scholarly journals Glucose-Specific Enzyme IIA Has Unique Binding Partners in The Vibrio cholerae Biofilm

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Bradley S. Pickering ◽  
Daniel R. Smith ◽  
Paula I. Watnick
Keyword(s):  
Adenylate Cyclase ◽  
Vibrio Cholerae ◽  
Carbon Storage ◽  
Biofilm Formation ◽  
Specific Enzyme ◽  
Microbial Physiology ◽  
Content Type ◽  
Multiprotein Complexes ◽  
Binding Partners ◽  
Carbon Storage Regulator

ABSTRACTGlucose-specific enzyme IIA (EIIAGlc) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIAGlcactivates transcription of the genes required forVibrio choleraebiofilm formation. While EIIAGlcmodulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIAGlcactivates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIAGlcin both planktonic and biofilm cells. A surprising number of novel EIIAGlcbinding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIAGlc, such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog ofEscherichia coliCsrD, was found to be a dominant binding partner of EIIAGlc. Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIAGlc. To explore the existence of multiprotein complexes centered on EIIAGlc, we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIAGlc, this analysis yielded many of the same proteins that copurified with EIIAGlc. We hypothesize that EIIAGlcserves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners.IMPORTANCEEIIAGlcis a global regulator of microbial physiology that acts through direct interactions with other proteins. This work represents the first demonstration that the protein partners of EIIAGlcare distinct in the microbial biofilm. Furthermore, it provides the first evidence that EIIAGlcmay exist in multiprotein complexes with its partners, setting the stage for an investigation of how the multiple partners of EIIAGlcinfluence one another. Last, it provides a connection between the phosphoenolpyruvate phosphotransferase (PTS) and Csr (Carbon storage regulator) regulatory systems. This work increases our understanding of the complexity of regulation by EIIAGlcand provides a link between the PTS and Csr networks, two global regulatory cascades that influence microbial physiology.

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
Mengting Shi ◽  
Yue Zheng ◽  
Xianghong Wang ◽  
Zhengjia Wang ◽  
Menghua Yang
Keyword(s):  
Mouse Model ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Type Species ◽  
Wild Type ◽  
Expression Library ◽  
Content Type ◽  
Genomic Expression ◽  
Infant Mouse ◽  
Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

scholarly journals Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2972-2985 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Rajesh G. Prabhu ◽  
Ying-Han Lin ◽  
Christine L. Miller ◽  
J. Seshu
Keyword(s):  
Borrelia Burgdorferi ◽  
Carbon Storage ◽  
Vertebrate Host ◽  
Acetyl Phosphate ◽  
Mobility Shift ◽  
Acetyl Coa ◽  
Site Specific ◽  
Content Type ◽  
Environmental Signals ◽  
Carbon Storage Regulator

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

scholarly journals Correction: Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation inVibrio choleraeand downregulate the expression of virulence genes

2018 ◽  
Vol 9 (39) ◽  
pp. 7715-7715
Author(s):  
Nicolas Perez-Soto ◽  
Lauren Moule ◽  
Daniel N. Crisan ◽  
Ignacio Insua ◽  
Leanne M. Taylor-Smith ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Synthetic Polymers ◽  
Cationic Polymers ◽  
Microbial Physiology

Correction for ‘Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation inVibrio choleraeand downregulate the expression of virulence genes’ by Nicolas Perez-Sotoet al.,Chem. Sci., 2017,8, 5291–5298.

scholarly journals A Subset of Exoribonucleases Serve as Degradative Enzymes for pGpG in c-di-GMP Signaling

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Mona W. Orr ◽  
Cordelia A. Weiss ◽  
Geoffrey B. Severin ◽  
Husan Turdiev ◽  
Soo-Kyoung Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacillus Anthracis ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Feedback Inhibition ◽  
Product Inhibition ◽  
Systematic Screening ◽  
Content Type ◽  
Genes Encoding ◽  
Mutant Phenotypes

ABSTRACT Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in Pseudomonas aeruginosa as the 3′-to-5′ exoribonuclease oligoribonuclease (Orn). Mutants of orn accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an orn ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from Vibrio cholerae and Bacillus anthracis, we found that only enzymes known to cleave oligoribonucleotides (orn and nrnA) rescued the P. aeruginosa Δorn mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (nrnA, nrnB, nrnC, and orn) can also hydrolyze pGpG. A B. subtilis nrnA nrnB mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides. IMPORTANCE The bacterial bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3′-to-5′ exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Pseudomonas aeruginosa. Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of Bacillus anthracis and Vibrio cholerae that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.

