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. 1990 Aug;172(8):4255–4262. doi: 10.1128/jb.172.8.4255-4262.1990

Rhizobium meliloti Fix L is an oxygen sensor and regulates R. meliloti nifA and fixK genes differently in Escherichia coli.

P de Philip 1, J Batut 1, P Boistard 1
PMCID: PMC213249  PMID: 2115865

Abstract

In Rhizobium meliloti, nif and fix genes, involved in nitrogen fixation during symbiosis with alfalfa, are under the control of two transcriptional regulators encoded by nifA and fixK. Expression of nifA and fixK is under the control of FixL/J, a two-component regulatory system. We showed, using Escherichia coli as a heterologous host, that FixL/J controls nifA and fixK expression in response to microaerobiosis. Furthermore, expression of the sensor gene fixL and of the activator gene fixJ under the control of two different promoters allowed us to show that FixL mediates microaerobic induction of nifA when the level of FixJ is low and aerobic repression of nifA when the level of FixJ is high. Similarly, activation of fixK occurred in microaerobiosis when the FixJ level was low in the presence of FixL. In contrast to nifA, fixK expression was not affected by FixL in aerated cultures when the level of FixJ was high. We conclude that R. meliloti FixL senses oxygen in the heterologous host E. coli consistent with the microaerobic induction of nifA and fixK in R. meliloti and that nifA and fixK promoters are differentially activated by FixJ in response to the oxygen signal.

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Selected References

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  1. Aguilar O. M., Reiländer H., Arnold W., Pühler A. Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product. J Bacteriol. 1987 Dec;169(12):5393–5400. doi: 10.1128/jb.169.12.5393-5400.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiba H., Nakasai F., Mizushima S., Mizuno T. Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. J Biochem. 1989 Jul;106(1):5–7. doi: 10.1093/oxfordjournals.jbchem.a122817. [DOI] [PubMed] [Google Scholar]
  3. Albright L. M., Huala E., Ausubel F. M. Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet. 1989;23:311–336. doi: 10.1146/annurev.ge.23.120189.001523. [DOI] [PubMed] [Google Scholar]
  4. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  5. Austin S., Henderson N., Dixon R. Requirements for transcriptional activation in vitro of the nitrogen-regulated glnA and nifLA promoters from Klebsiella pneumoniae: dependence on activator concentration. Mol Microbiol. 1987 Jul;1(1):92–100. doi: 10.1111/j.1365-2958.1987.tb00532.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Borkovich K. A., Kaplan N., Hess J. F., Simon M. I. Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1208–1212. doi: 10.1073/pnas.86.4.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buikema W. J., Klingensmith J. A., Gibbons S. L., Ausubel F. M. Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes. J Bacteriol. 1987 Mar;169(3):1120–1126. doi: 10.1128/jb.169.3.1120-1126.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. David M., Domergue O., Pognonec P., Kahn D. Transcription patterns of Rhizobium meliloti symbiotic plasmid pSym: identification of nifA-independent fix genes. J Bacteriol. 1987 May;169(5):2239–2244. doi: 10.1128/jb.169.5.2239-2244.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Debellé F., Sharma S. B. Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation. Nucleic Acids Res. 1986 Sep 25;14(18):7453–7472. doi: 10.1093/nar/14.18.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ditta G., Virts E., Palomares A., Kim C. H. The nifA gene of Rhizobium meliloti is oxygen regulated. J Bacteriol. 1987 Jul;169(7):3217–3223. doi: 10.1128/jb.169.7.3217-3223.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drummond M. H., Wootton J. C. Sequence of nifL from Klebsiella pneumoniae: mode of action and relationship to two families of regulatory proteins. Mol Microbiol. 1987 Jul;1(1):37–44. doi: 10.1111/j.1365-2958.1987.tb00524.x. [DOI] [PubMed] [Google Scholar]
  14. Earl C. D., Ronson C. W., Ausubel F. M. Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes. J Bacteriol. 1987 Mar;169(3):1127–1136. doi: 10.1128/jb.169.3.1127-1136.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fischer H. M., Bruderer T., Hennecke H. Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding. Nucleic Acids Res. 1988 Mar 25;16(5):2207–2224. doi: 10.1093/nar/16.5.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Forst S., Delgado J., Inouye M. Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6052–6056. doi: 10.1073/pnas.86.16.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forst S., Delgado J., Ramakrishnan G., Inouye M. Regulation of ompC and ompF expression in Escherichia coli in the absence of envZ. J Bacteriol. 1988 Nov;170(11):5080–5085. doi: 10.1128/jb.170.11.5080-5085.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  19. Friedrich M. J., Kadner R. J. Nucleotide sequence of the uhp region of Escherichia coli. J Bacteriol. 1987 Aug;169(8):3556–3563. doi: 10.1128/jb.169.8.3556-3563.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  21. Hertig C., Li R. Y., Louarn A. M., Garnerone A. M., David M., Batut J., Kahn D., Boistard P. Rhizobium meliloti regulatory gene fixJ activates transcription of R. meliloti nifA and fixK genes in Escherichia coli. J Bacteriol. 1989 Mar;171(3):1736–1738. doi: 10.1128/jb.171.3.1736-1738.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huala E., Ausubel F. M. The central domain of Rhizobium meliloti NifA is sufficient to activate transcription from the R. meliloti nifH promoter. J Bacteriol. 1989 Jun;171(6):3354–3365. doi: 10.1128/jb.171.6.3354-3365.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Igo M. M., Ninfa A. J., Silhavy T. J. A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation. Genes Dev. 1989 May;3(5):598–605. doi: 10.1101/gad.3.5.598. [DOI] [PubMed] [Google Scholar]
  24. Igo M. M., Silhavy T. J. EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro. J Bacteriol. 1988 Dec;170(12):5971–5973. doi: 10.1128/jb.170.12.5971-5973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iuchi S., Furlong D., Lin E. C. Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation. J Bacteriol. 1989 May;171(5):2889–2893. doi: 10.1128/jb.171.5.2889-2893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Iuchi S., Lin E. C. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1888–1892. doi: 10.1073/pnas.85.6.1888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Julliot J. S., Boistard P. Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti. Mol Gen Genet. 1979 Jun 20;173(3):289–298. doi: 10.1007/BF00268639. [DOI] [PubMed] [Google Scholar]
  28. Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kunst F., Debarbouille M., Msadek T., Young M., Mauel C., Karamata D., Klier A., Rapoport G., Dedonder R. Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J Bacteriol. 1988 Nov;170(11):5093–5101. doi: 10.1128/jb.170.11.5093-5101.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Magasanik B. Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet. 1982;16:135–168. doi: 10.1146/annurev.ge.16.120182.001031. [DOI] [PubMed] [Google Scholar]
  31. Melchers L. S., Regensburg-Tuïnk T. J., Bourret R. B., Sedee N. J., Schilperoort R. A., Hooykaas P. J. Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens. EMBO J. 1989 Jul;8(7):1919–1925. doi: 10.1002/j.1460-2075.1989.tb03595.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mizuno T., Mizushima S. Isolation and characterization of deletion mutants of ompR and envZ, regulatory genes for expression of the outer membrane proteins OmpC and OmpF in Escherichia coli. J Biochem. 1987 Feb;101(2):387–396. doi: 10.1093/oxfordjournals.jbchem.a121923. [DOI] [PubMed] [Google Scholar]
  33. Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ninfa A. J., Ninfa E. G., Lupas A. N., Stock A., Magasanik B., Stock J. Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5492–5496. doi: 10.1073/pnas.85.15.5492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parkinson J. S. Protein phosphorylation in bacterial chemotaxis. Cell. 1988 Apr 8;53(1):1–2. doi: 10.1016/0092-8674(88)90478-3. [DOI] [PubMed] [Google Scholar]
  36. Priefer U. B., Simon R., Pühler A. Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions. J Bacteriol. 1985 Jul;163(1):324–330. doi: 10.1128/jb.163.1.324-330.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Renalier M. H., Batut J., Ghai J., Terzaghi B., Gherardi M., David M., Garnerone A. M., Vasse J., Truchet G., Huguet T. A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus. J Bacteriol. 1987 May;169(5):2231–2238. doi: 10.1128/jb.169.5.2231-2238.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  39. Rosenberg C., Boistard P., Dénarié J., Casse-Delbart F. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet. 1981;184(2):326–333. doi: 10.1007/BF00272926. [DOI] [PubMed] [Google Scholar]
  40. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  41. Smith J. M., Rowsell E. H., Shioi J., Taylor B. L. Identification of a site of ATP requirement for signal processing in bacterial chemotaxis. J Bacteriol. 1988 Jun;170(6):2698–2704. doi: 10.1128/jb.170.6.2698-2704.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Szeto W. W., Nixon B. T., Ronson C. W., Ausubel F. M. Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol. 1987 Apr;169(4):1423–1432. doi: 10.1128/jb.169.4.1423-1432.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Szeto W. W., Zimmerman J. L., Sundaresan V., Ausubel F. M. A Rhizobium meliloti symbiotic regulatory gene. Cell. 1984 Apr;36(4):1035–1043. doi: 10.1016/0092-8674(84)90053-9. [DOI] [PubMed] [Google Scholar]
  45. Trageser M., Unden G. Role of cysteine residues and of metal ions in the regulatory functioning of FNR, the transcriptional regulator of anaerobic respiration in Escherichia coli. Mol Microbiol. 1989 May;3(5):593–599. doi: 10.1111/j.1365-2958.1989.tb00206.x. [DOI] [PubMed] [Google Scholar]
  46. Truchet G., Debellé F., Vasse J., Terzaghi B., Garnerone A. M., Rosenberg C., Batut J., Maillet F., Dénarié J. Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation. J Bacteriol. 1985 Dec;164(3):1200–1210. doi: 10.1128/jb.164.3.1200-1210.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Unden G., Guest J. R. Isolation and characterization of the Fnr protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli. Eur J Biochem. 1985 Jan 2;146(1):193–199. doi: 10.1111/j.1432-1033.1985.tb08638.x. [DOI] [PubMed] [Google Scholar]
  48. Virts E. L., Stanfield S. W., Helinski D. R., Ditta G. S. Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti. Proc Natl Acad Sci U S A. 1988 May;85(9):3062–3065. doi: 10.1073/pnas.85.9.3062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang E. A., Koshland D. E., Jr Receptor structure in the bacterial sensing system. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7157–7161. doi: 10.1073/pnas.77.12.7157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Waye M. M., Verhoeyen M. E., Jones P. T., Winter G. EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis. Nucleic Acids Res. 1985 Dec 9;13(23):8561–8571. doi: 10.1093/nar/13.23.8561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weston L. A., Kadner R. J. Identification of uhp polypeptides and evidence for their role in exogenous induction of the sugar phosphate transport system of Escherichia coli K-12. J Bacteriol. 1987 Aug;169(8):3546–3555. doi: 10.1128/jb.169.8.3546-3555.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weston L. A., Kadner R. J. Role of uhp genes in expression of the Escherichia coli sugar-phosphate transport system. J Bacteriol. 1988 Aug;170(8):3375–3383. doi: 10.1128/jb.170.8.3375-3383.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Winans S. C., Ebert P. R., Stachel S. E., Gordon M. P., Nester E. W. A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8278–8282. doi: 10.1073/pnas.83.21.8278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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