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.     

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.     

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.     

Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base‐pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base‐pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO‐domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO‐domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO‐domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.     

The extremophile Acidithiobacillus ferrooxidans possesses a c-di-GMP signalling pathway that could play a significant role during bioleaching of minerals

Aims: The primary goal of this study was to characterize the existence of a functional c‐di‐GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans. Methods and Results: A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c‐di‐GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c‐di‐GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c‐di‐GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells. Conclusions: At. ferrooxidans possesses a functional c‐di‐GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes. Significance and Impact of the Study: This is the first global study about the c‐di‐GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro‐organisms during the bioleaching process.     

Biofilm dispersal: deciding when it is better to travel

Bacteria live predominantly in biofilms, and the internal signal cyclic diguanylate (c‐di‐GMP) is a universal signal that governs the formation and the dispersal of these communities. Pseudomonas aeruginosa is one of the most important reference systems for studying bacterial biofilms and contains numerous diguanylate cyclases (DGCs) for synthesizing c‐di‐GMP and phosphodiesterases (PDEs) for degrading c‐di‐GMP. However, few studies have discerned how cells in biofilms respond to their environment to regulate c‐di‐GMP concentrations through this sophisticated network of enzymes. Basu Roy and Sauer (2014) provide insights on how cells disperse in response to an increase in nutrient levels. Their results show that the inner membrane protein NicD is a DGC that controls dispersal by sensing nutrient levels: when glutamate concentrations are increased, NicD is dephosphorylated, which increases c‐di‐GMP levels and leads to phosphorylation and processing of dispersal regulator BdlA. Processing of BdlA leads to activation of PDE DipA, which results in a net reduction of c‐di‐GMP and biofilm dispersal. These results suggest biofilm dispersal relies on surprisingly dynamic c‐di‐GMP concentrations as a result of a sophisticated interaction between DGCs and PDEs.     

Beyond antibiotic resistance: integrating conjugative elements of the SXT/R391 family that encode novel diguanylate cyclases participate to c‐di‐GMP signalling in Vibrio cholerae

In Vibrio cholerae, the second messenger bis‐(3′?5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) increases exopolysaccharides production and biofilm formation and decreases virulence and motility. As such, c‐di‐GMP is considered an important player in the transition from the host to persistence in the environment. c‐di‐GMP level is regulated through a complex network of more than 60 chromosomal genes encoding predicted diguanylate cyclases (DGCs) and phosphodiesterases. Herein we report the characterization of two additional DGCs, DgcK and DgcL, encoded by integrating conjugative elements (ICEs) belonging to the SXT/R391 family. SXT/R391 ICEs are self‐transmissible mobile elements that are widespread among vibrios and several species of enterobacteria. We found that deletion of dgcL increases the motility of V. cholerae, that overexpression of DgcK or DgcL modulates gene expression, biofilm formation and bacterial motility, and that a single amino acid change in the active site of either enzyme abolishes these phenotypes. We also show that DgcK and DgcL are able to synthesize c‐di‐GMP in vitro from GTP. DgcK was found to co‐purify with non‐covalently bound flavin mononucleotide (FMN). DgcL's enzymatic activity was augmented upon phosphorylation of its phosphorylatable response‐regulator domain suggesting that DgcL is part of a two‐component signal transduction system. Interestingly, we found orthologues of dgcK and dgcL in several SXT/R391 ICEs from two species of Vibrio originating from Asia, Africa and Central America. We propose that besides conferring usual antibiotic resistances, dgcKL‐bearing SXT/R391 ICEs could enhance the survival of vibrios in aquatic environments by increasing c‐di‐GMP level.