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. 1995 Jul;177(14):3979–3984. doi: 10.1128/jb.177.14.3979-3984.1995

Oxygen control of the Bradyrhizobium japonicum hemA gene.

K M Page 1, M L Guerinot 1
PMCID: PMC177127  PMID: 7608070

Abstract

The hemA gene of Bradyrhizobium japonicum, which encodes the first enzyme in the heme biosynthetic pathway, is regulated by oxygen. Up to ninefold induction of beta-galactosidase activity is seen when cultures of B. japonicum containing either a plasmid-encoded or a chromosomally integrated hemA-lacZ fusion are shifted to restricted aeration. The oxygen effect is mediated via the FixLJ two-component regulatory system, which regulates the expression of a number of genes involved in the nitrogen fixation process in response to low-oxygen conductions; oxygen induction is lost when the hemA-lacZ fusion is expressed in strains of B. japonicum carrying mutations in fixL or fixJ. The B. japonicum hemA promoter region contains a sequence identical to the Escherichia coli Fnr binding site (positions -46 to -33 relative to the hemA transcription start site). Fnr is a regulatory protein necessary for the oxygen-regulated expression of anaerobic respiratory genes. Activity of a hemA-lacZ fusion construct in which the Fnr box-like sequence was replaced with a BglII site is not induced in B. japonicum cultures grown under restricted aeration. The fnr homolog fixK is FixLJ dependent. Collectively, these data suggest a role for the rhizobial Fnr-like protein, FixK, in the regulation of hemA. Furthermore, the coregulation of hemA with symbiotically important genes via FixLJ is consistent with the idea that hemA is required in the nodule as well as under free-living conditions.

