Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Sep;171(9):4686–4693. doi: 10.1128/jb.171.9.4686-4693.1989

Subcellular localization of the nodD gene product in Rhizobium leguminosarum.

H R Schlaman 1, H P Spaink 1, R J Okker 1, B J Lugtenberg 1
PMCID: PMC210268  PMID: 2670892

Abstract

In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product. In order to localize NodD protein in R. leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product. Using these antibodies, we determined that the NodD protein is located exclusively in the cytoplasmic membrane of wild-type R. leguminosarum biovar viciae cells. This localization is independent of the presence of inducers. In a Rhizobium strain that overproduced the NodD protein, the protein was present both in the cytoplasmic membrane and the cytosol, indicating an influence of the protein abundance on its ultimate subcellular localization. It was estimated that 20 to 80 molecules of NodD protein were present per wild-type Rhizobium cell. A model which combines the localization and the DNA-binding properties of the NodD protein as well as the observed association of flavonoids with the cytoplasmic membrane is discussed.

Full text

PDF
4687

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appelbaum E. R., Thompson D. V., Idler K., Chartrain N. Rhizobium japonicum USDA 191 has two nodD genes that differ in primary structure and function. J Bacteriol. 1988 Jan;170(1):12–20. doi: 10.1128/jb.170.1.12-20.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beringer J. E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974 Sep;84(1):188–198. doi: 10.1099/00221287-84-1-188. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Djordjevic M. A., Redmond J. W., Batley M., Rolfe B. G. Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii. EMBO J. 1987 May;6(5):1173–1179. doi: 10.1002/j.1460-2075.1987.tb02351.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Egelhoff T. T., Fisher R. F., Jacobs T. W., Mulligan J. T., Long S. R. Nucleotide sequence of Rhizobium meliloti 1021 nodulation genes: nodD is read divergently from nodABC. DNA. 1985 Jun;4(3):241–248. doi: 10.1089/dna.1985.4.241. [DOI] [PubMed] [Google Scholar]
  6. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  7. Fisher R. F., Egelhoff T. T., Mulligan J. T., Long S. R. Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes. Genes Dev. 1988 Mar;2(3):282–293. doi: 10.1101/gad.2.3.282. [DOI] [PubMed] [Google Scholar]
  8. Fisher R. F., Swanson J. A., Mulligan J. T., Long S. R. Extended Region of Nodulation Genes in Rhizobium meliloti 1021. II. Nucleotide Sequence, Transcription Start Sites and Protein Products. Genetics. 1987 Oct;117(2):191–201. doi: 10.1093/genetics/117.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hong G. F., Burn J. E., Johnston A. W. Evidence that DNA involved in the expression of nodulation (nod) genes in Rhizobium binds to the product of the regulatory gene nodD. Nucleic Acids Res. 1987 Dec 10;15(23):9677–9690. doi: 10.1093/nar/15.23.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honma M. A., Ausubel F. M. Rhizobium meliloti has three functional copies of the nodD symbiotic regulatory gene. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8558–8562. doi: 10.1073/pnas.84.23.8558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horvath B., Bachem C. W., Schell J., Kondorosi A. Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product. EMBO J. 1987 Apr;6(4):841–848. doi: 10.1002/j.1460-2075.1987.tb04829.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  15. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  16. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  17. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  18. Miller V. L., Taylor R. K., Mekalanos J. J. Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein. Cell. 1987 Jan 30;48(2):271–279. doi: 10.1016/0092-8674(87)90430-2. [DOI] [PubMed] [Google Scholar]
  19. Mulligan J. T., Long S. R. Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6609–6613. doi: 10.1073/pnas.82.19.6609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  21. Recourt K., van Brussel A. A., Driessen A. J., Lugtenberg B. J. Accumulation of a nod gene inducer, the flavonoid naringenin, in the cytoplasmic membrane of Rhizobium leguminosarum biovar viciae is caused by the pH-dependent hydrophobicity of naringenin. J Bacteriol. 1989 Aug;171(8):4370–4377. doi: 10.1128/jb.171.8.4370-4377.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rossen L., Shearman C. A., Johnston A. W., Downie J. A. The nodD gene of Rhizobium leguminosarum is autoregulatory and in the presence of plant exudate induces the nodA,B,C genes. EMBO J. 1985 Dec 16;4(13A):3369–3373. doi: 10.1002/j.1460-2075.1985.tb04092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rostas K., Kondorosi E., Horvath B., Simoncsits A., Kondorosi A. Conservation of extended promoter regions of nodulation genes in Rhizobium. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1757–1761. doi: 10.1073/pnas.83.6.1757. [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. Schell M. A., Poser E. F. Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J Bacteriol. 1989 Feb;171(2):837–846. doi: 10.1128/jb.171.2.837-846.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schofield P. R., Watson J. M. DNA sequence of Rhizobium trifolii nodulation genes reveals a reiterated and potentially regulatory sequence preceding nodABC and nodFE. Nucleic Acids Res. 1986 Apr 11;14(7):2891–2903. doi: 10.1093/nar/14.7.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scott K. F. Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia). Nucleic Acids Res. 1986 Apr 11;14(7):2905–2919. doi: 10.1093/nar/14.7.2905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shearman C. A., Rossen L., Johnston A. W., Downie J. A. The Rhizobium leguminosarum nodulation gene nodF encodes a polypeptide similar to acyl-carrier protein and is regulated by nodD plus a factor in pea root exudate. EMBO J. 1986 Apr;5(4):647–652. doi: 10.1002/j.1460-2075.1986.tb04262.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spaink H. P., Okker R. J., Wijffelman C. A., Tak T., Goosen-de Roo L., Pees E., van Brussel A. A., Lugtenberg B. J. Symbiotic properties of rhizobia containing a flavonoid-independent hybrid nodD product. J Bacteriol. 1989 Jul;171(7):4045–4053. doi: 10.1128/jb.171.7.4045-4053.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Van der Meide P. H., Dubbeld M., Schellekens H. Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA. J Immunol Methods. 1985 May 23;79(2):293–305. doi: 10.1016/0022-1759(85)90109-7. [DOI] [PubMed] [Google Scholar]
  32. Wu H. C., Lai J. S., Hayashi S., Giam C. Z. Biogenesis of membrane lipoproteins in Escherichia coli. Biophys J. 1982 Jan;37(1):307–315. doi: 10.1016/S0006-3495(82)84679-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. You I. S., Ghosal D., Gunsalus I. C. Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product. J Bacteriol. 1988 Dec;170(12):5409–5415. doi: 10.1128/jb.170.12.5409-5415.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zaat S. A., Wijffelman C. A., Spaink H. P., van Brussel A. A., Okker R. J., Lugtenberg B. J. Induction of the nodA promoter of Rhizobium leguminosarum Sym plasmid pRL1JI by plant flavanones and flavones. J Bacteriol. 1987 Jan;169(1):198–204. doi: 10.1128/jb.169.1.198-204.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Maagd R. A., Lugtenberg B. Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol. 1986 Sep;167(3):1083–1085. doi: 10.1128/jb.167.3.1083-1085.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES