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. 1994 Nov;176(21):6717–6729. doi: 10.1128/jb.176.21.6717-6729.1994

Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation.

J Y Chun 1, G L Sexton 1, L E Roth 1, G Stacey 1
PMCID: PMC197029  PMID: 7961425

Abstract

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

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Selected References

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  1. Atkins C. A., Pate J. S., Shelp B. J. Effects of short-term n(2) deficiency on N metabolism in legume nodules. Plant Physiol. 1984 Nov;76(3):705–710. doi: 10.1104/pp.76.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bott M., Bolliger M., Hennecke H. Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain. Mol Microbiol. 1990 Dec;4(12):2147–2157. doi: 10.1111/j.1365-2958.1990.tb00576.x. [DOI] [PubMed] [Google Scholar]
  3. Bott M., Ritz D., Hennecke H. The Bradyrhizobium japonicum cycM gene encodes a membrane-anchored homolog of mitochondrial cytochrome c. J Bacteriol. 1991 Nov;173(21):6766–6772. doi: 10.1128/jb.173.21.6766-6772.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chua K. Y., Pankhurst C. E., Macdonald P. E., Hopcroft D. H., Jarvis B. D., Scott D. B. Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti. J Bacteriol. 1985 Apr;162(1):335–343. doi: 10.1128/jb.162.1.335-343.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cole M. A., Elkan G. H. Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum. Antimicrob Agents Chemother. 1973 Sep;4(3):248–253. doi: 10.1128/aac.4.3.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  7. Festl H., Ludwig W., Schleifer K. H. DNA hybridization probe for the Pseudomonas fluorescens group. Appl Environ Microbiol. 1986 Nov;52(5):1190–1194. doi: 10.1128/aem.52.5.1190-1194.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Fisher R. F., Long S. R. Rhizobium--plant signal exchange. Nature. 1992 Jun 25;357(6380):655–660. doi: 10.1038/357655a0. [DOI] [PubMed] [Google Scholar]
  10. Göttfert M., Grob P., Hennecke H. Proposed regulatory pathway encoded by the nodV and nodW genes, determinants of host specificity in Bradyrhizobium japonicum. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2680–2684. doi: 10.1073/pnas.87.7.2680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halverson L. J., Stacey G. Host recognition in the Rhizobium-soybean symbiosis : evidence for the involvement of lectin in nodulation. Plant Physiol. 1985 Mar;77(3):621–625. doi: 10.1104/pp.77.3.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Hotter G. S., Scott D. B. Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host. J Bacteriol. 1991 Jan;173(2):851–859. doi: 10.1128/jb.173.2.851-859.1991. [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. Kuykendall L. D., Elkan G. H. Rhizobium japonicum derivatives differing in nitrogen-fixing efficiency and carbohydrate utilization. Appl Environ Microbiol. 1976 Oct;32(4):511–519. doi: 10.1128/aem.32.4.511-519.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maier R. J., Graham L., Keefe R. G., Pihl T., Smith E. Bradyrhizobium japonicum mutants defective in nitrogen fixation and molybdenum metabolism. J Bacteriol. 1987 Jun;169(6):2548–2554. doi: 10.1128/jb.169.6.2548-2554.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nieuwkoop A. J., Banfalvi Z., Deshmane N., Gerhold D., Schell M. G., Sirotkin K. M., Stacey G. A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum. J Bacteriol. 1987 Jun;169(6):2631–2638. doi: 10.1128/jb.169.6.2631-2638.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Brian M. R., Kirshbom P. M., Maier R. J. Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture. J Bacteriol. 1987 Mar;169(3):1089–1094. doi: 10.1128/jb.169.3.1089-1094.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ramseier T. M., Winteler H. V., Hennecke H. Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes. J Biol Chem. 1991 Apr 25;266(12):7793–7803. [PubMed] [Google Scholar]
  20. Ronson C. W., Lyttleton P., Robertson J. G. C(4)-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4284–4288. doi: 10.1073/pnas.78.7.4284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roth L. E., Stacey G. Bacterium release into host cells of nitrogen-fixing soybean nodules: the symbiosome membrane comes from three sources. Eur J Cell Biol. 1989 Jun;49(1):13–23. [PubMed] [Google Scholar]
  22. Russell P., Schell M. G., Nelson K. K., Halverson L. J., Sirotkin K. M., Stacey G. Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum. J Bacteriol. 1985 Dec;164(3):1301–1308. doi: 10.1128/jb.164.3.1301-1308.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sadowsky M. J., Cregan P. B., Gottfert M., Sharma A., Gerhold D., Rodriguez-Quinones F., Keyser H. H., Hennecke H., Stacey G. The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):637–641. doi: 10.1073/pnas.88.2.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. So J. S., Hodgson A. L., Haugland R., Leavitt M., Banfalvi Z., Nieuwkoop A. J., Stacey G. Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation. Mol Gen Genet. 1987 Apr;207(1):15–23. doi: 10.1007/BF00331485. [DOI] [PubMed] [Google Scholar]
  26. Stacey G., Luka S., Sanjuan J., Banfalvi Z., Nieuwkoop A. J., Chun J. Y., Forsberg L. S., Carlson R. nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum. J Bacteriol. 1994 Feb;176(3):620–633. doi: 10.1128/jb.176.3.620-633.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thöny-Meyer L., Stax D., Hennecke H. An unusual gene cluster for the cytochrome bc1 complex in Bradyrhizobium japonicum and its requirement for effective root nodule symbiosis. Cell. 1989 May 19;57(4):683–697. doi: 10.1016/0092-8674(89)90137-2. [DOI] [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. Verma D. P., Ball S., Guérin C., Wanamaker L. Leghemoglobin biosynthesis in soybean root nodules. Characterization of the nascent and released peptides and the relative rate of synthesis of the major leghemoglobins. Biochemistry. 1979 Feb 6;18(3):476–483. doi: 10.1021/bi00570a016. [DOI] [PubMed] [Google Scholar]
  30. Vieira J., Messing J. New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene. 1991 Apr;100:189–194. doi: 10.1016/0378-1119(91)90365-i. [DOI] [PubMed] [Google Scholar]
  31. Ward L. J., Rockman E. S., Ball P., Jarvis B. D., Scott D. B. Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus. Mol Plant Microbe Interact. 1989 Sep-Oct;2(5):224–232. doi: 10.1094/mpmi-2-224. [DOI] [PubMed] [Google Scholar]
  32. Weaver C. D., Crombie B., Stacey G., Roberts D. M. Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26. Plant Physiol. 1991 Jan;95(1):222–227. doi: 10.1104/pp.95.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wilson K. J., Anjaiah V., Nambiar P. T., Ausubel F. M. Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation. J Bacteriol. 1987 May;169(5):2177–2186. doi: 10.1128/jb.169.5.2177-2186.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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