGF-β expression levels showed high expression in the jejunum of Sb and Bs groups. IL-10 expression level increased in the ileum and IL-6, TNFα, IL-10 and TGF-β expression levels increased in the jejunum of Sb group. Levels of IL-1 β, IL-17, and IL-4, increased merely in Sb group. Ileal cytokines IL-1β, IL-17 and IL-4concentration were noted higher in Sb group, and IL-1β, and IL-4 levels were up-regulated in Bs group. The results indicated that the INF-γ and IL-8 level decreased in Sb and BS groups. Serum IgA and sIgA level increased in both treatment groups. Our findings illustrated that S. boulardii and B. subtilis B10 may have a role to induce mucosal immunity by activating the TLRs and cytokines expressions in broilers.

  10. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs and cytokines expression patterns in jejunum and ileum of broilers.

    Science.gov (United States)

    Rajput, Imran Rashid; Ying, Huang; Yajing, Sun; Arain, Muhammad Asif; Weifen, Li; Ping, Li; Bloch, Dost Muhammad; Wenhua, Liu

    2017-01-01

    The present study was designed to evaluate the effects of Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) on intestinal epithelial Toll like receptors (TLR), and Cytokine expression response to understand the intestinal epithelial innate immune mechanism in broilers. A total of 300 birds (Sanhuang broilers) were allotted into three groups (n = 100) and each divided into five replications (n = 20). Control group (Ctr) birds were fed basal diet, broilers in experimental groups received (1×108cfu/kg feed) Sb and Bs respectively in addition to basal diet for 72 days. The result showed significant increase in mRNA expression level of TLR2, TLR4 and TLR15. Down streaming MyD88, TRAF6, TAB2 and NF-κB mRNA level noted higher, in the jejunum and ileum as compared to control group. Meanwhile, IL-6, TNFα, IL-10, TGF-β expression levels showed high expression in the jejunum of Sb and Bs groups. IL-10 expression level increased in the ileum and IL-6, TNFα, IL-10 and TGF-β expression levels increased in the jejunum of Sb group. Levels of IL-1 β, IL-17, and IL-4, increased merely in Sb group. Ileal cytokines IL-1β, IL-17 and IL-4concentration were noted higher in Sb group, and IL-1β, and IL-4 levels were up-regulated in Bs group. The results indicated that the INF-γ and IL-8 level decreased in Sb and BS groups. Serum IgA and sIgA level increased in both treatment groups. Our findings illustrated that S. boulardii and B. subtilis B10 may have a role to induce mucosal immunity by activating the TLRs and cytokines expressions in broilers.

  11. The Arabidopsis thaliana Homolog of the Helicase RTEL1 Plays Multiple Roles in Preserving Genome Stability[C][W

    Science.gov (United States)

    Recker, Julia; Knoll, Alexander; Puchta, Holger

    2014-01-01

    In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. PMID:25516598

  12. Menaquinone and iron are essential for complex colony development in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Gidi Pelchovich

    Full Text Available Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis.

  13. Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway

    Science.gov (United States)

    Donato, Verónica; Ayala, Facundo Rodríguez; Cogliati, Sebastián; Bauman, Carlos; Costa, Juan Gabriel; Leñini, Cecilia; Grau, Roberto

    2017-01-01

    Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis — the quorum-sensing pentapeptide CSF and nitric oxide (NO) — are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway. PMID:28134244

  14. Acid and base stress and transcriptomic responses in Bacillus subtilis.

    Science.gov (United States)

    Wilks, Jessica C; Kitko, Ryan D; Cleeton, Sarah H; Lee, Grace E; Ugwu, Chinagozi S; Jones, Brian D; BonDurant, Sandra S; Slonczewski, Joan L

    2009-02-01

    Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K(+)/H(+) antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.

  15. Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    International Nuclear Information System (INIS)

    Miyazaki, Nobuyoshi; Nagai, Kazuo; Tamura, Gakuzo

    1976-01-01

    A thermosensitive mutant ts 42, of Bacillus subtilis Marburg 168 thy trp2 which requires uracil, was examined as to the colony-forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in modified woese's medium. However, the cells retained the viability when sodium succinate or potassium chloride was added to the medium at that temperature, although uranil deficiency was unchanged. A little but significant incorporation of adenine-8- 14 C into RNA still continued even after the incorporation of N-acetyl- 3 H-D-glucosamine into the acid-insoluble fraction of the cells terminated in the modified Woese's medium at 48 0 C. Both incorporations as well as the increase of absorbance were slowed down in the presence of sodium succinate at 48 0 C. This mutant, ts42, was more sensitive to deoxycholate than the parent wild strain. The resoration of the colony-forming ability after the temperature shifted back from 48 0 to 37 0 C was suppressed by the addition of deoxycholate to the medium. However, the cells became resistant to deoxycholate when uracil had been added to the medium prior to the temperature shift. (Kobatake, H.)

  16. Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, N; Nagai, K; Tamura, G

    1976-01-01

    A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp/sub 2/ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese's (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-/sup 14/C into RNA still continued even after the incorporation of N-acetyl-/sup 3/H-D-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48/sup 0/C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48/sup 0/C. This mutant, ts-42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back to 37/sup 0/C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.

