The Phosphoenolpyruvate Phosphotransferase System Regulates Vibrio cholerae Biofilm Formation through Multiple Independent Pathways

ABSTRACT The bacterial phosphoenolpyruvate phosphotransferase system (PTS) is a highly conserved phosphotransfer cascade that participates in the transport and phosphorylation of selected carbohydrates and modulates many cellular functions in response to carbohydrate availability. It plays a role in the virulence of many bacterial pathogens. Components of the carbohydrate-specific PTS include the general cytoplasmic components enzyme I (EI) and histidine protein (HPr), the sugar-specific cytoplasmic components enzymes IIA (EIIA) and IIB (EIIB), and the sugar-specific membrane-associated multisubunit components enzymes IIC (EIIC) and IID (EIID). Many bacterial genomes also encode a parallel PTS pathway that includes the EI homolog EINtr, the HPr homolog NPr, and the EIIA homolog EIIANtr. This pathway is thought to be nitrogen specific because of the proximity of the genes encoding this pathway to the genes encoding the nitrogen-specific σ factor σ54. We previously reported that phosphorylation of HPr and FPr by EI represses Vibrio cholerae biofilm formation in minimal medium supplemented with glucose or pyruvate. Here we report two additional PTS-based biofilm regulatory pathways that are active in LB broth but not in minimal medium. These pathways involve the glucose-specific enzyme EIIA (EIIAGlc) and two nitrogen-specific EIIA homologs, EIIANtr1 and EIIANtr2. The presence of multiple, independent biofilm regulatory circuits in the PTS supports the hypothesis that the PTS and PTS-dependent substrates have a central role in sensing environments suitable for a surface-associated existence.

Download Full-text

Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01184-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4675-4683 ◽

Cited By ~ 29

Author(s):

Patrick Ymele-Leki ◽

Laetitia Houot ◽

Paula I. Watnick

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Diarrheal Disease ◽

Ectopic Expression ◽

Phosphotransferase System ◽

Content Type ◽

Regulatory Pathways ◽

B Subunit ◽

Multicomponent Signal ◽

Enzyme I

ABSTRACTVibrio choleraeis a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera.V. choleraeuse a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport byV. choleraerequires the mannitol-specific EII (EIIMtl), which is expressed only in the presence of mannitol. Here we show that mannitol activatesV. choleraebiofilm formation and transcription of thevpsbiofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EIIMtlis sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability ofV. choleraeto respond to environmental mannitol by forming a biofilm may play an important role in habitat selection.

Download Full-text

Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine

Infection and Immunity ◽

10.1128/iai.01356-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1482-1494 ◽

Cited By ~ 46

Author(s):

Laetitia Houot ◽

Sarah Chang ◽

Cedric Absalon ◽

Paula I. Watnick

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

System Control ◽

Cellular Functions ◽

Phosphotransferase System ◽

Germfree Mouse ◽

Abiotic Surfaces ◽

Carbohydrate Transport ◽

Mouse Intestine

ABSTRACT The bacterial phosphoenolpyruvate phosphotransferase system (PTS) is a highly conserved phosphotransfer cascade whose components modulate many cellular functions in response to carbohydrate availability. Here, we further elucidate PTS control of Vibrio cholerae carbohydrate transport and activation of biofilm formation on abiotic surfaces. We then define the role of the PTS in V. cholerae colonization of the adult germfree mouse intestine. We report that V. cholerae colonizes both the small and large intestines of the mouse in a distribution that does not change over the course of a month-long experiment. Because V. cholerae possesses many PTS-independent carbohydrate transporters, the PTS is not essential for bacterial growth in vitro. However, we find that the PTS is essential for colonization of the germfree adult mouse intestine and that this requirement is independent of PTS regulation of biofilm formation. Therefore, competition for PTS substrates may be a dominant force in the success of V. cholerae as an intestinal pathogen. Because the PTS plays a role in colonization of environmental surfaces and the mammalian intestine, we propose that it may be essential to successful transit of V. cholerae through its life cycle of pathogenesis and environmental persistence.

