DOCSLIB.ORG

The SOS Regulon of Enterococcus Faecalis Is Not Involved in Virulence Traits Carrilero, L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

The SOS regulon of Enterococcus faecalis is not involved in virulence traits Carrilero, L. 1, Matrat, S. 1, Thomas-Lopez, D. 1, Gutiérrez, B. 1, Santos-López, A. 1, L Ovejero, C. M1, Hidalgo, L1, Montero-Serra, N.1, Nes, I. F. 2, Gonzalez-Zorn, B. 1 1Departamento, Universidad Complutense de Madrid, Spain 2Laboratory of Microbial Gene Technology and Food Microbiology, The Norwegian University of Life Sciences, Ås, Norway. [email protected] Table 1 Differential Locus Gene Predicted function gene SOS box expression Introduction EF0064 Hypothetical protein x 1.12 Operon EF0065 EF0065 Luciferase family protein x 1.15 CGTACATATGTTCC Enterococcus faecalis is one of the most clinically relevant nosocomial pathogens in the developed EF0066 ruvA Holliday junction DNA helicase /1.17 GGAACATATGTACG Genes related with DNA processing EF0067 ruvB Holliday junction DNA helicase x 1.27 Operon EF0067 EF0682 DNA repair exonuclease family x 1.58 CGAACAAATTTTCG EF0683 Hypothetical protein x 1.56 Operon EF0682 world. Its genetic plasticity facilitates its rapid evolution which is concerning given its role as a carrier EF0684 Cmp-binding protein x 1.81 Operon EF0682 EF0760 amino acid ABC transporter , ATP binding protein x1.22 Operon EF0761 Genes related with metabolism EF0761 amino acid ABC transporter permease protein x 1.14 CGAACATATTTTCG 1 EF0762 uvrB Excinuclease ABC (subunit B) x 1.97 CGAAAATATGTTCG and disseminator of pathogenicity determinants. Some antibiotics are inducers of the SOS response , EF0763 uvrA Excinuclease ABC (subunit A) x 2.00 Operon EF0762 EF1067 Hypothetical protein x 3.25 CAAGCTTATCTTCG 2, 3 EF1080 Structural gene for UV resistance x 7.84 CGAACGTTTGTTCG Genes related with phages which promotes the recombination and horizontal transfer of virulence factors . For the first time, we EF1081 Hypothetical protein x 6.82 Operon EF1080 EF1082 Hypothetical protein x 4.23 Operon EF1080 EF1083 Hypothetical protein x 7.68 Operon EF1080 EF1084 Universal stress protein family x 7.76 Operon EF1080 identified the genes involved in the SOS response of Enterococcus faecalis to elucidate the relationship EF1085 Hypothetical protein x 12.00 Operon EF1080 EF1112 rexB Exonuclease x 1.34 CAAACACATGTTTC EF1113 rexA Exonuclease x 1.45 Operon EF1112 Genes related with aromatics aminoacids EF1114 Hypothetical protein x 1.46 Operon EF1112 between the SOS response and virulence. EF1162 Putative helicase x 1.58 TGAACGCATGATTG EF1163 L-asparaginase x 1.21 Operon EF1162 EF1164 HD domain-containing protein x 1.49 Operon EF1162 EF1343 Sugar ABC transporter permease / 3.43 Operon EF1345 Genes that rule SOS response EF1344 Sugar ABC transporter permease / 2.27 Operon EF1345 EF1345 Sugar ABC transporter sugar-binding protein / 1.74 CGAACACATTTTAG EF1347 glycosyl hydrolase family 13 / 2.39 CTAAAATGTGTTCG EF1348 glucan 1 6-alpha-glucosidase putative / 2.78 Operon EF1347 Methods EF1349 glycosyl hydrolase family 13 / 2.22 Operon EF1347 EF1417 Integrase phage 3 x 4.30 Operon EF1418 Site directed mutagenesis of recA and lexA, the genes responsible for the regulation of the SOS EF1418 Hypothetical protein x 3.83 AGAACATACGTTCT Figure 2 EF1537 XerD Integrase/recombinase /1.24 TGAACAAGTCTTTT EF1538 ScpA Segregation and condensation protein A /1.19 Operon EF1537 EF1561 aroE shikimate 5-dehydrogenase / 2.00 ACAACAAACGTTTG response, provided us with E. faecalis strains with the SOS response constitutively activated or EF1562 phospho-2-dehydro-3-deoxyheptonate aldolase putative / 2.36 Operon EF1561 LexA-box in EF1563 aroB 3-dehydroquinate synthase / 2.69 Operon EF1561 EF1564 aroC chorismate synthase / 3.46 Operon EF1561 Firmicutes inhibited. Furthermore a transcriptomic study with a bacteria-specific microarray was performed allowing EF1565 prephenate dehydrogenase putative / 2.39 Operon EF1561 EF1566 aroA 3-phosphoshikimate 1-carboxyvinyltransferase