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. 1997 May;179(10):3304–3309. doi: 10.1128/jb.179.10.3304-3309.1997

A [2Fe-2S] ferredoxin (FdVI) is essential for growth of the photosynthetic bacterium Rhodobacter capsulatus.

J Armengaud 1, C Meyer 1, Y Jouanneau 1
PMCID: PMC179111  PMID: 9150228

Abstract

The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a polypeptide having 34% similarity with HtrA, a serine protease found in enteric bacteria. ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when attempted on the wild-type strain B10. Disruption of fdxE could be achieved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdxE under the control of the nifH promoter grew only in N-free medium, thus demonstrating that fdxE expression is required for growth. Nevertheless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contained no detectable amount of FdVI. Taken together, the results indicate that FdVI is required for an essential metabolic function in R. capsulatus and that this FdVI dependence could be relieved by a single-mutation event. In accordance, FdVI biosynthesis was found to be constitutive in R. capsulatus.

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Selected References

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  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. Armengaud J., Meyer C., Jouanneau Y. Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a [2Fe-2S] ferredoxin. Biochem J. 1994 Jun 1;300(Pt 2):413–418. doi: 10.1042/bj3000413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barany F. Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene. 1985;37(1-3):111–123. doi: 10.1016/0378-1119(85)90263-x. [DOI] [PubMed] [Google Scholar]
  4. Beynon J., Cannon M., Buchanan-Wollaston V., Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. doi: 10.1016/0092-8674(83)90399-9. [DOI] [PubMed] [Google Scholar]
  5. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Bruschi M., Guerlesquin F. Structure, function and evolution of bacterial ferredoxins. FEMS Microbiol Rev. 1988 Apr-Jun;4(2):155–175. doi: 10.1111/j.1574-6968.1988.tb02741.x. [DOI] [PubMed] [Google Scholar]
  7. Duport C., Jouanneau Y., Vignais P. M. Transcriptional analysis and promoter mapping of the fdxA gene which encodes the 7Fe ferredoxin (FdII) of Rhodobacter capsulatus. Mol Gen Genet. 1992 Jan;231(2):323–328. doi: 10.1007/BF00279806. [DOI] [PubMed] [Google Scholar]
  8. Grabau C., Schatt E., Jouanneau Y., Vignais P. M. A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli. J Biol Chem. 1991 Feb 15;266(5):3294–3299. [PubMed] [Google Scholar]
  9. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  10. Hübner P., Masepohl B., Klipp W., Bickle T. A. nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol. 1993 Oct;10(1):123–132. doi: 10.1111/j.1365-2958.1993.tb00909.x. [DOI] [PubMed] [Google Scholar]
  11. Johnson K., Charles I., Dougan G., Pickard D., O'Gaora P., Costa G., Ali T., Miller I., Hormaeche C. The role of a stress-response protein in Salmonella typhimurium virulence. Mol Microbiol. 1991 Feb;5(2):401–407. doi: 10.1111/j.1365-2958.1991.tb02122.x. [DOI] [PubMed] [Google Scholar]
  12. Jouanneau Y., Meyer C., Gaillard J., Forest E., Gagnon J. Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus. J Biol Chem. 1993 May 15;268(14):10636–10644. [PubMed] [Google Scholar]
  13. Jouanneau Y., Meyer C., Gaillard J., Vignais P. M. Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus. Biochem Biophys Res Commun. 1990 Aug 31;171(1):273–279. doi: 10.1016/0006-291x(90)91388-9. [DOI] [PubMed] [Google Scholar]
  14. Jouanneau Y., Meyer C., Naud I., Klipp W. Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase. Biochim Biophys Acta. 1995 Nov 21;1232(1-2):33–42. doi: 10.1016/0005-2728(95)00106-x. [DOI] [PubMed] [Google Scholar]
  15. Lipinska B., Sharma S., Georgopoulos C. Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-independent mechanism of heat-inducible transcription. Nucleic Acids Res. 1988 Nov 11;16(21):10053–10067. doi: 10.1093/nar/16.21.10053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lipinska B., Zylicz M., Georgopoulos C. The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. J Bacteriol. 1990 Apr;172(4):1791–1797. doi: 10.1128/jb.172.4.1791-1797.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Moreno-Vivian C., Hennecke S., Pühler A., Klipp W. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus. J Bacteriol. 1989 May;171(5):2591–2598. doi: 10.1128/jb.171.5.2591-2598.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nagy P. L., Marolewski A., Benkovic S. J., Zalkin H. Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli. J Bacteriol. 1995 Mar;177(5):1292–1298. doi: 10.1128/jb.177.5.1292-1298.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nagy P. L., McCorkle G. M., Zalkin H. purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis. J Bacteriol. 1993 Nov;175(21):7066–7073. doi: 10.1128/jb.175.21.7066-7073.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Naud I., Meyer C., David L., Breton J., Gaillard J., Jouanneau Y. Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase. Eur J Biochem. 1996 Apr 15;237(2):399–405. doi: 10.1111/j.1432-1033.1996.0399k.x. [DOI] [PubMed] [Google Scholar]
  23. Naud I., Vinçon M., Garin J., Gaillard J., Forest E., Jouanneau Y. Purification of a sixth ferredoxin from Rhodobacter capsulatus. Primary structure and biochemical properties. Eur J Biochem. 1994 Jun 15;222(3):933–939. doi: 10.1111/j.1432-1033.1994.tb18942.x. [DOI] [PubMed] [Google Scholar]
  24. Pollock D., Bauer C. E., Scolnik P. A. Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter. Gene. 1988 May 30;65(2):269–275. doi: 10.1016/0378-1119(88)90463-5. [DOI] [PubMed] [Google Scholar]
  25. Saeki K., Suetsugu Y., Tokuda K., Miyatake Y., Young D. A., Marrs B. L., Matsubara H. Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus. J Biol Chem. 1991 Jul 15;266(20):12889–12895. [PubMed] [Google Scholar]
  26. 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]
  27. Schatt E., Jouanneau Y., Vignais P. M. Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol. 1989 Nov;171(11):6218–6226. doi: 10.1128/jb.171.11.6218-6226.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thöny B., Hennecke H. The -24/-12 promoter comes of age. FEMS Microbiol Rev. 1989 Dec;5(4):341–357. doi: 10.1016/0168-6445(89)90028-4. [DOI] [PubMed] [Google Scholar]
  29. Wang S. P., Chen Y. P., Ely B. A ferredoxin, designated FdxP, stimulates p-hydroxybenzoate hydroxylase activity in Caulobacter crescentus. J Bacteriol. 1995 May;177(10):2908–2911. doi: 10.1128/jb.177.10.2908-2911.1995. [DOI] [PMC free article] [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., Pierrard J., Hübner P. Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA. Gene. 1993 Oct 29;133(1):39–46. doi: 10.1016/0378-1119(93)90222-o. [DOI] [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]

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