1887

Abstract

, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH and CO. UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins () in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between and . The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that and H-NS acts to repress transcription of in an model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the gene was cloned and the 15·6 kDa H-NS was overexpressed and purified as a -His tail fusion. Using a gel shift assay, purified H-NS--His bound preferentially to a 609 bp DNA fragment containing the entire - intergenic region. H-NS and UreR were able to displace each other from the - intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 °C) on urease expression were shown in containing an knockout and where expression was increased at 37 °C. Increased transcription from p was seen in the knockout when temperature was increased from 25 to 37 °C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.

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2003-12-01
2020-07-13
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References

  1. Coker C., Bakare O. O., Mobley H. L.. 2000; H-NS is a repressor of the Proteus mirabilis urease transcriptional activator gene ureR . J Bacteriol182:2649–2653
    [Google Scholar]
  2. Colonna B., Casalino M., Fradiani P. A.. 7 other authors 1995; H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome. J Bacteriol177:4703–4712
    [Google Scholar]
  3. D'Orazio S. E., Collins C. M.. 1993; The plasmid-encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR, a member of the AraC family of transcriptional activators. J Bacteriol175:3459–3467
    [Google Scholar]
  4. D'Orazio S. E., Collins C. M.. 1995; UreR activates transcription at multiple promoters within the plasmid-encoded urease locus of the Enterobacteriaceae . Mol Microbiol16:145–155
    [Google Scholar]
  5. D'Orazio S. E., Thomas V., Collins C. M.. 1996; Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR. Mol Microbiol21:643–655
    [Google Scholar]
  6. Dorman C. J., Bhriain N. N., Higgins C. F.. 1990; DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri . Nature344:789–792
    [Google Scholar]
  7. Falconi M., Colonna B., Prosseda G., Micheli G., Gualerzi C. O.. 1998; Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J17:7033–7043
    [Google Scholar]
  8. Gardel C. L., Mekalanos J. J.. 1994; Regulation of cholera toxin by temperature, pH, and osmolarity. Methods Enzymol235:517–526
    [Google Scholar]
  9. Gendlina I., Gutman D. M., Thomas V., Collins C. M.. 2002; Urea-dependent signal transduction by the virulence regulator UreR. J Biol Chem277:37349–37358
    [Google Scholar]
  10. Griffith D. P.. 1979; Urease stones. Urol Res7:215–221
    [Google Scholar]
  11. Griffith D. P., Musher D. M., Itin C.. 1976; Urease. The primary cause of infection-induced urinary stones. Invest Urol13:346–350
    [Google Scholar]
  12. Hromockyj A. E., Tucker S. C., Maurelli A. T.. 1992; Temperature regulation of Shigella virulence: identification of the repressor gene virR , an analogue of hns , and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr). Mol Microbiol6:2113–2124
    [Google Scholar]
  13. Island M. D., Mobley H. L.. 1995; Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function. J Bacteriol177:5653–5660
    [Google Scholar]
  14. Johnson D. E., Russell R. G., Lockatell C. V., Zulty J. C., Warren J. W., Mobley H. L.. 1993; Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection. Infect Immun61:2748–2754
    [Google Scholar]
  15. Jones B. D., Mobley H. L.. 1987; Genetic and biochemical diversity of ureases of Proteus , Providencia , and Morganella species isolated from urinary tract infection. Infect Immun55:2198–2203
    [Google Scholar]
  16. Jones B. D., Mobley H. L.. 1988; Proteus mirabilis urease: genetic organization, regulation, and expression of structural genes. J Bacteriol170:3342–3349
    [Google Scholar]
  17. Jones B. D., Lockatell C. V., Johnson D. E., Warren J. W., Mobley H. L.. 1990; Construction of a urease-negative mutant of Proteus mirabilis : analysis of virulence in a mouse model of ascending urinary tract infection. Infect Immun58:1120–1123
    [Google Scholar]
  18. Koo H. S., Wu H. M., Crothers D. M.. 1986; DNA bending at adenine thymine tracts. Nature320:501–506
    [Google Scholar]
  19. Li X., Zhao H., Lockatell C. V., Drachenberg C. B., Johnson D. E., Mobley H. L.. 2002; Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection. Infect Immun70:389–394