putative / 2.91 Operon EF1561 EF1567 aroK shikimate kinase / 2.53 Operon EF1561 EF1568 chorismate mutase/prephenate dehydratase putattive / 1.84 Operon EF1561 the LexA-boxes to be identified in silico. Additionally, the resistance to ultraviolet (UV) radiation and EF1579 lexA Transcription repressor of SOS response x 1.31 CGAACGCTTGTTTG EF1686 Hypothetical protein x 9.00 CGAACACTCGTTCG EF1906 Hypothetical protein x 1.21 Operon EF1907 EF1907 maoC family protein x 1.21 AAATCGCGTGTTAG virulence of the different mutants were determined. EF2084 Hypothetical protein x 4.00 CGAACGAAAGTTCG LexA-box in EF2145 Integrase phage 5 x 5.16 CGAACATATATTCG E. faecalis V583 EF2415 Hypothetical protein x 2.73 Operon EF2416 EF2416 rpsU ribosomal protein S21 x 2.33 AAAACAGTCGTTCG EF2704 mutY A/G specific adenine glycosylase x 1.36 Operon EF2705 EF2705 recX Regulatory protein x 1.18 AGAATATATGTTCT EF2756 dinP DNA-damage-inducible protein x 2.14 AGAACGGACGTTCG Results EF3171 recA recombinase x 4.00 CGAATGTTTGTTCG Microarray experiments lead to the characterization of the SOS regulon. 54 genes have been identified, Table 1: Genes constituting the E. faecalis SOS regulon identified by microarray experiments and in silico analysis. Figure 2: LexA-box in Firmicutes and in Enterococcus faecalis. 37 being overexpressed and 17 repressed in the bacteria with the SOS response constitutively expressed. Some of these genes, for example those involved in DNA repair (recA, lexA, uvrBA), had Conclusions been previously identified as part of the SOS regulon of other bacteria. In contrast, some typical SOS This study identifies the SOS regulon of E. faecalis for the first time to date. The SOS response regulon genes, such as low fidelity polymerases, are not present in the Enterococcus faecalis SOS follows a typical system involving the proteins RecA and LexA, however a number of unique genes regulon. Genes involved in the metabolism of aromatic aminoacids, which are involved in the virulence of were identified as part of the regulon. This study also allowed for the precise identification of the E. Listeria spp., are suppressed in the SOS regulon of E. faecalis. The resistance to UV radiation was faecalis LexA-box consensus sequence. Interstit6 ngly, we did not observe any involvement of the increased when the SOS was expressed and there were no virulence differences between the WT strain SOS response in the virulence of E. faecalis. and the lexA- mutant in vivo. References Figure 1 Figure 1 A: Growth after exposure to different intensities of UV light. 1Miller C. et al. SOS response induction by b-lactams and bacterial defense against antibiotic lethality. Science. 2004. 305. - ind- A B WT, JH2-2 wild type; lexA , insertional mutant of lexA; lexA , mutant 1629-1631. with a LexA protein that can´t be cleaved; lexAcom, complemented 2Beaber J.W. et al. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature. 2004 Jan 1;427(6969):72-4. WT insertional lexA mutant; recA-, insertional mutant for recA. B: Virulence Epub 2003 Dec 21. LexA- assays performed in female BALB/c mice 3-6 weeks old. Mice were 3Aranda, J. et al. Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, Ind LexA - inoculated with 5x108 CFU of E. faecalis JH2-2 and individual CFU and virulence. 2011, Journal of Bacteriology, 193(15):3740-7. Com 4 LexA (black dots) in log10 and median (black bars) in kidneys at day 7 of Janky R. et al. Evaluation of phylogenetic footprint discovery for predicting bacterial cis-regulatory elements and revealing their RecA- mice are shown. WT, wild type; lexA-, insertional mutant for lexA. No evolution. BMC Bioinformatics. 2008. 9 (37). significant differences are observed in CFU (Man-Whitney test, α=0,05) between WT and lexA-, thus no differences in the virulence of FEMS Meeting Attendance Grant WT and lexA- strains were observed. FPU Grant .
Recommended publications
  • Examining the Mechanism of a Heavy- Metal ABC Exporter Dennis Hicks University of San Francisco, Dennishicks88@Gmail.Com

