1887

Abstract

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. choleraedksA 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.

Keyword(s): CsrBCD , DksA , Fis , HAP , HapR , RpoS and stationary phase
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2017-06-08
2019-10-24
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References

  1. Cashel M, Gentry DR, Hernandes VJ, Vinella D. The stringent response. In Neidhardt FC. (editor) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd ed. Washington, DC: American Society for Microbiology; 1996; pp.1458–1496
    [Google Scholar]
  2. Potrykus K, Cashel M. (p)ppGpp: still magical?. Annu Rev Microbiol 2008;62:35–51 [CrossRef][PubMed]
    [Google Scholar]
  3. Das B, Pal RR, Bag S, Bhadra RK. Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene. Mol Microbiol 2009;72:380–398 [CrossRef][PubMed]
    [Google Scholar]
  4. Atkinson GC, Tenson T, Hauryliuk V. The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS One 2011;6:e23479 [CrossRef][PubMed]
    [Google Scholar]
  5. Perederina A, Svetlov V, Vassylyeva MN, Tahirov TH, Yokoyama S et al. Regulation through the secondary channel:structural framework for ppGpp-DksA synergism during transcription. Cell 2004;118:297–309 [CrossRef][PubMed]
    [Google Scholar]
  6. Paul BJ, Barker MM, Ross W, Schneider DA, Webb C et al. DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell 2004;118:311–322 [CrossRef][PubMed]
    [Google Scholar]
  7. Rutherford ST, Lemke JJ, Vrentas CE, Gaal T, Ross W et al. Effects of DksA, GreA, and GreB on transcription initiation: insights into the mechanisms of factors that bind in the secondary channel of RNA polymerase. J Mol Biol 2007;366:1243–1257 [CrossRef][PubMed]
    [Google Scholar]
  8. Lennon CW, Ross W, Martin-Tumasz S, Toulokhonov I, Vrentas CE et al. Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation. Genes Dev 2012;26:2634–2646 [CrossRef][PubMed]
    [Google Scholar]
  9. Webb C, Moreno M, Wilmes-Riesenberg M, Curtiss R, Foster JW. Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium. Mol Microbiol 1999;34:112–123 [CrossRef][PubMed]
    [Google Scholar]
  10. Mogull SA, Runyen-Janecky LJ, Hong M, Payne SM. dksA is required for intercellular spread of Shigella flexneri via an RpoS-independent mechanism. Infect Immun 2001;69:5742–5751 [CrossRef][PubMed]
    [Google Scholar]
  11. Jude F, Köhler T, Branny P, Perron K, Mayer MP et al. Posttranscriptional control of quorum-sensing-dependent virulence genes by DksA in Pseudomonas aeruginosa. J Bacteriol 2003;185:3558–3566 [CrossRef][PubMed]
    [Google Scholar]
  12. Nakanishi N, Abe H, Ogura Y, Hayashi T, Tashiro K et al. ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes. Mol Microbiol 2006;61:194–205 [CrossRef][PubMed]
    [Google Scholar]
  13. Sharma AK, Payne SM. Induction of expression of hfq by DksA is essential for Shigella flexneri virulence. Mol Microbiol 2006;62:469–479 [CrossRef][PubMed]
    [Google Scholar]
  14. Pal RR, Das B, Dasgupta S, Bhadra RK. Genetic components of stringent response in Vibrio cholerae. Indian J Med Res 2011;133:212–217[PubMed]
    [Google Scholar]
  15. Pal RR, Bag S, Dasgupta S, Das B, Bhadra RK. Functional characterization of the stringent response regulatory gene dksA of Vibrio cholerae and its role in modulation of virulence phenotypes. J Bacteriol 2012;194:5638–5648 [CrossRef][PubMed]
    [Google Scholar]
  16. Branny P, Pearson JP, Pesci EC, Köhler T, Iglewski BH et al. Inhibition of quorum sensing by a Pseudomonas aeruginosa dksA homologue. J Bacteriol 2001;183:1531–1539 [CrossRef][PubMed]