    [Google Scholar]
  20. Lucht J. M., Dersch P., Kempf B., Bremer E.. 1994; Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli . J Biol Chem269:6578–6586
    [Google Scholar]
  21. Maurelli A. T., Sansonetti P. J.. 1988; Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc Natl Acad Sci U S A85:2820–2824
    [Google Scholar]
  22. McGee D. J., May C. A., Garner R. M., Himpsl J. M., Mobley H. L.. 1999; Isolation of Helicobacter pylori genes that modulate urease activity. J Bacteriol181:2477–2484
    [Google Scholar]
  23. Miller V. L., Mekalanos J. J.. 1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR . J Bacteriol170:2575–2583
    [Google Scholar]
  24. Mizuno T.. 1987; Random cloning of bent DNA segments from Escherichia coli chromosome and primary characterization of their structures. Nucleic Acids Res15:6827–6841
    [Google Scholar]
  25. Mobley H. L., Warren J. W.. 1987; Urease-positive bacteriuria and obstruction of long-term urinary catheters. J Clin Microbiol25:2216–2217
    [Google Scholar]
  26. Mobley H. L., Island M. D., Massad G.. 1994; Virulence determinants of uropathogenic Escherichia coli and Proteus mirabilis . Kidney Int Suppl47:S129–S136
    [Google Scholar]
  27. Munteanu M. G., Vlahovicek K., Parthasarathy S., Simon I., Pongor S.. 1998; Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena. Trends Biochem Sci23:341–347
    [Google Scholar]
  28. Nicholson E. B., Concaugh E. A., Foxall P. A., Island M. D., Mobley H. L.. 1993; Proteus mirabilis urease: transcriptional regulation by UreR. J Bacteriol175:465–473
    [Google Scholar]
  29. Nye M. B., Pfau J. D., Skorupski K., Taylor R. K.. 2000; Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade. J Bacteriol182:4295–4303
    [Google Scholar]
  30. Owen-Hughes T. A., Pavitt G. D., Santos D. S., Sidebotham J. M., Hulton C. S., Hinton J. C., Higgins C. F.. 1992; The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression. Cell71:255–265
    [Google Scholar]
  31. Platt T., Meuler-Hill B., Miller J.. 1972; Assays of β -galactosidase activity. In Experiments in Molecular Genetics Edited by Miller J. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  32. Poore C. A., Coker C., Dattelbaum J. D., Mobley H. L. T.. 2001; Identification of the domains of UreR, an AraC-like transcriptional regulator of the urease gene cluster in Proteus mirabilis . J Bacteriol183:4526–4535
    [Google Scholar]
  33. Porter M. E., Dorman C. J.. 1994; A role for H-NS in the thermo-osmotic regulation of virulence gene expression in Shigella flexneri . J Bacteriol176:4187–4191
    [Google Scholar]
  34. Prosseda G., Fradiani P. A., Di Lorenzo M., Falconi M., Micheli G., Casalino M., Nicoletti M., Colonna B.. 1998; A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli . Res Microbiol149:15–25
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  36. Spassky A., Rimsky S., Garreau H., Buc H.. 1984; H1a, an E. coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro. Nucleic Acids Res12:5321–5340
    [Google Scholar]
  37. Spurio R., Falconi M., Brandi A., Pon C. L., Gualerzi C. O.. 1997; The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending. EMBO J16:1795–1805
    [Google Scholar]
  38. Thomas V. J., Collins C. M.. 1999; Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons. Mol Microbiol31:1417–1428
    [Google Scholar]
  39. Thompson J. F., Landy A.. 1988; Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res16:9687–9705
    [Google Scholar]
  40. Tupper A. E., Owen-Hughes T. A., Ussery D. W., Santos D. S., Ferguson D. J., Sidebotham J. M., Hinton J. C., Higgins C. F.. 1994; The chromatin-associated protein H-NS alters DNA topology in vitro . EMBO J13:258–268
    [Google Scholar]
  41. Ulanovsky L., Bodner M., Trifonov E. N., Choder M.. 1986; Curved DNA: design, synthesis, and circularization. Proc Natl Acad Sci U S A83:862–866
    [Google Scholar]
  42. Wu H. M., Crothers D. M.. 1984; The locus of sequence-directed and protein-induced DNA bending. Nature308:509–513
    [Google Scholar]
  43. Yamada H., Yoshida T., Tanaka K., Sasakawa C., Mizuno T.. 1991; Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences. Mol Gen Genet230:332–336
    [Google Scholar]
  44. Zwieb C., Adhya S.. 1994; Improved plasmid vectors for the analysis of protein-induced DNA bending. Methods Mol Biol30:281–294
    [Google Scholar]
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