skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Mechanism underlying autoinducer recognition in the  Vibrio cholerae  DPO-VqmA quorum-sensing pathway
Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded, and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it to existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2’ position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone or Ala-AA, also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure–function analysis identifies key features required for VqmA activation and DNA binding and establishes that, while VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.  more » « less
Award ID(s):
1713731 1734030
PAR ID:
10135519
Author(s) / Creator(s):
; ; ; ; ; ;
Date Published:
Journal Name:
Journal of Biological Chemistry
ISSN:
0021-9258
Page Range / eLocation ID:
jbc.RA119.012104
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; (, 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
  2. ; ; ; (, 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
  3. ; ; ; (, PLOS Genetics)
    Matic, Ivan (Ed.)
    Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of theVibrioQS receptor-transcription factor, called VqmA, that monitors theVibrioQS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage geneqtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host genevqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequenceVibrio parahaemolyticusstrain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encodingvqmRandvqmAharbors a deletion encompassingvqmRand a portion of thevqmApromoter, inactivating that QS system. We discover thatV.parahaemolyticusstrain O3:K6 882 is also defective in its other QS systems, due to a mutation inluxO, encoding the central QS transcriptional regulator LuxO. Both thevqmR-vqmAandluxOmutations lockV.parahaemolyticusstrain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects inV.parahaemolyticusstrain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competentV.parahaemolyticusstrain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, inV.parahaemolyticusstrain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis. 
    more » « less
  4. ; ; ; (, 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
  5. ; ; ; (, Biochemistry)
    Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email