Identification of a cyclic-di-GMP-modulating response regulator that impacts biofilm formation in a model sulfate reducing bacterium

We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn2+-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth.

[1]  E. Pedroso,et al.  Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella , 2009, Proceedings of the National Academy of Sciences.

[2]  B. Kazmierczak,et al.  Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa , 2006, Molecular microbiology.

[3]  B. Peyton,et al.  Uranium immobilization by sulfate-reducing biofilms. , 2004, Environmental science & technology.

[4]  G. Hobbs,et al.  Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi , 2011, Infection and Immunity.

[5]  M. Auer,et al.  Exploring the role of CheA3 in Desulfovibrio vulgaris Hildenborough motility , 2014, Front. Microbiol..

[6]  Katherine H. Huang,et al.  Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion , 2005, Applied and Environmental Microbiology.

[7]  J. M. Dow,et al.  Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Michael Y. Galperin,et al.  Interplay of heritage and habitat in the distribution of bacterial signal transduction systems. , 2010, Molecular bioSystems.

[9]  Matthias Christen,et al.  Identification and Characterization of a Cyclic di-GMP-specific Phosphodiesterase and Its Allosteric Control by GTP* , 2005, Journal of Biological Chemistry.

[10]  Jinsong Chen,et al.  In situ long-term reductive bioimmobilization of Cr(VI) in groundwater using hydrogen release compound. , 2008, Environmental science & technology.

[11]  A. Paiardini,et al.  C-di-GMP Hydrolysis by Pseudomonas aeruginosa HD-GYP Phosphodiesterases: Analysis of the Reaction Mechanism and Novel Roles for pGpG , 2013, PloS one.

[12]  Peter Williams,et al.  IMG: the integrated microbial genomes database and comparative analysis system , 2011, Nucleic Acids Res..

[13]  Elizabeth M. Boon,et al.  Characterization of a diguanylate cyclase from Shewanella woodyi with cyclase and phosphodiesterase activities. , 2010, Molecular bioSystems.

[14]  Rekha Seshadri,et al.  The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough , 2004, Nature Biotechnology.

[15]  Jay D Keasling,et al.  Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state , 2012, BMC Genomics.

[16]  J. H. Boom,et al.  Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid , 1987, Nature.

[17]  L. Luo,et al.  GGDEF and EAL proteins play different roles in the control of Sinorhizobium meliloti growth, motility, exopolysaccharide production, and competitive nodulation on host alfalfa. , 2010, Acta biochimica et biophysica Sinica.

[18]  Regine Hengge,et al.  Principles of c-di-GMP signalling in bacteria , 2009, Nature Reviews Microbiology.

[19]  U. Jenal,et al.  Structural and mechanistic determinants of c-di-GMP signalling , 2009, Nature Reviews Microbiology.

[20]  J. Wall,et al.  Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems , 2013, Applied and Environmental Microbiology.

[21]  Zbigniew Lewandowski,et al.  Role of sulfate‐reducing bacteria in corrosion of mild steel: A review , 1995 .

[22]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[23]  G. Salmond,et al.  Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes. , 2014, Microbiology.

[24]  A. Spiers,et al.  The structure-function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain. , 2007, Microbiology.

[25]  Pei-Chi Hsu,et al.  GsmR, a response regulator with an HD‐related output domain in Xanthomonas campestris, is positively controlled by Clp and is involved in the expression of genes responsible for flagellum synthesis , 2013, The FEBS journal.

[26]  Michael Y. Galperin,et al.  Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger , 2013, Microbiology and Molecular Reviews.

[27]  M. Rohde,et al.  The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix , 2001, Molecular microbiology.

[28]  Thomas Bjarnsholt,et al.  Interactions in multispecies biofilms: do they actually matter? , 2014, Trends in microbiology.

[29]  J. Ghigo,et al.  A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli , 2006, Journal of bacteriology.

[30]  C. Jerez,et al.  The extremophile Acidithiobacillus ferrooxidans possesses a c‐di‐GMP signalling pathway that could play a significant role during bioleaching of minerals , 2012, Letters in applied microbiology.