    Examining the Mechanism of a Heavy- Metal ABC Exporter Dennis Hicks University of San Francisco, [email protected]

    The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Spring 3-31-2019 NaAtm1: Examining the mechanism of a Heavy- metal ABC Exporter Dennis Hicks University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biochemistry Commons Recommended Citation Hicks, Dennis, "NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter" (2019). Master's Theses. 1204. https://repository.usfca.edu/thes/1204 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter A thesis presented to the faculty of the Department of Chemistry at the University of San Francisco in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Written by Dennis Hicks Bachelor of Science in Biochemistry San Francisco State University August 2019 NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter Thesis written by Dennis Hicks This thesis is written under the guidance of the Faculty Advisory Committee, and approved by all its members, has been accepted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at the University of San Francisco Thesis Committee Janet G.
  • Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter

    Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter

    Cell Research (2017) 27:675-687. © 2017 IBCB, SIBS, CAS All rights reserved 1001-0602/17 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr Structure and mechanism of a group-I cobalt energy coupling factor transporter Zhihao Bao1, 2, 3, *, Xiaofeng Qi1, 3, *, Sen Hong1, 3, Ke Xu1, 3, Fangyuan He1, Minhua Zhang1, Jiugeng Chen1, Daiyin Chao1, Wei Zhao4, Dianfan Li4, Jiawei Wang5, Peng Zhang1 1National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 2Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 3University of Chinese Academy of Sciences, Beijing 100049, China; 4State Key Laboratory of Molecular Biology, National Center for Protein Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chi- nese Academy of Sciences, Shanghai 200031, China; 5State Key Laboratory of Membrane Biology, School of Life Sciences, Tsing- hua University, Beijing 100084, China Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently iden- tified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are mod- ular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A’. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transport- ers is very limited, partly due to a lack of transporter complex structural information.
  • A Flexible Microfluidic System for Single-Cell Transcriptome Profiling

    A Flexible Microfluidic System for Single-Cell Transcriptome Profiling

    www.nature.com/scientificreports OPEN A fexible microfuidic system for single‑cell transcriptome profling elucidates phased transcriptional regulators of cell cycle Karen Davey1,7, Daniel Wong2,7, Filip Konopacki2, Eugene Kwa1, Tony Ly3, Heike Fiegler2 & Christopher R. Sibley 1,4,5,6* Single cell transcriptome profling has emerged as a breakthrough technology for the high‑resolution understanding of complex cellular systems. Here we report a fexible, cost‑efective and user‑ friendly droplet‑based microfuidics system, called the Nadia Instrument, that can allow 3′ mRNA capture of ~ 50,000 single cells or individual nuclei in a single run. The precise pressure‑based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efciencies that compare favorably in the feld. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of diferent bufers and barcoded bead confgurations to facilitate diverse applications. Finally, by 3′ mRNA profling asynchronous human and mouse cells at diferent phases of the cell cycle, we demonstrate the system’s ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this provided supportive evidence for multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and fexible technology for future transcriptomic studies, and other related applications, at cell resolution. Single cell transcriptome profling has recently emerged as a breakthrough technology for understanding how cellular heterogeneity contributes to complex biological systems. Indeed, cultured cells, microorganisms, biopsies, blood and other tissues can be rapidly profled for quantifcation of gene expression at cell resolution.
  • Proquest Dissertations