scholarly journals Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

2015 ◽  
Vol 83 (3) ◽  
pp. 1199-1209 ◽  
Author(s):  
Kivanc Bilecen ◽  
Jiunn C. N. Fong ◽  
Andrew Cheng ◽  
Christopher J. Jones ◽  
David Zamorano-Sánchez ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Lipid A ◽  
Response Regulator ◽  
Regulatory Region ◽  
Regulatory System ◽  
Polymyxin B ◽  
Content Type ◽  
Two Component ◽  
Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

scholarly journals Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

2012 ◽  
Vol 81 (2) ◽  
pp. 608-617 ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Wei Li ◽  
Kate R. Fortney ◽  
Diane M. Janowicz ◽  
Sheila Ellinger ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stationary Phase ◽  
Carbon Storage ◽  
Stress Responses ◽  
Haemophilus Ducreyi ◽  
Content Type ◽  
Enhanced Sensitivity ◽  
Significant Survival ◽  
Carbon Storage Regulator ◽  
Mutant Phenotypes

ABSTRACTThe carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence.Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog ofcsrA. Here, we generated an unmarked, in-frame deletion mutant ofcsrAto assess its contribution toH. ducreyipathogenesis. In human inoculation experiments, thecsrAmutant was partially attenuated for pustule formation compared to its parent. Deletion ofcsrAresulted in decreased adherence ofH. ducreyito human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants ofH. ducreyiadherence to HFF cells, were downregulated in thecsrAmutant. Compared to its parent, thecsrAmutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. ThecsrAmutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation intranspartially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

scholarly journals Integration of Cyclic di-GMP and Quorum Sensing in the Control ofvpsTandaphAin Vibrio cholerae

2011 ◽  
Vol 193 (22) ◽  
pp. 6331-6341 ◽  
Author(s):  
Disha Srivastava ◽  
Rebecca C. Harris ◽  
Christopher M. Waters
Keyword(s):  
Quorum Sensing ◽  
Binding Site ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Virulence Gene ◽  
Transcriptional Activator ◽  
Transcriptional Regulators ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Signaling Systems

Vibrio choleraetransitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enableV. choleraeto alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism inV. choleraethat integrates c-di-GMP and QS to control the expression of two transcriptional regulators:aphA, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, andvpsT, a transcriptional activator that induces biofilm formation. Surprisingly,aphAexpression was induced by c-di-GMP. Activation of bothaphAandvpsTby c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest thatV. choleraecombines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators.

scholarly journals The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Jennifer K. Teschler ◽  
Andrew T. Cheng ◽  
Fitnat H. Yildiz
Keyword(s):  
Signal Transduction ◽  
Vibrio Cholerae ◽  
Histidine Kinase ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Systematic Analysis ◽  
Content Type ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels. IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.

scholarly journals The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

2016 ◽  
Vol 198 (6) ◽  
pp. 973-985 ◽  
Author(s):  
Andrew Rogers ◽  
Loni Townsley ◽  
Ana L. Gallego-Hernandez ◽  
Sinem Beyhan ◽  
Laura Kwuan ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Secretion System ◽  
Lon Protease ◽  
Human Pathogen ◽  
Type Vi Secretion System ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Infant Mouse ◽  
Type Vi Secretion

ABSTRACTThe presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogenVibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in alonAmutant compared to the wild type. We further demonstrated that alonAmutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence ofV. cholerae.IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogenV. choleraeis in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding ofV. choleraephysiology.

scholarly journals Cyclic di-GMP Positively Regulates DNA Repair in Vibrio cholerae

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Disha Srivastava ◽  
Amanda L. Ngouajio ◽  
Christopher M. Waters
Keyword(s):  
Dna Repair ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Binding Assays ◽  
Gene Encoding ◽  
Repair Gene ◽  
Content Type ◽  
Dna Repair Gene ◽  
High Concentrations

ABSTRACT In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade. IMPORTANCE Vibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

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