Download Full-text

Sugar-mediated regulation of a c-di-GMP phosphodiesterase in Vibrio cholerae

Nature Communications ◽

10.1038/s41467-019-13353-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Kyoo Heo ◽

Young-Ha Park ◽

Kyung-Ah Lee ◽

Joonwon Kim ◽

Hyeong-In Ham ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Carbon Sources ◽

Phosphotransferase System ◽

Host Colonization ◽

Host Immune Responses ◽

Gut Colonization ◽

Host Diet ◽

Underlying Mechanisms ◽

Cyclic Dinucleotide

AbstractBiofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIAGlc, a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIAGlc phosphorylation state, which in turn modulates the interaction of EIIAGlc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIAGlc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization.

Download Full-text

Quorum Sensing Controls Biofilm Formation in Vibrio cholerae through Modulation of Cyclic Di-GMP Levels and Repression of vpsT

Journal of Bacteriology ◽

10.1128/jb.01756-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2527-2536 ◽

Cited By ~ 266

Author(s):

Christopher M. Waters ◽

Wenyun Lu ◽

Joshua D. Rabinowitz ◽

Bonnie L. Bassler

Keyword(s):

Quorum Sensing ◽

Vibrio Cholerae ◽

Cell Density ◽

Biofilm Formation ◽

High Cell Density ◽

High Cell ◽

Chemical Signaling ◽

Signaling Systems ◽

Genes Encoding ◽

Low Cell Density

ABSTRACT Two chemical signaling systems, quorum sensing (QS) and 3′,5′-cyclic diguanylic acid (c-di-GMP), reciprocally control biofilm formation in Vibrio cholerae. QS is the process by which bacteria communicate via the secretion and detection of autoinducers, and in V. cholerae, QS represses biofilm formation. c-di-GMP is an intracellular second messenger that contains information regarding local environmental conditions, and in V. cholerae, c-di-GMP activates biofilm formation. Here we show that HapR, a major regulator of QS, represses biofilm formation in V. cholerae through two distinct mechanisms. HapR controls the transcription of 14 genes encoding a group of proteins that synthesize and degrade c-di-GMP. The net effect of this transcriptional program is a reduction in cellular c-di-GMP levels at high cell density and, consequently, a decrease in biofilm formation. Increasing the c-di-GMP concentration at high cell density to the level present in the low-cell-density QS state restores biofilm formation, showing that c-di-GMP is epistatic to QS in the control of biofilm formation in V. cholerae. In addition, HapR binds to and directly represses the expression of the biofilm transcriptional activator, vpsT. Together, our results suggest that V. cholerae integrates information about the vicinal bacterial community contained in extracellular QS autoinducers with the intracellular environmental information encoded in c-di-GMP to control biofilm formation.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00300-18 ◽

2018 ◽

Vol 200 (24) ◽

Cited By ~ 7

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.

Download Full-text

The Sodium-Driven Flagellar Motor Controls Exopolysaccharide Expression in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.186.15.4864-4874.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4864-4874 ◽

Cited By ~ 80

Author(s):

Crystal M. Lauriano ◽

Chandradipa Ghosh ◽

Nidia E. Correa ◽

Karl E. Klose

Keyword(s):

Vibrio Cholerae ◽

Gene Transcription ◽

Biofilm Formation ◽

Diarrheal Disease ◽

Response Regulator ◽

Gene Clusters ◽

Signaling Cascade ◽

Genes Encoding ◽

Life Threatening

ABSTRACT Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

Download Full-text

Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00466-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6646-6659 ◽

Cited By ~ 102

Author(s):

Jiunn C. N. Fong ◽

Fitnat H. Yildiz

Keyword(s):

Cyclic Amp ◽

Vibrio Cholerae ◽

Transcriptome Analysis ◽

Biofilm Formation ◽

Matrix Proteins ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Genes Encoding ◽

Camp Receptor ◽

Biofilm Matrix

ABSTRACT Vibrio cholerae is a facultative human pathogen. The ability of V. cholerae to form biofilms is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. Formation of mature biofilms requires the production of extracellular matrix components, including Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the transcriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) deletion mutants revealed that cAMP-CRP negatively regulates transcription of both VPS biosynthesis genes and genes encoding biofilm matrix proteins. Further mutational and expression analysis revealed that cAMP-CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. However, negative regulation of the genes encoding biofilm matrix proteins by cAMP-CRP can also occur independent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes encoding diguanylate cyclases (DGCs) and phosphodiesterases. Mutational and phenotypic analysis of the differentially regulated DGCs revealed that a DGC, CdgA, is responsible for the increase in biofilm formation in the Δcrp mutant, showing the connection between of cyclic di-GMP and cAMP signaling in V. cholerae.