    [Google Scholar]
  17. Miller MB, Skorupski K, Lenz DH, Taylor RK, Bassler BL. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 2002;110:303–314 [CrossRef][PubMed]
    [Google Scholar]
  18. Zhu J, Miller MB, Vance RE, Dziejman M, Bassler BL et al. Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 2002;99:3129–3134 [CrossRef][PubMed]
    [Google Scholar]
  19. Hammer BK, Bassler BL. Quorum sensing controls biofilm formation in Vibrio cholerae. Mol Microbiol 2003;50:101–104 [CrossRef][PubMed]
    [Google Scholar]
  20. Zhu J, Mekalanos JJ. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. Dev Cell 2003;5:647–656 [CrossRef][PubMed]
    [Google Scholar]
  21. Waters CM, Lu W, Rabinowitz JD, Bassler BL. Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT. J Bacteriol 2008;190:2527–2536 [CrossRef][PubMed]
    [Google Scholar]
  22. Finkelstein RA, Boesman-Finkelstein M, Chang Y, Häse CC. Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment. Infect Immun 1992;60:472–478[PubMed]
    [Google Scholar]
  23. Benitez JA, Silva AJ, Finkelstein RA. Environmental signals controlling production of hemagglutinin/protease in Vibrio cholerae. Infect Immun 2001;69:6549–6553 [CrossRef][PubMed]
    [Google Scholar]
  24. Nielsen AT, Dolganov NA, Otto G, Miller MC, Wu CY et al. RpoS controls the Vibrio cholerae mucosal escape response. PLoS Pathog 2006;2:e109 [CrossRef][PubMed]
    [Google Scholar]
  25. Yildiz FH, Schoolnik GK. Role of rpoS in stress survival and virulence of Vibrio cholerae. J Bacteriol 1998;180:773–781[PubMed]
    [Google Scholar]
  26. Silva AJ, Benitez JA. Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS. J Bacteriol 2004;186:6374–6382 [CrossRef][PubMed]
    [Google Scholar]
  27. Lenz DH, Miller MB, Zhu J, Kulkarni RV, Bassler BL. CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae. Mol Microbiol 2005;58:1186–1202 [CrossRef][PubMed]
    [Google Scholar]
  28. Lenz DH, Bassler BL. The small nucleoid protein Fis is involved in Vibrio cholerae quorum sensing. Mol Microbiol 2007;63:859–876 [CrossRef][PubMed]
    [Google Scholar]
  29. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG et al. Current Protocol in Molecular Biology New York: John Wiley and Sons; 1989
    [Google Scholar]
  30. das B, Bhadra RK. Molecular characterization of Vibrio cholerae ΔrelA ΔspoT double mutants. Arch Microbiol 2008;189:227–238 [CrossRef][PubMed]
    [Google Scholar]
  31. Miller JH. Experiments in Molecular Genetics Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1972
    [Google Scholar]
  32. Yildiz FH, Liu XS, Heydorn A, Schoolnik GK. Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant. Mol Microbiol 2004;53:497–515 [CrossRef][PubMed]
    [Google Scholar]
  33. Svenningsen SL, Tu KC, Bassler BL. Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing. EMBO J 2009;28:429–439 [CrossRef][PubMed]
    [Google Scholar]
  34. van Delden C, Iglewski BH. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Dis 1998;4:551–560 [CrossRef][PubMed]
    [Google Scholar]
  35. Jobling MG, Holmes RK. Characterization of HapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol Microbiol 1997;26:1023–1034 [CrossRef][PubMed]
    [Google Scholar]
  36. Tsou AM, Liu Z, Cai T, Zhu J. The VarS/VarA two-component system modulates the activity of the Vibrio cholerae quorum-sensing transcriptional regulator HapR. Microbiology 2011;157:1620–1628 [CrossRef][PubMed]
    [Google Scholar]
