The wing of the ToxR winged helix-turn-helix domain is required for DNA binding and activation of toxT and ompU

English version

Autoři: Sarah J. Morgan ^aff001; Emily L. French ^aff002; Sarah C. Plecha ^aff002; Eric S. Krukonis ^aff001
Působiště autorů: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, United States of America ^aff001; Division of Integrated Biomedical Sciences, University of Detroit Mercy School of Dentistry, Detroit, MI, United States of America ^aff002; Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, MI, United States of America ^aff003
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0221936

Souhrn

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT.

Klíčová slova:

Biology and life sciences – Genetics – Gene expression – DNA transcription – Biochemistry – Proteins – DNA-binding proteins – Cell biology – Cellular structures and organelles – Cell membranes – Membrane proteins – Outer membrane proteins – Microbiology – Medical microbiology – Microbial pathogens – Bacterial pathogens – Vibrio cholerae – Organisms – Bacteria – Vibrio – Research and analysis methods – Chemical characterization – Binding analysis – DNA binding assay – Immunologic techniques – Immunoassays – Enzyme-linked immunoassays – Extraction techniques – Protein extraction – Medicine and health sciences – Pathology and laboratory medicine – Pathogens – Physical sciences – Physics – Condensed matter physics – Solid state physics – Crystallography – Crystal structure

Zdroje

1. Goss TJ, Seaborn CP, Gray MD, Krukonis ES. Identification of the TcpP-Binding Site in the toxT Promoter of Vibrio cholerae and the Role of ToxR in TcpP-Mediated Activation. Infect Immun. 2010;78(10):4122–33. IAI.00566-10 [pii] doi: 10.1128/IAI.00566-10 20679441.

2. Krukonis ES, DiRita VJ. DNA binding and ToxR responsiveness by the wing domain of TcpP, an activator of virulence gene expression in Vibrio cholerae. Mol Cell. 2003;12(1):157–65. S1097276503002223 [pii]. 12887901.

3. Häse CC, Mekalanos JJ. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA. 1998;95:730–4. doi: 10.1073/pnas.95.2.730 9435261 PMCID: PMC18489

4. Goss TJ, Morgan SJ, French EL, Krukonis ES. ToxR recognizes a direct repeat element in the toxT, ompU, ompT, and ctxA promoters of Vibrio cholerae to regulate transcription. Infect Immun. 2013;81(3):884–95. Epub 2013/01/09. doi: 10.1128/IAI.00889-12 [pii]. 23297386; PubMed Central PMCID: PMC3584884.

5. Crawford JA, Kaper JB, DiRita VJ. Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae. Mol Microbiol. 1998;29:235–46. doi: 10.1046/j.1365-2958.1998.00925.x 9701817

6. Li CC, Crawford JA, DiRita VJ, Kaper JB. Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae. Mol Microbiol. 2000;35(1):189–203. mmi1699 [pii]. doi: 10.1046/j.1365-2958.2000.01699.x 10632889.

7. Martinez-Hackert E, Stock AM. Structural relationships in the OmpR family of winged-helix transcription factors. J Mol Biol. 1997;269:301–12. doi: 10.1006/jmbi.1997.1065 9199401

8. DiRita VJ, Mekalanos JJ. Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation. Cell. 1991;64:29–37. doi: 10.1016/0092-8674(91)90206-e 1898871

9. Ottemann KM, Mekalanos JJ. The ToxR protein of Vibrio cholerae forms homodimers and heterodimers. J Bacteriol. 1996;178:156–62. doi: 10.1128/jb.178.1.156-162.1996 8550410 PMCID: PMC177633

10. Fengler VH, Boritsch EC, Tutz S, Seper A, Ebner H, Roier S, et al. Disulfide Bond Formation and ToxR Activity in Vibrio cholerae. PLoS One. 2012;7(10):e47756. Epub 2012/11/13. doi: 10.1371/journal.pone.0047756 [pii]. 23144706; PubMed Central PMCID: PMC3483227.