Download Full-text

C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

10.1101/2021.03.01.433382 ◽

2021 ◽

Author(s):

Christopher Schubert ◽

Gottfried Unden

Keyword(s):

Escherichia Coli ◽

Nitrogen Assimilation ◽

Concerted Action ◽

Phosphotransferase System ◽

Regulatory Pathways ◽

E Coli ◽

Fumarate Respiration ◽

Genes Encoding ◽

Two Hybrid

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation. The aerobic C4-DC transporter DctA interacted with the regulator EIIAGlc of the E. coli glucose phosphotransferase system. It is suggested that EIIAGlc inhibits C4-DC uptake by DctA in the presence of the preferred substrate glucose.

Download Full-text

Oxygen depletion and nitric oxide stimulate type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

10.1101/2021.04.20.440694 ◽

2021 ◽

Author(s):

Hannah Q Hughes ◽

Kyle A Floyd ◽

Sajjad Hossain ◽

Sweta Anantharaman ◽

David T Kysela ◽

...

Keyword(s):

Nitric Oxide ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Bacterial Species ◽

Bacterial Pathogen ◽

Dna Uptake ◽

Dynamic Activity ◽

Regulatory Pathways ◽

Type Iv ◽

Microaerobic Conditions

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. Dynamic extension and retraction of pili is often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To study this question, we use the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we find that V. cholerae cells retract MSHA pili and detach from a surface in microaerobic conditions. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP (c-di-GMP) under microaerobic conditions to induce MSHA pilus retraction. CdpA contains a putative NO-sensing NosP domain, and we demonstrate that nitric oxide (NO) is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that microaerobic conditions result in endogenous production of NO (or an NO-like molecule) in V. cholerae. Together, these results describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. More broadly, these results show how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Download Full-text

Genome-Wide Mapping Reveals Complex Regulatory Activities of BfmR in Pseudomonas aeruginosa

Microorganisms ◽

10.3390/microorganisms9030485 ◽

2021 ◽

Vol 9 (3) ◽

pp. 485

Author(s):

Ke Fan ◽

Qiao Cao ◽

Lefu Lan

Keyword(s):

Pseudomonas Aeruginosa ◽

Chromatin Immunoprecipitation ◽

Biofilm Formation ◽

High Throughput Sequencing ◽

Response Regulator ◽

Cellular Functions ◽

Genome Wide ◽

Component Systems ◽

Genes Encoding ◽

Two Component Systems

BfmR is a response regulator that modulates diverse pathogenic phenotypes and induces an acute-to-chronic virulence switch in Pseudomonas aeruginosa, an important human pathogen causing serious nosocomial infections. However, the mechanisms of action of BfmR remain largely unknown. Here, using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we showed that 174 chromosomal regions of P. aeruginosa MPAO1 genome were highly enriched by coimmunoprecipitation with a C-terminal Flag-tagged BfmR. Integration of these data with global transcriptome analyses revealed that 172 genes in 106 predicted transcription units are potential targets for BfmR. We determined that BfmR binds to and modulates the promoter activity of genes encoding transcriptional regulators CzcR, ExsA, and PhoB. Intriguingly, BfmR bound to the promoters of a number of genes belong to either CzcR or PhoB regulon, or both, indicating that CzcRS and PhoBR two-component systems (TCSs) deeply feed into the BfmR-mediated regulatory network. In addition, we demonstrated that phoB is required for BfmR to promote the biofilm formation by P. aeruginosa. These results delineate the direct BfmR regulon and exemplify the complexity of BfmR-mediated regulation of cellular functions in P. aeruginosa.

Download Full-text