    Proquest Dissertations

    Division of Mycobacterial Research National Institute for Medical Research, Mill Hill London RecA expression and DNA damage in mycobacteria Nicola A Thomas June 1998 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Science, University College of London. ProQuest Number: U642358 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642358 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A bstr a ct Bacterial responses to DNA damage are highly conserved. One system, the SOS response, involves the co-ordinately induced expression of over 20 unlinked genes. The RecA protein plays a central role in the regulation of this response via its interaction with a repressor protein, LexA. The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (Davis et al, 1991) and a LexA homologue has recently been identified (Movahedzadeh, 1996). The intracellular lifestyle of pathogenic mycobacteria constantly exposes them to hostile agents such as hydrogen peroxide. Consequently, the response of mycobacteria to DNA damage is of great interest.
  • Using Viper, a Package for Virtual Inference of Protein-Activity by Enriched Regulon Analysis

    Using Viper, a Package for Virtual Inference of Protein-Activity by Enriched Regulon Analysis

    Using viper, a package for Virtual Inference of Protein-activity by Enriched Regulon analysis Mariano J. Alvarez1,2, Federico M. Giorgi1, and Andrea Califano1 1Department of Systems Biology, Columbia University, 1130 St. Nicholas Ave., New York 2DarwinHealth Inc, 3960 Broadway, New York May 19, 2021 1 Overview of VIPER Phenotypic changes effected by pathophysiological events are now routinely captured by gene expression pro- file (GEP) measurements, determining mRNA abundance on a genome-wide scale in a cellular population[8, 9]. In contrast, methods to measure protein abundance on a proteome-wide scale using arrays[11] or mass spectrometry[10] technologies are far less developed, covering only a fraction of proteins, requiring large amounts of tissue, and failing to directly capture protein activity. Furthermore, mRNA expression does not constitute a reliable predictor of protein activity, as it fails to capture a variety of post-transcriptional and post-translational events that are involved in its modulation. Even reliable measurements of protein abundance, for instance by low-throughput antibody based methods or by higher-throughput methods such as mass spectrometry, do not necessarily provide quantitative assessment of functional activity. For instance, enzymatic activity of signal transduction proteins, such as kinases, ubiquitin ligases, and acetyltransferases, is frequently modulated by post-translational modification events that do not affect total protein abundance. Similarly, transcription factors may require post-translationally mediated activation, nuclear translocation, and co-factor availability before they may regulate specific repertoires of their transcriptional targets. Fi- nally, most target-specific drugs affect the activity of their protein substrates rather than their protein or mRNA transcript abundance.
  • Reca and DNA Recombination: a Review of Molecular Mechanisms

    Reca and DNA Recombination: a Review of Molecular Mechanisms

    Biochemical Society Transactions (2019) 47 1511–1531 https://doi.org/10.1042/BST20190558 Review Article RecA and DNA recombination: a review of molecular mechanisms Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/47/5/1511/859295/bst-2019-0558.pdf by Tata Institute of Fundamental Research user on 20 January 2020 Elsa del Val1,2, William Nasser1, Hafid Abaibou2 and Sylvie Reverchon1 1Microbiologie, Adaptation et Pathogénie, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, 11 Avenue Jean Capelle, F-69621 Villeurbanne, France; 2Molecular Innovation Unit, Centre Christophe Mérieux, BioMérieux, 5 rue des Berges, 38024 Grenoble Cedex 01, France Correspondence: Sylvie Reverchon ([email protected]) or Hafid Abaibou ([email protected]) Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combin- ation of DNA synthesis, ligation and resolution.
  • Non-Canonical Functions of the Bacterial Sos Response

