Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Aug;178(15):4335–4343. doi: 10.1128/jb.178.15.4335-4343.1996

Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

R M Williams 1, S Rimsky 1, H Buc 1
PMCID: PMC178199  PMID: 8755860

Abstract

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.

Full Text

The Full Text of this article is available as a PDF (306.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barry T., Geary S., Hannify S., MacGearailt C., Shalloo M., Heery D., Gannon F., Powell R. Rapid mini-preparations of total RNA from bacteria. Nucleic Acids Res. 1992 Sep 25;20(18):4940–4940. doi: 10.1093/nar/20.18.4940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell A., Gaston K., Williams R., Chapman K., Kolb A., Buc H., Minchin S., Williams J., Busby S. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 1990 Dec 25;18(24):7243–7250. doi: 10.1093/nar/18.24.7243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bracco L., Kotlarz D., Kolb A., Diekmann S., Buc H. Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli. EMBO J. 1989 Dec 20;8(13):4289–4296. doi: 10.1002/j.1460-2075.1989.tb08615.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Busby S., Kotlarz D., Buc H. Deletion mutagenesis of the Escherichia coli galactose operon promoter region. J Mol Biol. 1983 Jun 25;167(2):259–274. doi: 10.1016/s0022-2836(83)80335-0. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dattananda C. S., Rajkumari K., Gowrishankar J. Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene. J Bacteriol. 1991 Dec;173(23):7481–7490. doi: 10.1128/jb.173.23.7481-7490.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dersch P., Schmidt K., Bremer E. Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation. Mol Microbiol. 1993 May;8(5):875–889. doi: 10.1111/j.1365-2958.1993.tb01634.x. [DOI] [PubMed] [Google Scholar]
  9. Finlay C. A., Hinds P. W., Levine A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083–1093. doi: 10.1016/0092-8674(89)90045-7. [DOI] [PubMed] [Google Scholar]
  10. Gowrishankar J. Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. J Bacteriol. 1985 Oct;164(1):434–445. doi: 10.1128/jb.164.1.434-445.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gowrishankar J. Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli. J Bacteriol. 1989 Apr;171(4):1923–1931. doi: 10.1128/jb.171.4.1923-1931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grabarek Z., Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem. 1990 Feb 15;185(1):131–135. doi: 10.1016/0003-2697(90)90267-d. [DOI] [PubMed] [Google Scholar]
  13. Göransson M., Sondén B., Nilsson P., Dagberg B., Forsman K., Emanuelsson K., Uhlin B. E. Transcriptional silencing and thermoregulation of gene expression in Escherichia coli. Nature. 1990 Apr 12;344(6267):682–685. doi: 10.1038/344682a0. [DOI] [PubMed] [Google Scholar]
  14. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  15. Lauder S. D., Kowalczykowski S. C. Negative co-dominant inhibition of recA protein function. Biochemical properties of the recA1, recA13 and recA56 proteins and the effect of recA56 protein on the activities of the wild-type recA protein function in vitro. J Mol Biol. 1993 Nov 5;234(1):72–86. doi: 10.1006/jmbi.1993.1564. [DOI] [PubMed] [Google Scholar]
  16. Lejeune P., Danchin A. Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1990 Jan;87(1):360–363. doi: 10.1073/pnas.87.1.360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lodge J., Williams R., Bell A., Chan B., Busby S. Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid. FEMS Microbiol Lett. 1990 Jan 15;55(1-2):221–225. doi: 10.1016/0378-1097(90)90199-z. [DOI] [PubMed] [Google Scholar]
  18. Lucht J. M., Dersch P., Kempf B., Bremer E. 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 Chem. 1994 Mar 4;269(9):6578–6578. [PubMed] [Google Scholar]
  19. McGovern V., Higgins N. P., Chiz R. S., Jaworski A. H-NS over-expression induces an artificial stationary phase by silencing global transcription. Biochimie. 1994;76(10-11):1019–1029. doi: 10.1016/0300-9084(94)90026-4. [DOI] [PubMed] [Google Scholar]