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Selected References

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  1. Anthamatten D., Hennecke H. The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti. Mol Gen Genet. 1991 Jan;225(1):38–48. doi: 10.1007/BF00282640. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Batut J., Daveran-Mingot M. L., David M., Jacobs J., Garnerone A. M., Kahn D. fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti. EMBO J. 1989 Apr;8(4):1279–1286. doi: 10.1002/j.1460-2075.1989.tb03502.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell A. I., Cole J. A., Busby S. J. Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter. Mol Microbiol. 1990 Oct;4(10):1753–1763. doi: 10.1111/j.1365-2958.1990.tb00553.x. [DOI] [PubMed] [Google Scholar]
  5. Chauhan S., O'Brian M. R. Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase is essential for symbiosis with soybean and contains a novel metal-binding domain. J Bacteriol. 1993 Nov;175(22):7222–7227. doi: 10.1128/jb.175.22.7222-7227.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Darie S., Gunsalus R. P. Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products. J Bacteriol. 1994 Sep;176(17):5270–5276. doi: 10.1128/jb.176.17.5270-5276.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. David M., Daveran M. L., Batut J., Dedieu A., Domergue O., Ghai J., Hertig C., Boistard P., Kahn D. Cascade regulation of nif gene expression in Rhizobium meliloti. Cell. 1988 Aug 26;54(5):671–683. doi: 10.1016/s0092-8674(88)80012-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Drolet M., Sasarman A. Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter. Mol Gen Genet. 1991 Apr;226(1-2):250–256. doi: 10.1007/BF00273610. [DOI] [PubMed] [Google Scholar]
  10. Eiglmeier K., Honoré N., Iuchi S., Lin E. C., Cole S. T. Molecular genetic analysis of FNR-dependent promoters. Mol Microbiol. 1989 Jul;3(7):869–878. doi: 10.1111/j.1365-2958.1989.tb00236.x. [DOI] [PubMed] [Google Scholar]
  11. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer H. M. Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev. 1994 Sep;58(3):352–386. doi: 10.1128/mr.58.3.352-386.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frustaci J. M., O'Brian M. R. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J Bacteriol. 1992 Jul;174(13):4223–4229. doi: 10.1128/jb.174.13.4223-4229.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilles-Gonzalez M. A., Ditta G. S., Helinski D. R. A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti. Nature. 1991 Mar 14;350(6314):170–172. doi: 10.1038/350170a0. [DOI] [PubMed] [Google Scholar]
  15. Green J., Guest J. R. Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. Mol Microbiol. 1994 May;12(3):433–444. doi: 10.1111/j.1365-2958.1994.tb01032.x. [DOI] [PubMed] [Google Scholar]
  16. Guerinot M. L., Chelm B. K. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1837–1841. doi: 10.1073/pnas.83.6.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gunsalus R. P. Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes. J Bacteriol. 1992 Nov;174(22):7069–7074. doi: 10.1128/jb.174.22.7069-7074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kahn D., David M., Domergue O., Daveran M. L., Ghai J., Hirsch P. R., Batut J. Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation. J Bacteriol. 1989 Feb;171(2):929–939. doi: 10.1128/jb.171.2.929-939.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kaminski P. A., Elmerich C. Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans. Mol Microbiol. 1991 Mar;5(3):665–673. doi: 10.1111/j.1365-2958.1991.tb00738.x. [DOI] [PubMed] [Google Scholar]
  20. Keng T., Guarente L. Constitutive expression of the yeast HEM1 gene is actually a composite of activation and repression. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9113–9117. doi: 10.1073/pnas.84.24.9113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keng T. HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Jun;12(6):2616–2623. doi: 10.1128/mcb.12.6.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Legocki R. P., Yun A. C., Szalay A. A. Expression of beta-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5806–5810. doi: 10.1073/pnas.81.18.5806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leong S. A., Williams P. H., Ditta G. S. Analysis of the 5' regulatory region of the gene for delta-aminolevulinic acid synthetase of Rhizobium meliloti. Nucleic Acids Res. 1985 Aug 26;13(16):5965–5976. doi: 10.1093/nar/13.16.5965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lois A. F., Weinstein M., Ditta G. S., Helinski D. R. Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen. J Biol Chem. 1993 Feb 25;268(6):4370–4375. [PubMed] [Google Scholar]
  29. Mandon K., Kaminski P. A., Elmerich C. Functional analysis of the fixNOQP region of Azorhizobium caulinodans. J Bacteriol. 1994 May;176(9):2560–2568. doi: 10.1128/jb.176.9.2560-2568.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McClung C. R., Somerville J. E., Guerinot M. L., Chelm B. K. Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase. Gene. 1987;54(1):133–139. doi: 10.1016/0378-1119(87)90355-6. [DOI] [PubMed] [Google Scholar]
  31. Nees D. W., Stein P. A., Ludwig R. A. The Azorhizobium caulinodans nifA gene: identification of upstream-activating sequences including a new element, the 'anaerobox'. Nucleic Acids Res. 1988 Oct 25;16(20):9839–9853. doi: 10.1093/nar/16.20.9839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Neidle E. L., Kaplan S. Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes. J Bacteriol. 1993 Apr;175(8):2292–2303. doi: 10.1128/jb.175.8.2292-2303.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Brian M. R., Maier R. J. Molecular aspects of the energetics of nitrogen fixation in Rhizobium-legume symbioses. Biochim Biophys Acta. 1989 May 30;974(3):229–246. doi: 10.1016/s0005-2728(89)80239-7. [DOI] [PubMed] [Google Scholar]
  34. Page K. M., Connolly E. L., Guerinot M. L. Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene. J Bacteriol. 1994 Mar;176(5):1535–1538. doi: 10.1128/jb.176.5.1535-1538.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Preisig O., Anthamatten D., Hennecke H. Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3309–3313. doi: 10.1073/pnas.90.8.3309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ratet P., Pawlowski K., Schell J., de Bruijn F. J. The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status. Mol Microbiol. 1989 Jun;3(6):825–838. doi: 10.1111/j.1365-2958.1989.tb00231.x. [DOI] [PubMed] [Google Scholar]
  37. Reyrat J. M., David M., Blonski C., Boistard P., Batut J. Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes. J Bacteriol. 1993 Nov;175(21):6867–6872. doi: 10.1128/jb.175.21.6867-6872.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sadowsky M. J., Tully R. E., Cregan P. B., Keyser H. H. Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-Specific Nodulation of Soybean. Appl Environ Microbiol. 1987 Nov;53(11):2624–2630. doi: 10.1128/aem.53.11.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sangwan I., O'brian M. R. Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules. Plant Physiol. 1992 Mar;98(3):1074–1079. doi: 10.1104/pp.98.3.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sangwan I., O'brian M. R. Evidence for an inter-organismic heme biosynthetic pathway in symbiotic soybean root nodules. Science. 1991 Mar 8;251(4998):1220–1222. doi: 10.1126/science.251.4998.1220. [DOI] [PubMed] [Google Scholar]
  41. Schlüter A., Patschkowski T., Unden G., Priefer U. B. The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription. Mol Microbiol. 1992 Nov;6(22):3395–3404. doi: 10.1111/j.1365-2958.1992.tb02207.x. [DOI] [PubMed] [Google Scholar]
  42. Schüddekopf K., Hennecke S., Liese U., Kutsche M., Klipp W. Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus. Mol Microbiol. 1993 May;8(4):673–684. doi: 10.1111/j.1365-2958.1993.tb01611.x. [DOI] [PubMed] [Google Scholar]
  43. Shapleigh J. P., Gennis R. B. Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides. Mol Microbiol. 1992 Mar;6(5):635–642. doi: 10.1111/j.1365-2958.1992.tb01511.x. [DOI] [PubMed] [Google Scholar]
  44. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Spiro S. The FNR family of transcriptional regulators. Antonie Van Leeuwenhoek. 1994;66(1-3):23–36. doi: 10.1007/BF00871630. [DOI] [PubMed] [Google Scholar]
  47. Squartini A., van Veen R. J., Regensburg-Tuink T., Hooykaas P. J., Nuti M. P. Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001. Mol Plant Microbe Interact. 1988 Mar;1(3):145–149. doi: 10.1094/mpmi-1-145. [DOI] [PubMed] [Google Scholar]
  48. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Waelkens F., Foglia A., Morel J. B., Fourment J., Batut J., Boistard P. Molecular genetic analysis of the Rhizobium meliloti fixK promoter: identification of sequences involved in positive and negative regulation. Mol Microbiol. 1992 Jun;6(11):1447–1456. doi: 10.1111/j.1365-2958.1992.tb00865.x. [DOI] [PubMed] [Google Scholar]
  50. Weinstein M., Lois A. F., Ditta G. S., Helinski D. R. Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter. Gene. 1993 Dec 8;134(2):145–152. doi: 10.1016/0378-1119(93)90088-k. [DOI] [PubMed] [Google Scholar]
  51. Weinstein M., Lois A. F., Monson E. K., Ditta G. S., Helinski D. R. Isolation of phosphorylation-deficient mutants of the Rhizobium meliloti two-component regulatory protein, FixJ. Mol Microbiol. 1992 Aug;6(15):2041–2049. doi: 10.1111/j.1365-2958.1992.tb01377.x. [DOI] [PubMed] [Google Scholar]
  52. Wright M. S., Eckert J. J., Biel S. W., Biel A. J. Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene. FEMS Microbiol Lett. 1991 Mar 1;62(2-3):339–342. doi: 10.1016/0378-1097(91)90181-9. [DOI] [PubMed] [Google Scholar]

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