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. 1997 Jun;179(11):3676–3682. doi: 10.1128/jb.179.11.3676-3682.1997

Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum.

M C Durmowicz 1, R J Maier 1
PMCID: PMC179164  PMID: 9171416

Abstract

In-frame deletion mutagenesis was used to study the roles of two Bradyrhizobium japonicum proteins, HoxX and HoxA, in hydrogenase biosynthesis; based on their sequences, these proteins were previously proposed to be sensor and regulator proteins, respectively, of a two-component regulatory system necessary for hydrogenase transcription. Deletion of the hoxX gene resulted in a strain that expressed only 30 to 40% of wild-type hydrogenase activity. The inactive unprocessed form of the hydrogenase large subunit accumulated in this strain, indicating a role for HoxX in posttranslational processing of the hydrogenase enzyme but not in transcriptional regulation. Strains containing a deletion of the hoxA gene or a double mutation (hoxX and hoxA) did not exhibit any hydrogenase activity under free-living conditions, and extracts from these strains were inactive in gel retardation assays with a 158-bp fragment of the DNA region upstream of the hupSL operon. However, bacteroids from root nodules formed by all three mutant types (hoxX, hoxA, and hoxX hoxA) exhibited hydrogenase activity comparable to that of wild-type bacteroids. Bacteroid extracts from all of these strains, including the wild type, failed to cause a shift of the hydrogenase upstream region used in our assay. It was shown that HoxA is a DNA-binding transcriptional activator of hydrogenase structural gene expression under free-living conditions but not under symbiotic conditions. Although symbiotic hydrogenase expression is still sigma54 dependent, a transcriptional activator other than HoxA functions presumably upstream of the HoxA binding site.

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Selected References

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  1. Anthamatten D., Scherb B., Hennecke H. Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes. J Bacteriol. 1992 Apr;174(7):2111–2120. doi: 10.1128/jb.174.7.2111-2120.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop P. E., Guevara J. G., Engelke J. A., Evans H. J. Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max. Plant Physiol. 1976 Apr;57(4):542–546. doi: 10.1104/pp.57.4.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black L. K., Maier R. J. IHF- and RpoN-dependent regulation of hydrogenase expression in Bradyrhizobium japonicum. Mol Microbiol. 1995 May;16(3):405–413. doi: 10.1111/j.1365-2958.1995.tb02406.x. [DOI] [PubMed] [Google Scholar]
  4. Eberz G., Friedrich B. Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus. J Bacteriol. 1991 Mar;173(6):1845–1854. doi: 10.1128/jb.173.6.1845-1854.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eskew D. L., Welch R. M., Cary E. E. A simple plant nutrient solution purification method for effective removal of trace metals using controlled pore glass-8-hydroxyquinoline chelation column chromatography. Plant Physiol. 1984 Sep;76(1):103–105. doi: 10.1104/pp.76.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fu C., Maier R. J. Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hupc mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme. Arch Microbiol. 1992;157(6):493–498. doi: 10.1007/BF00276768. [DOI] [PubMed] [Google Scholar]
  7. Gross R., Aricò B., Rappuoli R. Families of bacterial signal-transducing proteins. Mol Microbiol. 1989 Nov;3(11):1661–1667. doi: 10.1111/j.1365-2958.1989.tb00152.x. [DOI] [PubMed] [Google Scholar]
  8. Hom S. S., Novak P. D., Maier R. J. Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H(2). Appl Environ Microbiol. 1988 Feb;54(2):358–363. doi: 10.1128/aem.54.2.358-363.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Keefe R. G., Maier R. J. Purification and characterization of an O2-utilizing cytochrome-c oxidase complex from Bradyrhizobium japonicum bacteroid membranes. Biochim Biophys Acta. 1993 Nov 2;1183(1):91–104. doi: 10.1016/0005-2728(93)90008-4. [DOI] [PubMed] [Google Scholar]
  10. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  11. Kim H., Gabel C., Maier R. J. Expression of hydrogenase in Hupc strains of Bradyrhizobium japonicum. Arch Microbiol. 1993;160(1):43–50. doi: 10.1007/BF00258144. [DOI] [PubMed] [Google Scholar]
  12. Kim H., Maier R. J. Transcriptional regulation of hydrogenase synthesis by nickel in Bradyrhizobium japonicum. J Biol Chem. 1990 Nov 5;265(31):18729–18732. [PubMed] [Google Scholar]
  13. Kim H., Yu C., Maier R. J. Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen. J Bacteriol. 1991 Jul;173(13):3993–3999. doi: 10.1128/jb.173.13.3993-3999.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kullik I., Fritsche S., Knobel H., Sanjuan J., Hennecke H., Fischer H. M. Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN). J Bacteriol. 1991 Feb;173(3):1125–1138. doi: 10.1128/jb.173.3.1125-1138.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lambert G. R., Cantrell M. A., Hanus F. J., Russell S. A., Haddad K. R., Evans H. J. Intra- and interspecies transfer and expression of Rhizobium japonicum hydrogen uptake genes and autotrophic growth capability. Proc Natl Acad Sci U S A. 1985 May;82(10):3232–3236. doi: 10.1073/pnas.82.10.3232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lenz O., Schwartz E., Dernedde J., Eitinger M., Friedrich B. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation. J Bacteriol. 1994 Jul;176(14):4385–4393. doi: 10.1128/jb.176.14.4385-4393.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maier R. J., Merberg D. M. Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen. J Bacteriol. 1982 Apr;150(1):161–167. doi: 10.1128/jb.150.1.161-167.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Merberg D., O'Hara E. B., Maier R. J. Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen. J Bacteriol. 1983 Dec;156(3):1236–1242. doi: 10.1128/jb.156.3.1236-1242.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ninfa A. J., Bennett R. L. Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII. J Biol Chem. 1991 Apr 15;266(11):6888–6893. [PubMed] [Google Scholar]
  20. Palacios J. M., Murillo J., Leyva A., Ditta G., Ruiz-Argüeso T. Differential expression of hydrogen uptake (hup) genes in vegetative and symbiotic cells of Rhizobium leguminosarum. Mol Gen Genet. 1990 May;221(3):363–370. doi: 10.1007/BF00259401. [DOI] [PubMed] [Google Scholar]
  21. Rey L., Fernández D., Brito B., Hernando Y., Palacios J. M., Imperial J., Ruiz-Argüeso T. The hydrogenase gene cluster of Rhizobium leguminosarum bv. viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity. Mol Gen Genet. 1996 Sep 13;252(3):237–248. doi: 10.1007/BF02173769. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Stoker K., Reijnders W. N., Oltmann L. F., Stouthamer A. H. Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12. J Bacteriol. 1989 Aug;171(8):4448–4456. doi: 10.1128/jb.171.8.4448-4456.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Van Soom C., Verreth C., Sampaio M. J., Vanderleyden J. Identification of a potential transcriptional regulator of hydrogenase activity in free-living Bradyrhizobium japonicum strains. Mol Gen Genet. 1993 May;239(1-2):235–240. doi: 10.1007/BF00281623. [DOI] [PubMed] [Google Scholar]
  26. Van Soom C., de Wilde P., Vanderleyden J. HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum. Mol Microbiol. 1997 Mar;23(5):967–977. doi: 10.1046/j.1365-2958.1997.2781648.x. [DOI] [PubMed] [Google Scholar]

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