  20. Oppenheim A. B., Noff D. Deletion mapping of trans dominant mutations in the lambda repressor gene. Virology. 1975 Apr;64(2):553–556. doi: 10.1016/0042-6822(75)90132-4. [DOI] [PubMed] [Google Scholar]
  21. Perrin S., Gilliland G. Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer. Nucleic Acids Res. 1990 Dec 25;18(24):7433–7438. doi: 10.1093/nar/18.24.7433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shi X., Bennett G. N. Plasmids bearing hfq and the hns-like gene stpA complement hns mutants in modulating arginine decarboxylase gene expression in Escherichia coli. J Bacteriol. 1994 Nov;176(21):6769–6775. doi: 10.1128/jb.176.21.6769-6775.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shindo H., Iwaki T., Ieda R., Kurumizaka H., Ueguchi C., Mizuno T., Morikawa S., Nakamura H., Kuboniwa H. Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli. FEBS Lett. 1995 Feb 27;360(2):125–131. doi: 10.1016/0014-5793(95)00079-o. [DOI] [PubMed] [Google Scholar]
  24. Spassky A., Buc H. C. Physico-chemical properties of a DNA binding protein: Escherichia coli factor H1. Eur J Biochem. 1977 Nov 15;81(1):79–90. doi: 10.1111/j.1432-1033.1977.tb11929.x. [DOI] [PubMed] [Google Scholar]
  25. Spurio R., Dürrenberger M., Falconi M., La Teana A., Pon C. L., Gualerzi C. O. Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy. Mol Gen Genet. 1992 Jan;231(2):201–211. doi: 10.1007/BF00279792. [DOI] [PubMed] [Google Scholar]
  26. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  27. Thein S. L., Hinton J. A simple and rapid method of direct sequencing using Dynabeads. Br J Haematol. 1991 Sep;79(1):113–115. doi: 10.1111/j.1365-2141.1991.tb08016.x. [DOI] [PubMed] [Google Scholar]
  28. 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. The chromatin-associated protein H-NS alters DNA topology in vitro. EMBO J. 1994 Jan 1;13(1):258–268. doi: 10.1002/j.1460-2075.1994.tb06256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ueguchi C., Kakeda M., Mizuno T. Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription. Mol Gen Genet. 1993 Jan;236(2-3):171–178. doi: 10.1007/BF00277109. [DOI] [PubMed] [Google Scholar]
  30. Ueguchi C., Mizuno T. The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor. EMBO J. 1993 Mar;12(3):1039–1046. doi: 10.1002/j.1460-2075.1993.tb05745.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ussery D. W., Hinton J. C., Jordi B. J., Granum P. E., Seirafi A., Stephen R. J., Tupper A. E., Berridge G., Sidebotham J. M., Higgins C. F. The chromatin-associated protein H-NS. Biochimie. 1994;76(10-11):968–980. doi: 10.1016/0300-9084(94)90022-1. [DOI] [PubMed] [Google Scholar]
  32. Yamada H., Muramatsu S., Mizuno T. An Escherichia coli protein that preferentially binds to sharply curved DNA. J Biochem. 1990 Sep;108(3):420–425. doi: 10.1093/oxfordjournals.jbchem.a123216. [DOI] [PubMed] [Google Scholar]
  33. Yamada H., Yoshida T., Tanaka K., Sasakawa C., Mizuno T. Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences. Mol Gen Genet. 1991 Nov;230(1-2):332–336. doi: 10.1007/BF00290685. [DOI] [PubMed] [Google Scholar]
  34. Zhang A., Belfort M. Nucleotide sequence of a newly-identified Escherichia coli gene, stpA, encoding an H-NS-like protein. Nucleic Acids Res. 1992 Dec 25;20(24):6735–6735. doi: 10.1093/nar/20.24.6735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhang A., Derbyshire V., Salvo J. L., Belfort M. Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro. RNA. 1995 Oct;1(8):783–793. [PMC free article] [PubMed] [Google Scholar]
  36. Zhang A., Rimsky S., Reaban M. E., Buc H., Belfort M. Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics. EMBO J. 1996 Mar 15;15(6):1340–1349. [PMC free article] [PubMed] [Google Scholar]
  37. Zhou Y. H., Zhang X. P., Ebright R. H. Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res. 1991 Nov 11;19(21):6052–6052. doi: 10.1093/nar/19.21.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zuber F., Kotlarz D., Rimsky S., Buc H. Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H-NS. Mol Microbiol. 1994 Apr;12(2):231–240. doi: 10.1111/j.1365-2958.1994.tb01012.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES