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. 1990 Jun;58(6):1879–1885. doi: 10.1128/iai.58.6.1879-1885.1990

Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling.

J E Galán 1, R Curtiss 3rd 1
PMCID: PMC258738  PMID: 2160435

Abstract

The ability to enter intestinal epithelial cells is an essential virulence factor of salmonellae. We have previously cloned a group of genes (invA, B, C, and D) that allow S. typhimurium to penetrate tissue culture cells (J. E. Galán and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). Transcriptional and translational cat and phoA fusions to invA (the proximal gene in the invABC operon) were constructed, and their expression was studied by measuring the levels of alkaline phosphatase or chloramphenicol acetyltransferase activity in mutants grown under different conditions. It was found that when strains containing the fusions were grown on media with high osmolarity, a condition known to increase DNA superhelicity, the level of invA transcription was approximately eightfold higher than that in strains grown on media with low osmolarity. The osmoinducibility of invA was independent of ompR, which controls the osmoinducibility of other genes. Strains grown in high-osmolarity media in the presence of subinhibitory concentrations of gyrase inhibitors (novobiocin or coumermycin A1), which reduce the level of DNA supercoiling, showed reduced expression of invA. Nevertheless, invA was poorly expressed in topA mutants of S. typhimurium, which have increased DNA superhelicity. In all cases, the differential expression of the invasion genes was correlated with the ability of S. typhimurium to penetrate tissue culture cells. These results taken together indicate that expression of S. typhimurium invasion genes is affected by changes in DNA supercoiling and suggest that this may represent a way in which this organism regulates the expression of these genes.

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Selected References

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  1. Balke V. L., Gralla J. D. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli. J Bacteriol. 1987 Oct;169(10):4499–4506. doi: 10.1128/jb.169.10.4499-4506.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cairney J., Booth I. R., Higgins C. F. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system. J Bacteriol. 1985 Dec;164(3):1224–1232. doi: 10.1128/jb.164.3.1224-1232.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dorman C. J., Barr G. C., Ni Bhriain N., Higgins C. F. DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression. J Bacteriol. 1988 Jun;170(6):2816–2826. doi: 10.1128/jb.170.6.2816-2826.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dorman C. J., Chatfield S., Higgins C. F., Hayward C., Dougan G. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect Immun. 1989 Jul;57(7):2136–2140. doi: 10.1128/iai.57.7.2136-2140.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elsinghorst E. A., Baron L. S., Kopecko D. J. Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5173–5177. doi: 10.1073/pnas.86.13.5173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fields P. I., Groisman E. A., Heffron F. A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science. 1989 Feb 24;243(4894 Pt 1):1059–1062. doi: 10.1126/science.2646710. [DOI] [PubMed] [Google Scholar]
  8. Finlay B. B., Gumbiner B., Falkow S. Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer. J Cell Biol. 1988 Jul;107(1):221–230. doi: 10.1083/jcb.107.1.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Finlay B. B., Heffron F., Falkow S. Epithelial cell surfaces induce Salmonella proteins required for bacterial adherence and invasion. Science. 1989 Feb 17;243(4893):940–943. doi: 10.1126/science.2919285. [DOI] [PubMed] [Google Scholar]
  10. Finlay B. B., Starnbach M. N., Francis C. L., Stocker B. A., Chatfield S., Dougan G., Falkow S. Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer. Mol Microbiol. 1988 Nov;2(6):757–766. doi: 10.1111/j.1365-2958.1988.tb00087.x. [DOI] [PubMed] [Google Scholar]
  11. Franco R. J., Drlica K. Gyrase inhibitors can increase gyrA expression and DNA supercoiling. J Bacteriol. 1989 Dec;171(12):6573–6579. doi: 10.1128/jb.171.12.6573-6579.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galán J. E., Curtiss R., 3rd Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6383–6387. doi: 10.1073/pnas.86.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Galán J. E., Curtiss R., 3rd Virulence and vaccine potential of phoP mutants of Salmonella typhimurium. Microb Pathog. 1989 Jun;6(6):433–443. doi: 10.1016/0882-4010(89)90085-5. [DOI] [PubMed] [Google Scholar]
  14. Giannella R. A., Washington O., Gemski P., Formal S. B. Invasion of HeLa cells by Salmonella typhimurium: a model for study of invasiveness of Salmonella. J Infect Dis. 1973 Jul;128(1):69–75. doi: 10.1093/infdis/128.1.69. [DOI] [PubMed] [Google Scholar]
  15. Gibson M. M., Ellis E. M., Graeme-Cook K. A., Higgins C. F. OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium. Mol Gen Genet. 1987 Apr;207(1):120–129. doi: 10.1007/BF00331499. [DOI] [PubMed] [Google Scholar]
  16. Goldstein E., Drlica K. Regulation of bacterial DNA supercoiling: plasmid linking numbers vary with growth temperature. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4046–4050. doi: 10.1073/pnas.81.13.4046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gottesman S. Bacterial regulation: global regulatory networks. Annu Rev Genet. 1984;18:415–441. doi: 10.1146/annurev.ge.18.120184.002215. [DOI] [PubMed] [Google Scholar]
  18. Graeme-Cook K. A., May G., Bremer E., Higgins C. F. Osmotic regulation of porin expression: a role for DNA supercoiling. Mol Microbiol. 1989 Sep;3(9):1287–1294. doi: 10.1111/j.1365-2958.1989.tb00279.x. [DOI] [PubMed] [Google Scholar]
  19. Hall M. N., Silhavy T. J. The ompB locus and the regulation of the major outer membrane porin proteins of Escherichia coli K12. J Mol Biol. 1981 Feb 15;146(1):23–43. doi: 10.1016/0022-2836(81)90364-8. [DOI] [PubMed] [Google Scholar]
  20. Higgins C. F., Dorman C. J., Stirling D. A., Waddell L., Booth I. R., May G., Bremer E. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell. 1988 Feb 26;52(4):569–584. doi: 10.1016/0092-8674(88)90470-9. [DOI] [PubMed] [Google Scholar]
  21. Hoiseth S. K., Stocker B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981 May 21;291(5812):238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  22. Isberg R. R., Leong J. M. Cultured mammalian cells attach to the invasin protein of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6682–6686. doi: 10.1073/pnas.85.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Isberg R. R., Voorhis D. L., Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769–778. doi: 10.1016/0092-8674(87)90335-7. [DOI] [PubMed] [Google Scholar]