11. Dziejman M, Kolmar H, Fritz HJ, Mekalanos JJ. ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation. Mol Microbiol. 1999;31(1):305–17. doi: 10.1046/j.1365-2958.1999.01173.x 9987131.

12. Dziejman M, Mekalanos JJ. Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor. Mol Microbiol. 1994;13(3):485–94. doi: 10.1111/j.1365-2958.1994.tb00443.x 7997165.

13. Ottemann KM, Mekalanos JJ. Analysis of Vibrio cholerae ToxR function by construction of novel fusion proteins. Mol Microbiol. 1995;15(4):719–31. doi: 10.1111/j.1365-2958.1995.tb02380.x 7783643

14. Matson JS, DiRita VJ. Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae. Proc Natl Acad Sci USA. 2005;102(45):16403–8. doi: 10.1073/pnas.0505818102 16254052 PMCID: PMC1283431

15. Beck NA, Krukonis ES, DiRita VJ. TcpH Influences Virulence Gene Expression in Vibrio cholerae by Inhibiting Degradation of the Transcription Activator TcpP. J Bacteriol. 2004;186(24):8309–16. doi: 10.1128/JB.186.24.8309-8316.2004 15576780 PMCID: PMC532408

16. Teoh WP, Matson JS, DiRita VJ. Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp). Mol Microbiol. 2015;97(5):822–31. Epub 2015/05/23. doi: 10.1111/mmi.13069 25999037.

17. Morgan SJ, French EL, Thomson JJ, Seaborn CP, Shively CA, Krukonis ES. Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae. J Bacteriol. 2015;198(3):498–509. Epub 2015/11/18. doi: 10.1128/JB.00338-15 [pii]. 26574510; PubMed Central PMCID: PMC4719457.

18. Almagro-Moreno S, Kim TK, Skorupski K, Taylor RK. Proteolysis of Virulence Regulator ToxR Is Associated with Entry of Vibrio cholerae into a Dormant State. PLoS Genet. 2015;11(4):e1005145. Epub 2015/04/08. doi: 10.1371/journal.pgen.1005145 [pii]. 25849031; PubMed Central PMCID: PMC4388833.

19. Midgett CR, Almagro-Moreno S, Pellegrini M, Taylor RK, Skorupski K, Kull FJ. Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS. Mol Microbiol. 2017;105(2):258–72. doi: 10.1111/mmi.13699 28464377; PubMed Central PMCID: PMC5498992.

20. Almagro-Moreno S, Root MZ, Taylor RK. Role of ToxS in the proteolytic cascade of virulence regulator ToxR in Vibrio cholerae. Mol Microbiol. 2015;98(5):963–76. doi: 10.1111/mmi.13170 26316386.

21. Blanco AG, Sola M, Gomis-Ruth FX, Coll M. Tandem DNA Recognition by PhoB, a Two-Component Signal Transduction Transcriptional Activator. Structure. 2002;10(5):701–13. 12015152

22. Kenney LJ. Structure/function relationships in OmpR and other winged-helix transcription factors. Curr Opin Microbiol. 2002;5(2):135–41. S1369527402003107 [pii]. 11934608.

23. Maris AE, Walthers D, Mattison K, Byers N, Kenney LJ. The response regulator OmpR oligomerizes via beta-sheets to form head-to-head dimers. J Mol Biol. 2005;350(5):843–56. S0022-2836(05)00612-1 [pii] doi: 10.1016/j.jmb.2005.05.057 15979641.

24. Martinez-Hackert E, Stock AM. The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor. Structure. 1997;5(1):109–24. 9016718.

25. Yamane T, Okamura H, Ikeguchi M, Nishimura Y, Kidera A. Water-mediated interactions between DNA and PhoB DNA-binding/transactivation domain: NMR-restrained molecular dynamics in explicit water environment. Proteins. 2008;71(4):1970–83. doi: 10.1002/prot.21874 18186481.