  17. Thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Nagai, K; Some, H; Tamura, G

    1976-01-01

    Thermonsensitive division mutants were derived from Bacillus subtilis Marburg 168 thy trp/sub 2/ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48/sup 0/C was investigated. In the absence of uracil, the mutant cells grew normally at 37/sup 0/C and stopped dividing after temperature shift to 48/sup 0/C resulting in filaments of two to four times length of normal rods. The total cell number after the temperature shift increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48/sup 0/C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48/sup 0/C or when uracil was introduced to the culture at 48/sup 0/C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48/sup 0/C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature. No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.

  18. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis.

    Science.gov (United States)

    Elsholz, Alexander K W; Birk, Marlene S; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis . We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

  19. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    Science.gov (United States)

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics. PMID:28748186

  20. Higher antibiotic yielding mutants of bacillus subtilis by gamma radiation

    International Nuclear Information System (INIS)

    Ahmad, M.S.; Shaukat, G.A.; Malik, M.A.

    1987-01-01

    When Bacillus Subtilis AECL69 was grown in malt extract-pepetone-molasses-sugar (MPMS) medium, it could produce antibiotic substance(s) with antibacterial and antifungal properties in the culture fluid. The bacterial cells grown in MPMS medium were washed and suspended into distilled water and irradiated with gamma rays in Gammacell 220 at different doses. Higher antibiotic yielding isolates (plus mutants) were obtained from cell pollutions irradiated at 15 Kr. These gamma rays-induced plus mutants showed simultaneous higher production of antibacterial as well as antifungal activity. (author)

  1. Hyper production of alkaline protease by mutagenized bacillus subtilis

    International Nuclear Information System (INIS)

    Qureshi, A.M.; Tanseem, F.

    2010-01-01

    The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

  2. Sticking together: building a biofilm the Bacillus subtilis way

    Science.gov (United States)

    Vlamakis, Hera; Chai, Yunrong; Beauregard, Pascale; Losick, Richard; Kolter, Roberto

    2014-01-01

    Preface Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long-served as a robust model organism to examine the molecular mechanisms of biofilm formation and a number of studies have revealed that this process is subject to a number of integrated regulatory pathways. In this Review, we focus on the molecular mechanisms controlling biofilm assembly and briefly summarize the current state of knowledge regarding their disassembly. We also discuss recent progress that has expanded our understanding of biofilm formation on plant roots, which are a natural habitat for this soil bacterium. PMID:23353768

  3. Bacterial determinants of the social behavior of Bacillus subtilis.

    Science.gov (United States)

    Romero, Diego

    2013-09-01

    Bacteria utilize sophisticated cellular machinery to sense environmental changes and coordinate the most appropriate response. Fine sensors located on cell surfaces recognize a myriad of triggers and initiate genetic cascades leading to activation or repression of certain groups of genes. Structural elements such as pilli, exopolysaccharides and flagella are also exposed at the cell surface and contribute to modulating the intimate interaction with surfaces and host cells. This review will cover the latest advances in our understanding of the biology and functionality of these bacterial determinants within the context of biofilm formation of Bacillus subtilis. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. Production and applications of biosurfactant from Bacillus subtilis MUV4

    Directory of Open Access Journals (Sweden)

    Aran H-Kittikun

    2008-04-01

    Full Text Available Bacillus subtilis MUV4 produced biosurfactant in shake-flask culture (200 rpm at 30oC with modified Mckeen medium containing 1% glucose as a carbon source, 1% monosodium glutamate and 0.3% yeast extract as nitrogen sources. The supernatant of B. subtilis MUV4 reduced the surface tension of the medium from 53.50 mN/m to 33.50 mN/m after 48 h of cultivation. The yield of crude biosurfactant from B. subtilis MUV4 after precipitating the supernatant with 6N HCl was 0.652 g/L. Growth kinetics studies showed the specific growth rate (μ of 0.14 h-1, yield of biomass to substrate (Yx/s of 0.713, yield of product to substrate (Yp/s of 0.072 and yield of product to biomass (Yp/x of 0.101. Moreover, B. subtilis MUV4 produced 0.30 g/L crude biosurfactant after 96 h of cultivation in the fermentor with agitation rate of 200 rpm without aeration and uncontrolled pH condition. The crude biosurfactant was dissolved in methanol and dried by vacuum evaporator (crude methanol. The supernatant, the crude biosurfactant and the crude methanol retained the biosurfactant activity over the pH range of 1-6, 7-10 and 4-10, respectively and the emulsion stability at 24 h (E24 at pH 7 were 66.67%, 33.33% and 33.33%, respectively. The supernatant and the crude biosurfactant showed surface tension activity at 4oC, room temperature (30±2oC and 50oC after incubation for 5 h. However, only crude methanol still retained surface tension activity after 100oC for 5 h. The surface tension activity of the supernatant and the crude biosurfactant was stable in 3-10% (w/v NaCl while crude methanol showed stability in 3-20% (w/v NaCl. However, all samples lost emulsion stability when NaCl concentration was higher than 5% (w/v. With sand pack column technique, crude methanol enhanced the recovery of crude oil and kerosene oil by 41.85% and 75.00%, respectively. In hydrocarbon degradation application study, the crude biosurfactant was added to the culture medium containing 0.3% crude oil

  5. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Wiedermannova, J.; Sudzinova, Z.; Koval, T.; Rabatinova, A.; Sanderova, H.; Ramaniuk, O.; Rittich, S.; Dohnálek, Jan; Fu, Z.; Halada, P.; Lewis, P.; Krasny, L.