  24. Jovanovich S. B., Lebowitz J. Estimation of the effect of coumermycin A1 on Salmonella typhimurium promoters by using random operon fusions. J Bacteriol. 1987 Oct;169(10):4431–4435. doi: 10.1128/jb.169.10.4431-4435.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kennedy E. P. Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1092–1095. doi: 10.1073/pnas.79.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kier L. D., Weppelman R. M., Ames B. N. Regulation of nonspecific acid phosphatase in Salmonella: phoN and phoP genes. J Bacteriol. 1979 Apr;138(1):155–161. doi: 10.1128/jb.138.1.155-161.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knapp S., Mekalanos J. J. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J Bacteriol. 1988 Nov;170(11):5059–5066. doi: 10.1128/jb.170.11.5059-5066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kranz R. G., Haselkorn R. Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6805–6809. doi: 10.1073/pnas.83.18.6805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kretschmer P. J., Cohen S. N. Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element. J Bacteriol. 1977 May;130(2):888–899. doi: 10.1128/jb.130.2.888-899.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  31. Lenny A. B., Margolin P. Locations of the opp and supX genes of Salmonella typhimurium and Escherichia coli. J Bacteriol. 1980 Aug;143(2):747–752. doi: 10.1128/jb.143.2.747-752.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Liu S. L., Ezaki T., Miura H., Matsui K., Yabuuchi E. Intact motility as a Salmonella typhi invasion-related factor. Infect Immun. 1988 Aug;56(8):1967–1973. doi: 10.1128/iai.56.8.1967-1973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maurelli A. T., Blackmon B., Curtiss R., 3rd Temperature-dependent expression of virulence genes in Shigella species. Infect Immun. 1984 Jan;43(1):195–201. doi: 10.1128/iai.43.1.195-201.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maurelli A. T., Sansonetti P. J. Genetic determinants of Shigella pathogenicity. Annu Rev Microbiol. 1988;42:127–150. doi: 10.1146/annurev.mi.42.100188.001015. [DOI] [PubMed] [Google Scholar]
  36. May G., Faatz E., Villarejo M., Bremer E. Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12. Mol Gen Genet. 1986 Nov;205(2):225–233. doi: 10.1007/BF00430432. [DOI] [PubMed] [Google Scholar]
  37. Meiklejohn A. L., Gralla J. D. Activation of the lac promoter and its variants. Synergistic effects of catabolite activator protein and supercoiling in vitro. J Mol Biol. 1989 Jun 20;207(4):661–673. doi: 10.1016/0022-2836(89)90236-2. [DOI] [PubMed] [Google Scholar]
  38. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  39. Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Miller V. L., Falkow S. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect Immun. 1988 May;56(5):1242–1248. doi: 10.1128/iai.56.5.1242-1248.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Miller V. L., Finlay B. B., Falkow S. Factors essential for the penetration of mammalian cells by Yersinia. Curr Top Microbiol Immunol. 1988;138:15–39. [PubMed] [Google Scholar]
  42. Mroczenski-Wildey M. J., Di Fabio J. L., Cabello F. C. Invasion and lysis of HeLa cell monolayers by Salmonella typhi: the role of lipopolysaccharide. Microb Pathog. 1989 Feb;6(2):143–152. doi: 10.1016/0882-4010(89)90017-x. [DOI] [PubMed] [Google Scholar]
  43. Mukai F. H., Margolin P. ANALYSIS OF UNLINKED SUPPRESSORS OF AN O degrees MUTATION IN SALMONELLA. Proc Natl Acad Sci U S A. 1963 Jul;50(1):140–148. doi: 10.1073/pnas.50.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Overbye K. M., Margolin P. Role of the supX gene in ultraviolet light-induced mutagenesis in Salmonella typhimurium. J Bacteriol. 1981 Apr;146(1):170–178. doi: 10.1128/jb.146.1.170-178.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pruss G. J. DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. J Mol Biol. 1985 Sep 5;185(1):51–63. doi: 10.1016/0022-2836(85)90182-2. [DOI] [PubMed] [Google Scholar]
  47. Pruss G. J., Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. doi: 10.1016/0092-8674(89)90574-6. [DOI] [PubMed] [Google Scholar]
  48. Pruss G. J., Drlica K. DNA supercoiling and suppression of the leu-500 promoter mutation. J Bacteriol. 1985 Nov;164(2):947–949. doi: 10.1128/jb.164.2.947-949.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Richardson S. M., Higgins C. F., Lilley D. M. The genetic control of DNA supercoiling in Salmonella typhimurium. EMBO J. 1984 Aug;3(8):1745–1752. doi: 10.1002/j.1460-2075.1984.tb02041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. SCHNEIDER H. A., ZINDER N. D. Nutrition of the host and natural resistance to infection. V. An improved assay employing genetic markers in the double strain inoculation test. J Exp Med. 1956 Feb 1;103(2):207–223. doi: 10.1084/jem.103.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  52. Small P. L., Isberg R. R., Falkow S. Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells. Infect Immun. 1987 Jul;55(7):1674–1679. doi: 10.1128/iai.55.7.1674-1679.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  54. Takeuchi A. Electron microscope studies of experimental Salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium. Am J Pathol. 1967 Jan;50(1):109–136. [PMC free article] [PubMed] [Google Scholar]
  55. Tse-Dinh Y. C., Beran R. K. Multiple promoters for transcription of the Escherichia coli DNA topoisomerase I gene and their regulation by DNA supercoiling. J Mol Biol. 1988 Aug 20;202(4):735–742. doi: 10.1016/0022-2836(88)90554-2. [DOI] [PubMed] [Google Scholar]
  56. Yamamoto N., Droffner M. L. Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2077–2081. doi: 10.1073/pnas.82.7.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]

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