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. 1991 Dec 1;88(23):10749–10753. doi: 10.1073/pnas.88.23.10749

A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression.

B Toussaint 1, C Bosc 1, P Richaud 1, A Colbeau 1, P M Vignais 1
PMCID: PMC53008  PMID: 1961742

Abstract

A gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2.

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Selected References

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

  1. Alias A., Cejudo F. J., Chabert J., Willison J. C., Vignais P. M. Nucleotide sequence of wild-type and mutant nifR4 (ntrA) genes of Rhodobacter capsulatus: identification of an essential glycine residue. Nucleic Acids Res. 1989 Jul 11;17(13):5377–5377. doi: 10.1093/nar/17.13.5377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonnefoy E., Rouvière-Yaniv J. HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments. EMBO J. 1991 Mar;10(3):687–696. doi: 10.1002/j.1460-2075.1991.tb07998.x. [DOI] [PMC free article] [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. Colbeau A., Godfroy A., Vignais P. M. Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata. Biochimie. 1986 Jan;68(1):147–155. doi: 10.1016/s0300-9084(86)81079-3. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drlica K., Rouviere-Yaniv J. Histonelike proteins of bacteria. Microbiol Rev. 1987 Sep;51(3):301–319. doi: 10.1128/mr.51.3.301-319.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Friedman D. I. Integration host factor: a protein for all reasons. Cell. 1988 Nov 18;55(4):545–554. doi: 10.1016/0092-8674(88)90213-9. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Haugland R. A., Hanus F. J., Cantrell M. A., Evans H. J. Rapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum. Appl Environ Microbiol. 1983 Mar;45(3):892–897. doi: 10.1128/aem.45.3.892-897.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Hübner P., Willison J. C., Vignais P. M., Bickle T. A. Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol. 1991 May;173(9):2993–2999. doi: 10.1128/jb.173.9.2993-2999.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones R., Haselkorn R. The DNA sequence of the Rhodobacter capsulatus ntrA, ntrB and ntrC gene analogues required for nitrogen fixation. Mol Gen Genet. 1989 Feb;215(3):507–516. doi: 10.1007/BF00427050. [DOI] [PubMed] [Google Scholar]
  15. Kimura M., Wilson K. S. On the DNA binding protein II from Bacillus stearothermophilus. II. The amino acid sequence and its relation to those of homologous proteins from other prokaryotes. J Biol Chem. 1983 Mar 25;258(6):4007–4011. [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. Kranz R. G., Haselkorn R. Characterization of nif regulatory genes in Rhodopseudomonas capsulata using lac gene fusions. Gene. 1985;40(2-3):203–215. doi: 10.1016/0378-1119(85)90043-5. [DOI] [PubMed] [Google Scholar]
  18. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laine B., Kmiecik D., Sautiere P., Biserte G., Cohen-Solal M. Complete amino-acid sequences of DNA-binding proteins HU-1 and HU-2 from Escherichia coli. Eur J Biochem. 1980 Feb;103(3):447–461. doi: 10.1111/j.1432-1033.1980.tb05968.x. [DOI] [PubMed] [Google Scholar]
  20. Leclerc M., Colbeau A., Cauvin B., Vignais P. M. Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus. Mol Gen Genet. 1988 Sep;214(1):97–107. doi: 10.1007/BF00340186. [DOI] [PubMed] [Google Scholar]
  21. Lee E. C., MacWilliams M. P., Gumport R. I., Gardner J. F. Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site. J Bacteriol. 1991 Jan;173(2):609–617. doi: 10.1128/jb.173.2.609-617.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lutz S., Böhm R., Beier A., Böck A. Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli. Mol Microbiol. 1990 Jan;4(1):13–20. doi: 10.1111/j.1365-2958.1990.tb02010.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Mechulam Y., Fayat G., Blanquet S. Sequence of the Escherichia coli pheST operon and identification of the himA gene. J Bacteriol. 1985 Aug;163(2):787–791. doi: 10.1128/jb.163.2.787-791.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miller H. I., Kirk M., Echols H. SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6754–6758. doi: 10.1073/pnas.78.11.6754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  27. Richaud P., Colbeau A., Toussaint B., Vignais P. M. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus. J Bacteriol. 1991 Sep;173(18):5928–5932. doi: 10.1128/jb.173.18.5928-5932.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Römermann D., Warrelmann J., Bender R. A., Friedrich B. An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation. J Bacteriol. 1989 Feb;171(2):1093–1099. doi: 10.1128/jb.171.2.1093-1099.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tanaka I., Appelt K., Dijk J., White S. W., Wilson K. S. 3-A resolution structure of a protein with histone-like properties in prokaryotes. Nature. 1984 Aug 2;310(5976):376–381. doi: 10.1038/310376a0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. White S. W., Appelt K., Wilson K. S., Tanaka I. A protein structural motif that bends DNA. Proteins. 1989;5(4):281–288. doi: 10.1002/prot.340050405. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yang C. C., Nash H. A. The interaction of E. coli IHF protein with its specific binding sites. Cell. 1989 Jun 2;57(5):869–880. doi: 10.1016/0092-8674(89)90801-5. [DOI] [PubMed] [Google Scholar]
  36. 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]

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