    2014-01-01

    Roč. 42, č. 8 (2014), s. 5155-5163 ISSN 0305-1048 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GAP302/11/0855 Institutional support: RVO:86652036 Keywords : RICHIA-COLI * BACTERIAL HOMOLOG * DELTA-SUBUNIT Subject RIV: CE - Biochemistry Impact factor: 9.112, year: 2014

  6. Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto).

    Science.gov (United States)

    Azimirad, Masoumeh; Alebouyeh, Masoud; Naji, Tahereh

    2017-03-01

    Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.

  7. DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

    DEFF Research Database (Denmark)

    Pettersson, Olof Joakim; Aagaard, Lars; Andrejeva, Diana

    2014-01-01

    RNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat m......RNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration...... in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events...

  8. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy.

    Science.gov (United States)

    Feng, Yue; Liu, Song; Jiao, Yun; Gao, Hui; Wang, Miao; Du, Guocheng; Chen, Jian

    2017-02-01

    L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (-28: A → G, -13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K m and k cat of the ASN were 5.29 mM and 54.4 s -1 , respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

  9. PRODUKSI ANTIBIOTIKA OLEH Bacillus subtilis M10 DALAM MEDIA UREA-SORBITOL

    Directory of Open Access Journals (Sweden)

    Supartono Supartono

    2012-04-01

    Full Text Available PRODUCTION OF ANTIBIOTICS BY Bacillus subtilis M10 IN UREA-SORBITOL MEDIUM. Infection diseases still become the main health problems that suffered by people in Indonesia. Besides, there were many pathogen bacteria found to be resistant to the some antibiotics. Therefore, the efforts to get a new antibiotic require to be done continuously. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibit Serratia marcescens ATCC 27117 growth. To make efficient the local strain, mutation on Bacillus subtilis BAC4 was done by using acridine orange and a mutant cell of Bacillus subtilis M10 that overproduction for producing antibiotic was obtained. Nevertheless, the production kinetics of antibiotic by this mutant has not been reported. The objective of this research was to study the production kinetics of antibiotic by Bacillus subtilis M10 mutant. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with agar absorption method using Serratia marcescens ATCC 27117 as bacteria assay. Research result provided that Bacillus subtilis M10 mutant with overproduction of antibiotic produced an antibiotic since 8th hour’s fermentation and optimum of it production was at 14th hours after inoculation.  Penyakit infeksi masih menjadi masalah yang utama diderita oleh masyarakat Indonesia. Di samping itu, banyak bakteri patogen yang ditemukan resisten terhadap beberapa antibiotika. Oleh karena itu, upaya-upaya untuk mendapatkan antibiotika baru perlu dilakukan secara terus-menerus. Suatu galur lokal baru Bacillus subtilis BAC4 teridentifikasi memproduksi senyawa antibiotika yang menghambat pertumbuhan Serratia marcescens ATCC27117. Untuk memberdayakan galur tersebut, terhadap Bacillus subtilis BAC4 dilakukan mutasi dengan larutan akridin oranye dan diperoleh mutan Bacillus subtilis M10 yang memproduksi antibiotika berlebihan. Namun, kinetika produksi antibiotika oleh Bacillus

  10. RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Tomonori Kato

    2010-11-01

    Full Text Available We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV, and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacies of thismethod: 1 inmore clinically relevant situations including metastatic diseases, 2 on other tumor types and other animal species, and 3 on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP-activated bone marrow-derived DCs (rSeV/dF-GFP-DC, consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I (rSeV/dF-RIGIC-DC, an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies.

  11. Anatomy of the bacitracin resistance network in Bacillus subtilis.

    Science.gov (United States)

    Radeck, Jara; Gebhard, Susanne; Orchard, Peter Shevlin; Kirchner, Marion; Bauer, Stephanie; Mascher, Thorsten; Fritz, Georg

    2016-05-01

    Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well-characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle-inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage-shock proteins LiaIH. Our systems-level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks. © 2016 John Wiley & Sons Ltd.

  12. Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis.

    Science.gov (United States)

    Yüksel, Melih; Power, Jeffrey J; Ribbe, Jan; Volkmann, Thorsten; Maier, Berenike

    2016-01-01

    In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off.

  13. A Combinatorial Kin Discrimination System in Bacillus subtilis.

    Science.gov (United States)

    Lyons, Nicholas A; Kraigher, Barbara; Stefanic, Polonca; Mandic-Mulec, Ines; Kolter, Roberto

    2016-03-21

    Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis, but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell-surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. A major protein component of the Bacillus subtilis biofilm matrix.

