DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

The RNA chaperone Hfq acting as a hexamer, is a known mediator of post-transcriptional regulation expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here, for the first time we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

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In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 39-diphosphate 59-triphosphate (pppGpp) and guanosine 39,59-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp 0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N-and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.