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. 1993 Oct;175(20):6499–6504. doi: 10.1128/jb.175.20.6499-6504.1993

Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit.

B Toussaint 1, I Delic-Attree 1, R De Sury D'Aspremont 1, L David 1, M Vinçon 1, P M Vignais 1
PMCID: PMC206759  PMID: 8407826

Abstract

We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup- mutant IR4, which is mutated in the himA gene (coding for the alpha subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.

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Selected References

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  1. Bonnefoy E., Rouvière-Yaniv J. HU, the major histone-like protein of E. coli, modulates the binding of IHF to oriC. EMBO J. 1992 Dec;11(12):4489–4496. doi: 10.1002/j.1460-2075.1992.tb05550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bourmeyster N., Stasia M. J., Garin J., Gagnon J., Boquet P., Vignais P. V. Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum. Biochemistry. 1992 Dec 29;31(51):12863–12869. doi: 10.1021/bi00166a022. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Cannon W., Charlton W., Buck M. Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters. J Mol Biol. 1991 Aug 20;220(4):915–931. doi: 10.1016/0022-2836(91)90363-b. [DOI] [PubMed] [Google Scholar]
  5. Claverie-Martin F., Magasanik B. Positive and negative effects of DNA bending on activation of transcription from a distant site. J Mol Biol. 1992 Oct 20;227(4):996–1008. doi: 10.1016/0022-2836(92)90516-m. [DOI] [PubMed] [Google Scholar]
  6. Claverie-Martin F., Magasanik B. Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1631–1635. doi: 10.1073/pnas.88.5.1631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colbeau A., Kelley B. C., Vignais P. M. Hydrogenase activity in Rhodopseudomonas capsulata: relationship with nitrogenase activity. J Bacteriol. 1980 Oct;144(1):141–148. doi: 10.1128/jb.144.1.141-148.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colbeau A., Richaud P., Toussaint B., Caballero F. J., Elster C., Delphin C., Smith R. L., Chabert J., Vignais P. M. Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants. Mol Microbiol. 1993 Apr;8(1):15–29. doi: 10.1111/j.1365-2958.1993.tb01199.x. [DOI] [PubMed] [Google Scholar]
  9. Colbeau A., Vignais P. M. Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2. J Bacteriol. 1992 Jul;174(13):4258–4264. doi: 10.1128/jb.174.13.4258-4264.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flamm E. L., Weisberg R. A. Primary structure of the hip gene of Escherichia coli and of its product, the beta subunit of integration host factor. J Mol Biol. 1985 May 25;183(2):117–128. doi: 10.1016/0022-2836(85)90206-2. [DOI] [PubMed] [Google Scholar]
  11. Gober J. W., Shapiro L. Integration host factor is required for the activation of developmentally regulated genes in Caulobacter. Genes Dev. 1990 Sep;4(9):1494–1504. doi: 10.1101/gad.4.9.1494. [DOI] [PubMed] [Google Scholar]
  12. Goodrich J. A., Schwartz M. L., McClure W. R. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 1990 Sep 11;18(17):4993–5000. doi: 10.1093/nar/18.17.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haluzi H., Goitein D., Koby S., Mendelson I., Teff D., Mengeritsky G., Giladi H., Oppenheim A. B. Genes coding for integration host factor are conserved in gram-negative bacteria. J Bacteriol. 1991 Oct;173(19):6297–6299. doi: 10.1128/jb.173.19.6297-6299.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hillmer P., Gest H. H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J Bacteriol. 1977 Feb;129(2):724–731. doi: 10.1128/jb.129.2.724-731.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoover T. R., Santero E., Porter S., Kustu S. The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons. Cell. 1990 Oct 5;63(1):11–22. doi: 10.1016/0092-8674(90)90284-l. [DOI] [PubMed] [Google Scholar]
  16. Klemme J. H. Untersuchungen zur Photoautotrophie mit molekularem Wasserstoff bei neuisolierten schwefelfreien Purpurbakterien. Arch Mikrobiol. 1968;64(1):29–42. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lee E. C., Hales L. M., Gumport R. I., Gardner J. F. The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities. EMBO J. 1992 Jan;11(1):305–313. doi: 10.1002/j.1460-2075.1992.tb05053.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marrs B. Genetic recombination in Rhodopseudomonas capsulata. Proc Natl Acad Sci U S A. 1974 Mar;71(3):971–973. doi: 10.1073/pnas.71.3.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nash H. A., Granston A. E. Similarity between the DNA-binding domains of IHF protein and TFIID protein. Cell. 1991 Dec 20;67(6):1037–1038. doi: 10.1016/0092-8674(91)90280-c. [DOI] [PubMed] [Google Scholar]
  21. Nash H. A., Robertson C. A. Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination. J Biol Chem. 1981 Sep 10;256(17):9246–9253. [PubMed] [Google Scholar]
  22. Nikolov D. B., Hu S. H., Lin J., Gasch A., Hoffmann A., Horikoshi M., Chua N. H., Roeder R. G., Burley S. K. Crystal structure of TFIID TATA-box binding protein. Nature. 1992 Nov 5;360(6399):40–46. doi: 10.1038/360040a0. [DOI] [PubMed] [Google Scholar]
  23. Rosenfeld J., Capdevielle J., Guillemot J. C., Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem. 1992 May 15;203(1):173–179. doi: 10.1016/0003-2697(92)90061-b. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Santero E., Hoover T. R., North A. K., Berger D. K., Porter S. C., Kustu S. Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter. J Mol Biol. 1992 Oct 5;227(3):602–620. doi: 10.1016/0022-2836(92)90211-2. [DOI] [PubMed] [Google Scholar]
  26. Schneider G. J., Sayre M. H., Geiduschek E. P. DNA-bending properties of TF1. J Mol Biol. 1991 Oct 5;221(3):777–794. doi: 10.1016/0022-2836(91)80175-t. [DOI] [PubMed] [Google Scholar]
  27. Spiro S., Guest J. R. FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev. 1990 Aug;6(4):399–428. doi: 10.1111/j.1574-6968.1990.tb04109.x. [DOI] [PubMed] [Google Scholar]
  28. Toussaint B., Bosc C., Richaud P., Colbeau A., Vignais P. M. A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10749–10753. doi: 10.1073/pnas.88.23.10749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vignais P. M., Colbeau A., Willison J. C., Jouanneau Y. Hydrogenase, nitrogenase, and hydrogen metabolism in the photosynthetic bacteria. Adv Microb Physiol. 1985;26:155–234. doi: 10.1016/s0065-2911(08)60397-5. [DOI] [PubMed] [Google Scholar]
  30. Weaver P. F., Wall J. D., Gest H. Characterization of Rhodopseudomonas capsulata. Arch Microbiol. 1975 Nov 7;105(3):207–216. doi: 10.1007/BF00447139. [DOI] [PubMed] [Google Scholar]
  31. Willison J. C., Madern D., Vignais P. M. Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. Biochem J. 1984 Apr 15;219(2):593–600. doi: 10.1042/bj2190593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  33. de Lorenzo V., Herrero M., Metzke M., Timmis K. N. An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J. 1991 May;10(5):1159–1167. doi: 10.1002/j.1460-2075.1991.tb08056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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