    Science.gov (United States)

    Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

    2006-02-01

    Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

  15. DNA repair in ultraviolet-irradiated spores of Bacillus subtilis

    International Nuclear Information System (INIS)

    Wang, T.C.V.

    1976-01-01

    It has been shown previously by others that at least two independent repair mechanisms are present in Bacillus subtilis for removing ''spore photoproduct'' from DNA of ultraviolet (254 nm)-irradiated spores after germination. One of these, designated as ''spore repair,'' is shown in this study to restore ''spore photoproduct'' to two thymine residues, leaving the DNA backbone intact at the end of the process in vivo. The circumstances under which this repair can occur and some characteristics of its energy requirements have been clarified. The second repair process is identified as excision repair, which can excise both ''spore photoproduct'' from DNA of irradiated spores and cyclobutane-type pyrimidine dimers from DNA of irradiated vegetative cells. In this study it is shown that the gene hcr 1 affects an enzyme activity for the incision step initiating this repair, while the gene hcr 42 affects a step subsequent to incision in the mechanism. In addition a third, independent repair system, termed ''germinative excision repair,'' is discovered and shown to be specific for excising only cyclobutane-type pyrimidine dimers but not ''spore photoproduct.'' This repair system is responsible for the observed high ultraviolet-resistance and temporary capacity for host cell reactivation on recently germinated spores of Bacillus subtilis HCR - strains

  16. Analysis of Spo0M function in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Luz Adriana Vega-Cabrera

    Full Text Available Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell.

  17. DNA Repair and Genome Maintenance in Bacillus subtilis

    Science.gov (United States)

    Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

  18. The ESX system in Bacillus subtilis mediates protein secretion.

    Directory of Open Access Journals (Sweden)

    Laura A Huppert

    Full Text Available Esat-6 protein secretion systems (ESX or Ess are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.

  19. Pulsed dielectric barrier discharge for Bacillus subtilis inactivation in water

    Science.gov (United States)

    Hernández-Arias, A. N.; Rodríguez-Méndez, B. G.; López-Callejas, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Peña-Eguiluz, R.; Barocio, S. R.; Muñoz-Castro, A. E.; de la Piedad Beneitez, A.

    2012-06-01

    The inactivation of Bacillus subtilis bacteria in water has been experimentally studied by means of a pulsed dielectric barrier discharge (PDBD) in a coaxial reactor endowed with an alumina dielectric. The plasma source is capable of operating at atmospheric pressure with gas, water or hybrid gas-liquid media at adjustable 25 kV pulses, 30 μs long and at a 500 Hz frequency. In order to evaluate the inactivation efficiency of the system, a set of experiments were designed on the basis of oxygen flow control. The initial data have showed a significant bacterial rate reduction of 103-107 CFU/mL. Additional results proved that applying an oxygen flow for a few seconds during the PDBD treatment inactivates the Bacillus subtilis population with 99.99% effectiveness. As a reference, without gas flow but with the same exposure times, this percentage is reduced to ~90%. The analysis of the relationship between inactivation rate and chemical species in the discharge has been carried out using optical emission spectroscopy as to identifying the main reactive species. Reactive oxygen species such as atomic oxygen and ozone tuned out to be the dominant germicidal species. Some proposed inactivation mechanisms of this technique are discussed.

  20. Evidence for differentiation of cell wall poles in Bacillus subtilis

    International Nuclear Information System (INIS)

    Sonnenfeld, E.M.

    1985-01-01

    Previous data have suggested that the chromosome of Bacillus subtilis was found to the cell surface at polar regions. A significant corollary of DNA attachment to cell poles is the role of the cell wall in chromosome segregation. This project was mainly concerned with visualizing the DNA-cell wall association through autoradiography. The origin and terminus of replication were labelled with ( 3 H)-thymidine using a temperature-sensitive DNA initiation mutant. It was found that most of the radioactivity was associated with cell poles. Ultrastructural analyses of cell walls stained with dilute cationized ferritin showed that the polar area contained a site of dense electronegativity. It is not immediately apparent why cell wall poles would contain an area with a high concentration of negative charge. This finding may be related to the cell pole functioning as the site of chromosome attachment. An additional observation encountered in this study was that cell wall exhibited asymmetry with regard to negative charge, the outside surface being more electronegative than the inside. A significant consequence of this finding is that both teichoic acid and muramyl peptides are situated perpendicularly to the cell surface. This favored arrangement may facilitate cell separation during the division process due to opposition of like charges at septa. The results of this work provide further convincing evidence that the cell wall of B. subtilis is differentiated

  1. Pulsed dielectric barrier discharge for Bacillus subtilis inactivation in water

    International Nuclear Information System (INIS)

    Hernández-Arias, A N; López-Callejas, R; De la Piedad Beneitez, A; Rodríguez-Méndez, B G; Valencia-Alvarado, R; Mercado-Cabrera, A; Peña-Eguiluz, R; Barocio, S R; Muñoz-Castro, A E