    Non-Canonical Functions of the Bacterial Sos Response

    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Non-Canonical Functions Of The aB cterial Sos Response Amanda Samuels University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Microbiology Commons Recommended Citation Samuels, Amanda, "Non-Canonical Functions Of The aB cterial Sos Response" (2019). Publicly Accessible Penn Dissertations. 3253. https://repository.upenn.edu/edissertations/3253 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3253 For more information, please contact [email protected]. Non-Canonical Functions Of The aB cterial Sos Response Abstract DNA damage is a pervasive environmental threat, as such, most bacteria encode a network of genes called the SOS response that is poised to combat genotoxic stress. In the absence of DNA damage, the SOS response is repressed by LexA, a repressor-protease. In the presence of DNA damage, LexA undergoes a self-cleavage reaction relieving repression of SOS-controlled effector genes that promote bacterial survival. However, depending on the bacterial species, the SOS response has an expanded role beyond DNA repair, regulating genes involved in mutagenesis, virulence, persistence, and inter-species competition. Despite a plethora of research describing the significant consequences of the SOS response, it remains unknown what physiologic environments induce and require the SOS response for bacterial survival. In Chapter 2, we utilize a commensal E. coli strain, MP1, and established that the SOS response is critical for sustained colonization of the murine gut. Significantly, in evaluating the origin of the genotoxic stress, we found that the SOS response was nonessential for successful colonization in the absence of the endogenous gut microbiome, suggesting that competing microbes might be the source of genotoxic stress.
  • Toolbox Model of Evolution of Prokaryotic Metabolic Networks and Their Regulation

    Toolbox Model of Evolution of Prokaryotic Metabolic Networks and Their Regulation

    Toolbox model of evolution of prokaryotic metabolic networks and their regulation Sergei Maslova,1, Sandeep Krishnab, Tin Yau Panga,c, and Kim Sneppenb aDepartment of Condensed Matter Physics and Materials Science, Brookhaven National Laboratory, Upton, NY 11973; bNiels Bohr Institute, Blegdamsvej 17, DK-2100 Copenhagen, Denmark; and cDepartment of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794-3800 Edited by David J. Lipman, National Institutes of Health, Bethesda, MD, and approved April 16, 2009 (received for review March 23, 2009) It has been reported that the number of transcription factors A simple evolutionary model explains both these empirical encoded in prokaryotic genomes scales approximately quadrati- observations in a unified framework based on modular functional cally with their total number of genes. We propose a conceptual design of prokaryotic metabolic networks and their regulation. explanation of this finding and illustrate it using a simple model in which metabolic and regulatory networks of prokaryotes are Toolbox View of Metabolic Networks shaped by horizontal gene transfer of coregulated metabolic Metabolic networks are composed of many semiautonomous func- pathways. Adapting to a new environmental condition monitored tional modules corresponding to traditional metabolic pathways (8) by a new transcription factor (e.g., learning to use another nutri- or their subunits (9). Constituent genes of such evolutionary ent) involves both acquiring new enzymes and reusing some of the modules tend to cooccur (be either all present or all absent) in enzymes already encoded in the genome. As the repertoire of genomes (9, 10). These pathways overlap with each other to form branched, interconnected metabolic networks.
  • An Improved Bind-N-Seq Strategy to Determine Protein-DNA Interactions Validated Using the Bacterial Transcriptional Regulator Yipr Shi-Qi An1,2* , Miguel A

    An Improved Bind-N-Seq Strategy to Determine Protein-DNA Interactions Validated Using the Bacterial Transcriptional Regulator Yipr Shi-Qi An1,2* , Miguel A

    An et al. BMC Microbiology (2020) 20:1 https://doi.org/10.1186/s12866-019-1672-7 METHODOLOGY ARTICLE Open Access An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR Shi-qi An1,2* , Miguel A. Valvano2, Yan-hua Yu3, Jeremy S. Webb1 and Guillermo Lopez Campos2 Abstract Background: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid,andsimplifiedBind-n-seqprotocolcoupledwithauser-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well- known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). Results: The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes.
  • Making Informed Decisions: Regulatory Interactions Between Two-Component Systems

    Making Informed Decisions: Regulatory Interactions Between Two-Component Systems

    Review TRENDS in Microbiology Vol.11 No.8 August 2003 359 Making informed decisions: regulatory interactions between two-component systems Jetta J.E. Bijlsma and Eduardo A. Groisman Department of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, Campus Box 8230, 660 S. Euclid Avenue, St Louis, MO 63110, USA Bacteria experience a multitude of stresses in the com- phosphorylate from small molecular weight phosphoryl plex niches they inhabit. These stresses are denoted by donors, such as acetyl phosphate and carbamoyl phos- specific signals that typically alter the activity of individ- phate [3,4]. In addition, the vast majority of sensor kinases ual two-component regulatory systems. Processing of exhibit phosphatase activity towards their cognate multiple signals by different two-component systems response regulators. Although most two-component sys- occurs when multiple sensors interact with a single tems entail a single His-to-Asp phosphotransfer event response regulator, by phosphatases interrupting phos- between a histidine kinase and a response regulator, there phoryl transfer in phosphorelays and through transcrip- is a subset that consists of a three-step His-Asp-His-Asp tional and post-transcriptional mechanisms. Gaining phosphorelay (Fig. 1b) [1,2]. The extra phosphorylatable insight into these mechanisms provides an understand- domains in a phosphorelay can be present in separate ing at the molecular level of bacterial adaptation to ever proteins or be part of multi-domain (i.e.
  • Testing the DNA Repair Hypothesis in Bacillus Subtilis and Haemophilus Intuenzae

    Testing the DNA Repair Hypothesis in Bacillus Subtilis and Haemophilus Intuenzae

    Copyright 0 1993 by the Genetics Society of America Evolution of Natural Transformation: Testing the DNA Repair Hypothesis in Bacillus subtilis and Haemophilus intuenzae Rosemary J. Redfield' Department of Biochemistry, University of British Columbia, Vancouver, BritishColumbia V6T lZ3, Canada Manuscript received July 23, 1992 Accepted for publication December 15, 1992 ABSTRACT The hypothesis that the primary function of bacterial transformation is DNA repair was tested in the naturallytransformable bacteria Bacillus subtilis and Haemophilusinjluenzae by determining whether competence for transformation is regulated by DNA damage. Accordingly, DNA damage was induced by mitomycin C and by ultraviolet radiation at doses that efficiently induced a known damage-inducible gene fusion, and the ability of the damaged cultures to transform was monitored. Experiments were carried out both under conditions wherecells do not normally become competent and under competence-inducing conditions.No induction or enhancement of competence by damage was seen in either organism. These experiments strongly suggest thatthe regulation of competence does not involve a response to DNA damage, andthus that explanations other than DNA repair must be sought for the evolutionaryfunctions of natural transformation systems. ANY groups of bacteria have evolved mecha- noncompetent cells) are provided with homologous M nisms of natural transformation which enable DNA after the cell's own DNA has been damaged by them to take upfree DNA fragmentsfrom their UV irradiation, the proportion of transformants is environments. If this DNA is homologous and genet- increased amongthe survivors (MICHOD, WOJCIE- ically marked, recombinant pregnancy are often pro- CHOWSKI and HOELZER1988). The favored interpre- duced (STEWARTand CARLSON1986). Most research- tation was that the cells that took up DNA survived ers have assumed thattransformation evolved for damage better than the cells that did not.
  • Molecular Analysis of the Reca Gene and SOS Box of the Purple Non-Sulfur Bacterium Rhodopseudomonas Palustris No

    Molecular Analysis of the Reca Gene and SOS Box of the Purple Non-Sulfur Bacterium Rhodopseudomonas Palustris No

    Microbiology (1999), 145, 1275-1 285 Printed in Great Britain Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 Valerie Dumay,t Masayuki lnui and Hideaki Yukawa Author for correspondence: Hideaki Yukawa. Tel: +81 774 75 2308. Fax: + 81 774 75 2321. e-mail : yukawa(&rite.or.jp ~~~ ~~ Molecular Microbiology The red gene of the purple non-sulfur bacterium Rhodopseudomonas and Research palustris no. 7 was isolated by a PCR-based method and sequenced. The Institute of Innovative Technology for the Earth, complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 9-2 Kizugawadai, Kizu-cho, 363 amino acids which is most closely related to the RecA proteins from Soraku-gun, Kyoto 619- species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. 0292, Japan palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no.