  37. Edwards AN, Patterson-Fortin LM, Vakulskas CA, Mercante JW, Potrykus K et al. Circuitry linking the Csr and stringent response global regulatory systems. Mol Microbiol 2011;80:1561–1580 [CrossRef][PubMed]
    [Google Scholar]
  38. Azam TA, Ishihama A. Twelve species of the nucleoid-associated protein from Escherichia coli sequence recognition specificity and DNA binding affinity. J Biol Chem 1999;274:33105–33113 [CrossRef][PubMed]
    [Google Scholar]
  39. Mallik P, Paul BJ, Rutherford ST, Gourse RL, Osuna R. DksA is required for growth phase-dependent regulation, growth rate-dependent control, and stringent control of fis expression in Escherichia coli. J Bacteriol 2006;188:5775–5782 [CrossRef][PubMed]
    [Google Scholar]
  40. Mukhopadhyay S, Audia JP, Roy RN, Schellhorn HE. Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two-component regulator. Mol Microbiol 2000;37:371–381 [CrossRef][PubMed]
    [Google Scholar]
  41. Lange R, Fischer D, Hengge-Aronis R. Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli. J Bacteriol 1995;177:4676–4680 [CrossRef][PubMed]
    [Google Scholar]
  42. Balandina A, Claret L, Hengge-Aronis R, Rouviere-Yaniv J. The Escherichia coli histone-like protein HU regulates rpoS translation. Mol Microbiol 2001;39:1069–1079 [CrossRef][PubMed]
    [Google Scholar]
  43. Barth M, Marschall C, Muffler A, Fischer D, Hengge-Aronis R. Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of sigma S and many sigma S-dependent genes in Escherichia coli. J Bacteriol 1995;177:3455–3464 [CrossRef][PubMed]
    [Google Scholar]
  44. Schweder T, Lee KH, Lomovskaya O, Matin A. Regulation of Escherichia coli starvation sigma factor (sigma S) by ClpXP protease. J Bacteriol 1996;178:470–476 [CrossRef][PubMed]
    [Google Scholar]
  45. Brown L, Gentry D, Elliott T, Cashel M. DksA affects ppGpp induction of RpoS at a translational level. J Bacteriol 2002;184:4455–4465 [CrossRef][PubMed]
    [Google Scholar]
  46. Joelsson A, Kan B, Zhu J. Quorum sensing enhances the stress response in Vibrio cholerae. Appl Environ Microbiol 2007;73:3742–3746 [CrossRef][PubMed]
    [Google Scholar]
  47. Albertson NH, Nyström T, Kjelleberg S. Exoprotease activity of two marine bacteria during starvation. Appl Environ Microbiol 1990;56:218–223[PubMed]
    [Google Scholar]
  48. Gentry DR, Hernandez VJ, Nguyen LH, Jensen DB, Cashel M. Synthesis of the stationary-phase sigma factor sigma S is positively regulated by ppGpp. J Bacteriol 1993;175:7982–7989 [CrossRef][PubMed]
    [Google Scholar]
  49. Hirsch M, Elliott T. Role of ppGpp in rpoS stationary-phase regulation in Escherichia coli. J Bacteriol 2002;184:5077–5087 [CrossRef][PubMed]
    [Google Scholar]
  50. Song M, Kim HJ, Kim EY, Shin M, Lee HC et al. ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1. J Biol Chem 2004;279:34183–34190 [CrossRef][PubMed]
    [Google Scholar]
  51. Holley CL, Zhang X, Fortney KR, Ellinger S, Johnson P et al. DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans. Infect Immun 2015;83:3281–3292 [CrossRef][PubMed]
    [Google Scholar]
  52. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 2000;406:477–483 [CrossRef][PubMed]
    [Google Scholar]
  53. Haralalka S, Nandi S, Bhadra RK. Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors. J Bacteriol 2003;185:4672–4682 [CrossRef][PubMed]
    [Google Scholar]
  54. Chowdhury G, Bhadra RK, Bag S, Pazhani GP, Das B et al. Rugose atypical Vibrio cholerae O1 El Tor responsible for 2009 cholera outbreak in India. J Med Microbiol 2016;65:1130–1136 [CrossRef][PubMed]
    [Google Scholar]
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