    2012-01-01

    The inactivation of Bacillus subtilis bacteria in water has been experimentally studied by means of a pulsed dielectric barrier discharge (PDBD) in a coaxial reactor endowed with an alumina dielectric. The plasma source is capable of operating at atmospheric pressure with gas, water or hybrid gas-liquid media at adjustable 25 kV pulses, 30 μs long and at a 500 Hz frequency. In order to evaluate the inactivation efficiency of the system, a set of experiments were designed on the basis of oxygen flow control. The initial data have showed a significant bacterial rate reduction of 10 3 -10 7 CFU/mL. Additional results proved that applying an oxygen flow for a few seconds during the PDBD treatment inactivates the Bacillus subtilis population with 99.99% effectiveness. As a reference, without gas flow but with the same exposure times, this percentage is reduced to ∼90%. The analysis of the relationship between inactivation rate and chemical species in the discharge has been carried out using optical emission spectroscopy as to identifying the main reactive species. Reactive oxygen species such as atomic oxygen and ozone tuned out to be the dominant germicidal species. Some proposed inactivation mechanisms of this technique are discussed.

  2. The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres.

    Science.gov (United States)

    Mendez-Bermudez, Aaron; Hidalgo-Bravo, Alberto; Cotton, Victoria E; Gravani, Athanasia; Jeyapalan, Jennie N; Royle, Nicola J

    2012-11-01

    Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN- ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN- ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.

  3. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

    Science.gov (United States)

    Du, Xin-Jun; Zhou, Tian-Jiao; Li, Ping; Wang, Shuo

    2017-08-01

    Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Antiviral drug resistance and helicase-primase inhibitors of herpes simplex virus.

    Science.gov (United States)

    Field, Hugh J; Biswas, Subhajit

    2011-02-01

    A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase-primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    Science.gov (United States)

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  6. Mutation and Methylation Analysis of the Chromodomain-Helicase-DNA Binding 5 Gene in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kylie L. Gorringe

    2008-11-01

    Full Text Available Chromodomain, helicase, DNA binding 5 (CHD5 is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04. The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

  7. AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms.

    Directory of Open Access Journals (Sweden)

    Chloe Girard

    2015-07-01

    Full Text Available Meiotic crossovers (COs generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1 as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.

  8. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Science.gov (United States)

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-02

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  9. Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

    Science.gov (United States)

    Shiraki, K

    2017-11-01

    Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy. Copyright 2017 Clarivate Analytics.

  10. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

    Science.gov (United States)

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

    2013-09-03

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

  11. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

    Science.gov (United States)

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

    2013-01-01

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

  12. Inhibition of quorum sensing-mediated virulence in Serratia marcescens by Bacillus subtilis R-18.

    Science.gov (United States)

    Devi, Kannan Rama; Srinivasan, Subramaniyan; Ravi, Arumugam Veera

    2018-04-13

    Serratia marcescens is an opportunistic human pathogen causing various nosocomial infections, most importantly urinary tract infections (UTIs). It exhibits increased resistance towards the conventional antibiotics. This study was aimed to evaluate the anti-virulence effect of a rhizosphere soil bacterium Bacillus subtilis strain R-18 against the uropathogen S. marcescens. First, the bacterial cell-free culture supernatant (CFCS) of B. subtilis strain R-18 was evaluated for its quorum sensing inhibitory (QSI) potential against biomarker strain Chromobacterium violaceum and the test pathogen S. marcescens. The B. subtilis R-18 CFCS effectively inhibited the quorum sensing (QS)-mediated violacein pigment production in C. violaceum and prodigiosin pigment production in S. marcescens. Furthermore, B. subtilis R-18 CFCS was successively extracted with different solvent systems. Of these solvents, B. subtilis R-18 petroleum ether (PE) extract showed inhibition in biofilm formation, protease, lipase, and hemolysin productions in S. marcescens. Fourier transform infrared spectroscopic (FT-IR) analysis revealed the alterations in the cellular components of bacterial cell pellets obtained from B. subtilis R-18 PE extract treated and untreated S. marcescens. The differential gene expression study further validated the downregulation of virulence-associated genes. Characterization of the active principle in B. subtilis R-18 PE extract by gas chromatography-mass spectrometry (GC-MS) analysis showed the presence of multiple compounds with therapeutic values, which could possibly reduce the QS-dependent phenotypes in S. marcescens. Copyright © 2018. Published by Elsevier Ltd.

  13. Cost-effective medium for the production of mosquito pupicidal lipopeptide from Bacillus subtilis subsp. subtilis (VCRC B471).

    Science.gov (United States)

    Bhuvaneswari, S; Manonmani, A M; Geetha, I

    2015-03-01

    A cyclic lipopeptide (CLP), surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit mosquitocidal activity. The present study was carried out to enhance the surfactin level using low cost material in the production medium. Two carbon sources, glucose and common sugar, and two nitrogen sources, ammonium nitrate and soya were used in the study. Different concentrations of 'C' and 'N' sources were used in the production medium to enhance the production of surfactin. A new medium (SS7) containing 2% sugar, 6% soya and 0.5% common salt with micronutrients was designed which was found to enhance the production of surfactin. The crude mosquitocidal metabolite (CMM) produced in this medium was 3 g/l which was two times higher than that obtained using synthetic medium NYSM. The LC50 dosage of the CMM to the pupal stages of An. stephensi (2.3 μg/ml) was comparable to that obtained with CMM from the conventional medium. The newly designed cost-effective medium designated as sugar soya medium (SSM) enhanced the production of surfactin and the cost of production was estimated as [symbol: see text] 6 per litre, which is six times lesser than that of the conventional medium. Replacement of sodium chloride with cooking salt further reduced the cost of the medium.

  14. High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris.

    Science.gov (United States)

    Tang, Wei; Li, Zhezhe; Li, Chunhua; Yu, Xianhong; Wang, Fei; Wan, Xin; Wang, Yaping; Ma, Lixin

    2016-06-01

    Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  16. High Pressure Germination of Bacillus subtilis Spores with Alterations in Levels and Types of Germination Proteins

    Science.gov (United States)

    2014-01-01

    1ITLE AND SUBTITLE 5a CONTRACTNUMBER High pressure germination of Bacillus subtilis spores with W911NF-09-l-0286 alterations in levels and types of...A moderate high pressure (mHP) of 150 megaPascals (MPa) triggers germination of Bacillus subtilis spores via germinant receptors (GRs), while...germination by a very high pressure (vHP) of550 MPa is GR-independent. The mHP and vHP germination of Bacillus subtilis spores with different levels ofGRs

  17. The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

    Science.gov (United States)

    Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

    2003-01-01

    Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

  18. Bacillus subtilis generates a major specific deletion in pAM beta 1.

    OpenAIRE

    van der Lelie, D; Venema, G

    1987-01-01

    pAM beta 1, a 26.5-kilobase plasmid originally isolated from Streptococcus faecalis, was conjugally transferred from Streptococcus lactis to Bacillus subtilis. No conjugal transfer of pAM beta 1 from B. subtilis to S. lactis was observed. In addition, pAM beta 1 which had been reintroduced in S. lactis after cycling through B. subtilis had lost its conjugal transferability to Streptococcus cremoris, although under the same conditions noncycled pAM beta 1 was transferred at high efficiency. Re...

  19. RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation

    Science.gov (United States)

    2013-01-01

    Background The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. Results A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. Conclusion The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains. PMID:24079885

  20. RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation.

    Science.gov (United States)

    Wiegand, Sandra; Dietrich, Sascha; Hertel, Robert; Bongaerts, Johannes; Evers, Stefan; Volland, Sonja; Daniel, Rolf; Liesegang, Heiko

    2013-10-01

    The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains.

  1. Ribosomal protein L10(L12)4 autoregulates expression of the Bacillus subtilis rplJL operon by a transcription attenuation mechanism.

    Science.gov (United States)

    Yakhnin, Helen; Yakhnin, Alexander V; Babitzke, Paul

    2015-08-18

    Ribosomal protein genes are often controlled by autoregulatory mechanisms in which a protein encoded in the operon can either bind to newly synthesized rRNA during rapid growth or to a similar target in its mRNA during poor growth conditions. The rplJL operon encodes the ribosomal L10(L12)4 complex. In Escherichia coli L10(L12)4 represses its translation by binding to the rplJL leader transcript. We identified three RNA structures in the Bacillus subtilis rplJL leader transcript that function as an anti-antiterminator, antiterminator or intrinsic terminator. Expression studies with transcriptional and translational fusions indicated that L10(L12)4 represses rplJL expression at the transcriptional level. RNA binding studies demonstrated that L10(L12)4 stabilizes the anti-antiterminator structure, while in vitro transcription results indicated that L10(L12)4 promotes termination. Disruption of anti-antiterminator, antiterminator or terminator function by competitor oligonucleotides in vitro and by mutations in vivo demonstrated that each structure functions as predicted. Thus, rplJL expression is regulated by an autogenous transcription attenuation mechanism in which L10(L12)4 binding to the anti-antiterminator structure promotes termination. We also found that translation of a leader peptide increases rplJL expression, presumably by inhibiting Rho-dependent termination. Thus, the rplJL operon of B. subtilis is regulated by transcription attenuation and antitermination mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Adaptation of Bacillus subtilis to Life at Extreme Potassium Limitation.

    Science.gov (United States)

    Gundlach, Jan; Herzberg, Christina; Hertel, Dietrich; Thürmer, Andrea; Daniel, Rolf; Link, Hannes; Stülke, Jörg

