Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae

Type VI secretion is critical for Vibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V. cholerae type VI secretion system genes are encoded in one large and two small clusters. In V. cholerae, type VI secretion is controlled by quorum sensing, the cell–cell communication process that enables bacteria to orchestrate group behaviours. The quorum‐sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum‐sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs (Qrr sRNAs) that function between LuxO and HapR in the quorum‐sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA‐dependent regulation of the type VI secretion system is conserved in pandemic and non‐pandemic V. cholerae strains.     

Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing

Quorum sensing is a mechanism of cell‐to‐cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell‐population density. At the core of the Vibrio cholerae quorum‐sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1–4 (Qrr1–4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild‐type quorum‐sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA‐activator LuxO and one involving the sRNA‐target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1–4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum‐sensing‐regulated target genes.     

Comparative structural analysis of two proteins belonging to quorum sensing system in Vibrio cholerae

Vibrio cholerae uses quorum sensing communication system to interact with other bacteria and for gauzing environmental parameters. This organism dwells equally well in both human host and aquatic environments. Quorum sensing regulates multitude of activities and is one of the lucrative targets presently pursued for drug design in bacteria to encounter virulence. Histidine phosphotransfer protein LuxU and response regulator LuxO of V. cholerae are known to play important roles in biofilms and virulence machinery. In the present study, we used computational methods to model LuxU and LuxO and simulated the interactions of LuxO and LuxU. Since no structural details of the proteins were available, we employed homology modeling to construct the three-dimensional structures and then performed molecular dynamics simulations to study dynamic behavior of the LuxO and LuxU from V. cholerae. The modeled proteins were validated and subjected to molecular docking analyses. This allowed us to predict the binding modes of the proteins to elucidate probable sites of interference.     

Functional determinants of the quorum‐sensing non‐coding RNAs and their roles in target regulation

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1–5, control quorum‐sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem‐loops: the first stem‐loop is crucial for base pairing with a subset of targets. This stem‐loop also protects the Qrr sRNAs from RNase E‐mediated degradation. The second stem‐loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem‐loop plays an accessory role in base pairing and stability. The fourth stem‐loop functions as a rho‐independent terminator. In the quorum‐sensing regulon, Qrr sRNAs‐controlled genes are the most rapid to respond to quorum‐sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.     

Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54

The bioluminescent marine bacterium Vibrio harveyi controls light production (lux) by an elaborate quorum-sensing circuit. V. harveyi produces and responds to two different autoinducer signals (AI-1 and AI-2) to modulate the luciferase structural operon (luxCDABEGH) in response to changes in cell-population density. Unlike all other Gram-negative quorum-sensing organisms, V. harveyi regulates quorum sensing using a two-component phosphorylation-dephosphorylation cascade. Each autoinducer is recognized by a cognate hybrid sensor kinase (called LuxN and LuxQ). Both sensors transduce information to a shared phosphorelay protein called LuxU, which in turn conveys the signal to the response regulator protein LuxO. Phospho-LuxO is responsible for repression of luxCDABEGH expression at low cell density. In the present study, we demonstrate that LuxO functions as an activator protein via interaction with the alternative sigma factor, sigma54 (encoded by rpoN). Our results suggest that LuxO, together with sigma54, activates the expression of a negative regulator of luminescence. We also show that phenotypes other than lux are regulated by LuxO and sigma54, demonstrating that in Vibrio harveyi, quorum sensing controls multiple processes.     

Comparative structural analysis of two proteins belonging to quorum sensing system in Vibrio cholerae

Vibrio cholerae uses quorum sensing communication system to interact with other bacteria and for gauzing environmental parameters. This organism dwells equally well in both human host and aquatic environments. Quorum sensing regulates multitude of activities and is one of the lucrative targets presently pursued for drug design in bacteria to encounter virulence. Histidine phosphotransfer protein LuxU and response regulator LuxO of V. cholerae are known to play important roles in biofilms and virulence machinery. In the present study, we used computational methods to model LuxU and LuxO and simulated the interactions of LuxO and LuxU. Since no structural details of the proteins were available, we employed homology modeling to construct the three-dimensional structures and then performed molecular dynamics simulations to study dynamic behavior of the LuxO and LuxU from V. cholerae. The modeled proteins were validated and subjected to molecular docking analyses. This allowed us to predict the binding modes of the proteins to elucidate probable sites of interference.     

Sequence and Function of LuxU: a Two-Component Phosphorelay Protein That Regulates Quorum Sensing in Vibrio harveyi

Vibrio harveyi regulates the expression of bioluminescence (lux) in response to cell density, a phenomenon known as quorum sensing. In V. harveyi, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.     

Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.     

Solution structure and dynamics of LuxU from Vibrio harveyi, a phosphotransferase protein involved in bacterial quorum sensing

The marine bacterium Vibrio harveyi controls its bioluminescence by a process known as quorum sensing. In this process, autoinducer molecules are detected by membrane-bound sensor kinase/response regulator proteins (LuxN and LuxQ) that relay a signal via a series of protein phosphorylation reactions to another response regulator protein, LuxO. Phosphorylated LuxO indirectly represses the expression of the proteins responsible for bioluminescence. Integral to this quorum sensing process is the function of the phosphotransferase protein, LuxU. LuxU acts to shuttle the phosphate from the membrane-bound proteins, LuxN and LuxQ, to LuxO. LuxU is a 114 amino acid residue monomeric protein. Solution NMR was used to determine the three-dimensional structure of LuxU. LuxU contains a four-helix bundle topology with the active-site histidine residue (His58) located on alpha-helix C and exposed to solution. The active site represents a cluster of positively charged residues located on an otherwise hydrophobic protein face. NMR spin-relaxation experiments identify a collection of flexible residues localized on the same region of LuxU as His58. The studies described here represent the first structural characterization of an isolated, monomeric bacterial phosphotransferase protein.     

The role of small RNAs in quorum sensing

Quorum sensing is a form of cell-cell signaling in bacteria that provides information regarding population density, species composition, and environmental and metabolic signals. It enables community-wide coordination of gene expression, and presumably benefits group behaviors. Multiple regulatory small RNAs (sRNAs) act centrally in quorum sensing, integrating signals with other environmental stimuli, to produce an appropriate output.