[31]  J. M. Dow,et al.  HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa. , 2009, Environmental microbiology.

[32]  R. Thauer,et al.  Growth of Desulfovibrio species on Hydrogen and Sulphate as Sole Energy Source , 1981 .

[33]  A. Arkin,et al.  Deletion of the Desulfovibrio vulgaris Carbon Monoxide Sensor Invokes Global Changes in Transcription , 2012, Journal of bacteriology.

[34]  Michael Y. Galperin.  Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations , 2006, Journal of bacteriology.

[35]  Paramvir S. Dehal,et al.  Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium , 2011, Genome Biology.

[36]  E V Koonin,et al.  The HD domain defines a new superfamily of metal-dependent phosphohydrolases. , 1998, Trends in biochemical sciences.

[37]  A. Arkin,et al.  New Family of Tungstate-Responsive Transcriptional Regulators in Sulfate-Reducing Bacteria , 2013, Journal of bacteriology.

[38]  Mark Gomelsky,et al.  Cyclic Diguanylate Is a Ubiquitous Signaling Molecule in Bacteria: Insights into Biochemistry of the GGDEF Protein Domain , 2005, Journal of bacteriology.

[39]  J. Keasling,et al.  HipA-Triggered Growth Arrest and β-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis , 2013, Journal of bacteriology.

[40]  Inna Dubchak,et al.  Reconstruction Of Regulatory And Metabolic Pathways In Metal-Reducing delta-Proteobacteria , 2004 .

[41]  Christopher L. Hemme,et al.  How sulphate-reducing microorganisms cope with stress: lessons from systems biology , 2011, Nature Reviews Microbiology.

[42]  J. M. Dow,et al.  Cell–cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris , 2010, Proceedings of the National Academy of Sciences.

[43]  Judy D. Wall,et al.  Effect of the Deletion of qmoABC and the Promoter-Distal Gene Encoding a Hypothetical Protein on Sulfate Reduction in Desulfovibrio vulgaris Hildenborough , 2010, Applied and Environmental Microbiology.

[44]  B. Giese,et al.  Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. , 2004, Genes & development.

[45]  M. Fields,et al.  Biofilm formation in Desulfovibrio vulgaris Hildenborough is dependent upon protein filaments. , 2007, Environmental microbiology.

[46]  Andrew J. Schmidt,et al.  The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains , 2005, Journal of bacteriology.

[47]  Inna Dubchak,et al.  MicrobesOnline: an integrated portal for comparative and functional genomics , 2009, Nucleic Acids Res..

[48]  Inna Dubchak,et al.  Reconstruction of regulatory and metabolic pathways in metal-reducing δ-proteobacteria , 2004, Genome Biology.

[49]  Steven D. Brown,et al.  New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20 , 2013, Applied and Environmental Microbiology.

[50]  U. Römling,et al.  GGDEF and EAL domains inversely regulate cyclic di‐GMP levels and transition from sessility to motility , 2004, Molecular microbiology.

[51]  Lu-Yun Lian,et al.  A simple method for improving protein solubility and long-term stability. , 2004, Journal of the American Chemical Society.

[52]  J. M. Dow,et al.  Retracted: Dynamic complex formation between HD‐GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris , 2012, Molecular microbiology.

[53]  Christopher L. Hemme,et al.  Hydrogen peroxide-induced oxidative stress responses in Desulfovibrio vulgaris Hildenborough. , 2010, Environmental microbiology.

[54]  A. Arkin,et al.  Transcription Factor Family-Based Reconstruction of Singleton Regulons and Study of the Crp/Fnr, ArsR, and GntR Families in Desulfovibrionales Genomes , 2012, Journal of bacteriology.

[55]  David A. Stahl,et al.  Rapid evolution of stability and productivity at the origin of a microbial mutualism , 2010, Proceedings of the National Academy of Sciences.

[56]  N. Luscombe,et al.  Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity , 2010, Nucleic acids research.