    2017-07-05

    Potassium is the most abundant metal ion in every living cell. This ion is essential due to its requirement for the activity of the ribosome and many enzymes but also because of its role in buffering the negative charge of nucleic acids. As the external concentrations of potassium are usually low, efficient uptake and intracellular enrichment of the ion is necessary. The Gram-positive bacterium Bacillus subtilis possesses three transporters for potassium, KtrAB, KtrCD, and the recently discovered KimA. In the absence of the high-affinity transporters KtrAB and KimA, the bacteria were unable to grow at low potassium concentrations. However, we observed the appearance of suppressor mutants that were able to overcome the potassium limitation. All these suppressor mutations affected amino acid metabolism, particularly arginine biosynthesis. In the mutants, the intracellular levels of ornithine, citrulline, and arginine were strongly increased, suggesting that these amino acids can partially substitute for potassium. This was confirmed by the observation that the supplementation with positively charged amino acids allows growth of B. subtilis even at the extreme potassium limitation that the bacteria experience if no potassium is added to the medium. In addition, a second class of suppressor mutations allowed growth at extreme potassium limitation. These mutations result in increased expression of KtrAB, the potassium transporter with the highest affinity and therefore allow the acquisition and accumulation of the smallest amounts of potassium ions from the environment. IMPORTANCE Potassium is essential for every living cell as it is required for the activity for many enzymes and for maintaining the intracellular pH by buffering the negative charge of the nucleic acids. We have studied the adaptation of the soil bacterium Bacillus subtilis to life at low potassium concentrations. If the major high-affinity transporters are missing, the bacteria are unable to grow

  3. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  4. Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth.

    Science.gov (United States)

    Leite, Hianna Almeida Câmara; Silva, Anderson Barbosa; Gomes, Fábio Pinto; Gramacho, Karina Peres; Faria, José Cláudio; de Souza, Jorge Teodoro; Loguercio, Leandro Lopes

    2013-03-01

    Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif(R)) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif(R) variants systemically colonized the entire cacao seedlings in 15-20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings' height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif(R) variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings

  5. Dietary Corn Bran Fermented by Bacillus subtilis MA139 Decreased Gut Cellulolytic Bacteria and Microbiota Diversity in Finishing Pigs

    Directory of Open Access Journals (Sweden)

    Ping Liu

    2017-12-01

    Full Text Available Solid-state fermentation of feedstuffs by Bacillus subtilis MA139 can reduce insoluble dietary fiber content in vitro and improve growth performance in pigs. This study was conducted to investigate the effects of dietary corn bran (CB fermented by B. subtilis on growth performance and gut microbiota composition in finishing pigs. A total of 60 finishing pigs were allocated to 3 dietary treatments consisting of a control (CON diet, a 10% CB diet, and a 10% fermented CB (FCB diet in a 21 d feeding trial. Growth performance and nutrient digestibility were evaluated. Fecal samples were determined for bacterial community diversity by 16S rRNA gene amplicon sequencing. The dietary CB and FCB did not affect growth performance of finishing pigs. The digestibility of organic matter was decreased in both CB and FCB treatments compared with CON group (P < 0.05. The α-diversity for bacterial community analysis of Chao 1 in FCB treatment was lower than CON treatment (P < 0.05. The Fibrobacteres phylum belongs to cellulolytic bacteria was isolated, and their relative abundance in CB group showed no difference between CON and FCB treatments. The abundance of Lachnospiraceae_NK4A136_group in CB treatment was higher than CON and FCB groups (P < 0.05, whereas the population of norank_f_Prevotellaceae was higher in FCB group compared to CON and CB groups (P < 0.05. In conclusion, dietary FCB decreased the abundance of bacterial communities, particularly the population of bacteria related to cellulolytic degradation.

  6. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs.

    Science.gov (United States)

    Rajput, Imran Rashid; Hussain, Altaf; Li, Ya Li; Zhang, Xiaoping; Xu, Xin; Long, Mao Yu; You, Dong Yu; Li, Wei Fen

    2014-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a critical role to activate immune response. They may be targeted for immunomodulation by microbes, including probiotics. In this study, chicken bone marrow dendrite cells (chi-BMDCs) were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10 (Bs), co-culture of Sb + Bs and phosphate buffer saline (PBS) as a control group (Ctr) at 3, 6, and 12 h intervals. Results revealed that treatment groups modulated the phenotype and biological functions of chi-BMDCs. Scan electron microscopy showed attachment of probiotics on the surface of chi-BMDCs. Additionally transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC-II, CD40, CD80 and CD86 up-regulated in stimulated groups. Furthermore, toll-like receptors TLR1, TLR2, TLR4, and chicken specific TLR15 expressions were improved and downstream associated factors MyD88, TRAF6, TAB1, and NFκ-B mRNA levels increased in all treatment groups as compared to control. Surprisingly, NFκ-B response was noted significant higher in LPS treatment among all groups. Moreover, IL-1β, IL-17, IL-4, TGF-β, and IL-10 production levels were found higher, and lower concentration of INF-γ and IL-8 were observed in Sb, Bs, and Sb + Bs treatment groups. In contrast, LPS groups showed prominent increase in IL-12, INF-γ, and IL-8 concentration levels as compared to control group. Altogether, these results emphasize a potentially important role of Saccharomyces boulardii and Bacillus subtilis B10 in modulating immunological functions of chi-BMDCs by targeting specific toll like receptors (TLRs) and associated factors. The role of probiotics on chi-BMDCs functionality in a non-mammalian species have been presented for the first time. © 2013 Wiley Periodicals, Inc.