26. Morgan SJ, Felek S, Gadwal S, Koropatkin NM, Perry JW, Bryson AB, et al. The two faces of ToxR: activator of ompU, co-regulator of toxT in Vibrio cholerae. Mol Microbiol. 2011;81(1):113–28. doi: 10.1111/j.1365-2958.2011.07681.x 21542860 PMCID: PMC3124598

27. Dorman CJ, Dorman MJ. Control of virulence gene transcription by indirect readout in Vibrio cholerae and Salmonella enterica serovar Typhimurium. Environ Microbiol. 2017. doi: 10.1111/1462-2920.13838 28631437.

28. Harrison-McMonagle P, Denissova N, Martinez-Hackert E, Ebright RH, Stock AM. Orientation of OmpR monomers within an OmpR:DNA complex determined by DNA affinity cleaving. J Mol Biol. 1999;285(2):555–66. doi: 10.1006/jmbi.1998.2375 9878429

29. Littlefield O, Nelson HCM. A new use for the 'wing' of the 'winged' helix-turn-helix motif in the HSF–DNA cocrystal. Nature Struct Biol. 1999;6(5):464–70. doi: 10.1038/8269 10331875

30. Gupta S, Pathak A, Sinha A, Sarkar D. Mycobacterium tuberculosis PhoP recognizes two adjacent direct-repeat sequences to form head-to-head dimers. J Bacteriol. 2009;191(24):7466–76. Epub 2009/10/13. doi: 10.1128/JB.00669-09 [pii]. 19820095; PubMed Central PMCID: PMC2786591.

31. Rhee JE, Sheng W, Morgan LK, Nolet R, Liao X, Kenney LJ. Amino acids important for DNA recognition by the response regulator OmpR. J Biol Chem. 2008;283(13):8664–77. M705550200 [pii] doi: 10.1074/jbc.M705550200 18195018.

32. Yang M, Liu Z, Hughes C, Stern AM, Wang H, Zhong Z, et al. Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence. Proc Natl Acad Sci USA. 2013;110(6):2348–53. Epub 2013/01/24. doi: 10.1073/pnas.1218039110 [pii]. 23341592; PubMed Central PMCID: PMC3568309.

33. Fan F, Liu Z, Jabeen N, Birdwell LD, Zhu J, Kan B. Enhanced interaction of Vibrio cholerae virulence regulators TcpP and ToxR under oxygen-limiting conditions. Infect Immun. 2014;82(4):1676–82. Epub 2014/02/05. IAI.01377-13 [pii] doi: 10.1128/IAI.01377-13 24491579.

34. Krukonis ES, Yu RR, Dirita VJ. The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol. 2000;38(1):67–84. mmi2111 [pii]. doi: 10.1046/j.1365-2958.2000.02111.x 11029691.

35. Nye MB, Pfau JD, Skorupski K, Taylor RK. Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade. J Bacteriol. 2000;182:4295–303. doi: 10.1128/jb.182.15.4295-4303.2000 10894740 PMCID: PMC101945

36. Haas BL, Matson JS, DiRita VJ, Biteen JS. Single-molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane-bound virulence regulator TcpP to the toxT promoter. Mol Microbiol. 2015;96(1):4–13. doi: 10.1111/mmi.12834 25318589.

37. Crawford JA, Krukonis ES, DiRita VJ. Membrane localization of the ToxR winged-helix domain is required for TcpP-mediated virulence gene activation in Vibrio cholerae. Mol Microbiol. 2003;47(5):1459–73. doi: 10.1046/j.1365-2958.2003.03398.x 12603748

38. Osorio CR, Klose KE. A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species. J Bacteriol. 2000;182(2):526–8. doi: 10.1128/jb.182.2.526-528.2000 10629204.

39. Skorupski K, Taylor RK. Broad-host-range positive selection vectors for allelic exchange. Gene. 1996;169:47–52. doi: 10.1016/0378-1119(95)00793-8 8635748

40. Miller JH. Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 1972.

41. Miller VL, Taylor RK, Mekalanos JJ. Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein. Cell. 1987;48:271–9. doi: 10.1016/0092-8674(87)90430-2 3802195

The wing of the ToxR winged helix-turn-helix domain is required for DNA binding and activation of toxT and ompU

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