More Like this

1. Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

   https://doi.org/10.1371/journal.pgen.1009550  

   Duddy, Olivia P. ; Huang, Xiuliang ; Silpe, Justin E. ; Bassler, Bonnie L. ( July 2021 , PLOS Genetics)

   Crosson, Sean (Ed.)

   Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae , one quorum-sensing receptor and transcription factor, called VqmA (VqmA Vc ), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmA Phage that activates transcription of the phage gene qtip , and Qtip launches the phage lytic program. Curiously, VqmA Phage can activate vqmR expression but VqmA Vc cannot activate expression of qtip . Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmA Phage and A192, the equivalent residue in VqmA Vc , in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmA Phage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmA Phage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmA Phage and VqmA Vc . A second, targeted genetic screen revealed a region located in the VqmA Vc DNA-binding domain that is necessary to prevent VqmA Vc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmA Vc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmA Vc from binding the qtip promoter. 

   more » « less
2. The Vibrio cholerae Quorum-Sensing Protein VqmA Integrates Cell Density, Environmental, and Host-Derived Cues into the Control of Virulence

   https://doi.org/10.1128/mBio.01572-20  

   Mashruwala, Ameya A. ; Bassler, Bonnie L. ( August 2020 , mBio)

   Buchrieser, Carmen (Ed.)

   ABSTRACT Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 autoinducer, while it continues to produce the DPO autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae . Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output. IMPORTANCE Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae , the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine. 

   more » « less
3. An autoinducer-independent RhlR quorum-sensing receptor enables analysis of RhlR regulation

   McCready, A. R. ; Paczkowski, J. E. ; Cong, J-P. ; Bassler, B. L. ( July 2019 , PLOS pathogens)

   Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification. 

   more » « less
4. Regulatory small RNA, Qrr2 is expressed independently of sigma factor-54 and can function as the sole Qrr sRNA to control quorum sensing in Vibrio parahaemolyticus

   https://doi.org/10.1128/JB.00350-21  

   Tague, J.G. ; Hong, J. ; Kalburge, S.S. ; Boyd, E.F. ( October 2021 , Journal of Bacteriology)

   null (Ed.)

   Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR. 

   more » « less
5. Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

   https://doi.org/10.1128/JB.00380-18  

   Muzhingi, Itai ; Prado, Cecilia ; Sylla, Mariame ; Diehl, Frances F. ; Nguyen, Duy K. ; Servos, Mariah M. ; Flores Ramos, Stephany ; Purdy, Alexandra E. ; Stock, Ann M. ( December 2018 , Journal of Bacteriology)

   ABSTRACT Vibrio cholerae controls the pathogenicity of interactions with arthropod hosts via the activity of the CrbS/R two-component system. This signaling pathway regulates the consumption of acetate, which in turn alters the relative virulence of interactions with arthropods, including Drosophila melanogaster . CrbS is a histidine kinase that links a transporter-like domain to its signaling apparatus via putative STAC and PAS domains. CrbS and its cognate response regulator are required for the expression of acetyl coenzyme A (acetyl-CoA) synthetase (product of acs ), which converts acetate to acetyl-CoA. We demonstrate that the STAC domain of CrbS is required for signaling in culture; without it, acs transcription is reduced in LB medium, and V. cholerae cannot grow on acetate minimal media. However, the strain remains virulent toward Drosophila and expresses acs similarly to the wild type during infection. This suggests that there is a unique signal or environmental variable that modulates CrbS in the gastrointestinal tract of Drosophila . Second, we present evidence in support of CrbR, the response regulator that interacts with CrbS, binding directly to the acs promoter, and we identify a region of the promoter that CrbR may target. We further demonstrate that nutrient signals, together with the cAMP receptor protein (CRP)-cAMP system, control acs transcription, but regulation may occur indirectly, as CRP-cAMP activates the expression of the crbS and crbR genes. Finally, we define the role of the Pta-AckA system in V. cholerae and identify redundancy built into acetate excretion pathways in this pathogen. IMPORTANCE CrbS is a member of a unique family of sensor histidine kinases, as its structure suggests that it may link signaling to the transport of a molecule. However, mechanisms through which CrbS senses and communicates information about the outside world are unknown. In the Vibrionaceae , orthologs of CrbS regulate acetate metabolism, which can, in turn, affect interactions with host organisms. Here, we situate CrbS within a larger regulatory framework, demonstrating that crbS is regulated by nutrient-sensing systems. Furthermore, CrbS domains may play various roles in signaling during infection and growth in culture, suggesting a unique mechanism of host recognition. Finally, we define the roles of additional pathways in acetate flux, as a foundation for further studies of this metabolic nexus point. 

   more » « less