  7. Transcriptional regulation of the Bacillus subtilis menp1 promoter.

    Science.gov (United States)

    Qin, X; Taber, H W

    1996-02-01

    The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone. The menp1 promoter previously was shown to be the primary cis element for menFD gene expression. In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase. The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization. Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added.

  8. Thinking about Bacillus subtilis as a multicellular organism.

    Science.gov (United States)

    Aguilar, Claudio; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2007-12-01

    Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.

  9. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms.

    Science.gov (United States)

    Romero, Diego; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

    2010-02-02

    Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

  10. Mutagenic effect of tritated water on spores of Bacillus subtilis

    International Nuclear Information System (INIS)

    Tanooka, H.; Munakata, N.

    1978-01-01

    The mutagenic effect of tritiated water was observed with spores of Bacillus subtilis polA strain suspended in 50 mCi/ml of tritiated water for various intervals. Dose rate given by tritium beta particles to spore core was estimated to be 400 rad/hr from some assumptions and E. coli data computed by Bockrath et al. and Sands et al. The initial mutation rate was 4.2 x 10 -9 mutants/rad, as compared with 2.4 x 10 -9 mutants/rad for 60 Co γ rays and 3.3 x 10 -9 mutants/rad for 30-kVp x rays. The mutagenic effect of tritiated water on spores is most likely due to beta particle ionizing radiation damage

  11. Functional requirements of cellular differentiation: lessons from Bacillus subtilis.

    Science.gov (United States)

    Narula, Jatin; Fujita, Masaya; Igoshin, Oleg A

    2016-12-01

    Successful execution of differentiation programs requires cells to assess multitudes of internal and external cues and respond with appropriate gene expression programs. Here, we review how Bacillus subtilis sporulation network deals with these tasks focusing on the lessons generalizable to other systems. With feedforward loops controlling both production and activation of downstream transcriptional regulators, cells achieve ultrasensitive threshold-like responses. The arrangement of sporulation network genes on the chromosome and transcriptional feedback loops allow coordination of sporulation decision with DNA-replication. Furthermore, to assess the starvation conditions without sensing specific metabolites, cells respond to changes in their growth rates with increased activity of sporulation master regulator. These design features of the sporulation network enable cells to robustly decide between vegetative growth and sporulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Bioproperties of potent nattokinase from Bacillus subtilis YJ1.

    Science.gov (United States)

    Yin, Li-Jung; Lin, Hsin-Hung; Jiang, Shann-Tzong

    2010-05-12

    Fibrinolytic enzyme activity was observed during cultivation of Bacillus subtilis YJ1 in a medium containing 1% skim milk, 1% rice husk, 0.5% NaCl, and 0.25% glucose. It was purified to electrophoretical homogeneity after CM-sepharose FF chromatography. The specific activity and yield were 1791.9 FU/mg and 9.5%, respectively. This purified fibrinolytic enzyme had M of 27.5 kDa, optimal temperature and pH at 50 degrees C and 8.5, respectively. It was stable at pH 6.0-10.0 and 10-40 degrees C and inhibited by Fe(3+), Hg(2+), Cu(2+), Zn(2+), and PMSF. Compared the N terminal of amino acids and full DNA sequence with those in NCBI, it was considered to be a nattokinase.

  13. Proteins that interact with GTP during sporulation of Bacillus subtilis

    International Nuclear Information System (INIS)

    Mitchell, C.; Vary, J.C.

    1989-01-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [α- 32 P]GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either [α- 32 P]GTP or [γ- 32 P]GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms

  14. Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach.

    Science.gov (United States)

    Thakur, Roshan Singh; Desingu, Ambika; Basavaraju, Shivakumar; Subramanya, Shreelakshmi; Rao, Desirazu N; Nagaraju, Ganesh

    2014-09-05

    The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

    Czech Academy of Sciences Publication Activity Database

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, I.; Krásný, Libor; Hocek, Michal

    2016-01-01

    Roč. 44, č. 7 (2016), s. 3000-3012 ISSN 0305-1048 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 ; RVO:61388971 Keywords : Escherichia coli RNA * Bacillus subtilis * restriction endonucleases Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (MBU-M) Impact factor: 10.162, year: 2016 http://nar.oxfordjournals.org/content/44/7/3000.full

  16. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  17. Molecular analysis of Phr peptide processing in Bacillus subtilis.

    Science.gov (United States)

    Stephenson, Sophie; Mueller, Christian; Jiang, Min; Perego, Marta

    2003-08-01

    In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

  18. A bioinformatic survey of RNA-binding proteins in Plasmodium.

    Science.gov (United States)

    Reddy, B P Niranjan; Shrestha, Sony; Hart, Kevin J; Liang, Xiaoying; Kemirembe, Karen; Cui, Liwang; Lindner, Scott E

    2015-11-02

    The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5% of all annotated genes. Almost 90% (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27% of RBPs have elevated expression in gametocytes, while 47 and 24% have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post

  19. The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance.

    Directory of Open Access Journals (Sweden)

    Amit Dipak Amin

    2010-11-01

    Full Text Available The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout

  20. Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

    Science.gov (United States)

    Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

    2016-09-01

    In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.