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. 1991 May;173(9):2872–2878. doi: 10.1128/jb.173.9.2872-2878.1991

Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4'-phosphopantetheine prosthetic group.

O Geiger 1, H P Spaink 1, E P Kennedy 1
PMCID: PMC207868  PMID: 2019559

Abstract

Rhizobium species produce a protein product of the nodF gene that has a limited but recognizable homology to the well-characterized acyl carrier protein (ACP) of Escherichia coli. NodF functions together with NodE in generating a host-specific response to the plant host in the interchange of signals leading to the effective nodulation of roots (H.P. Spaink, J. Weinman, M.A. Djordjevic, C.A. Wijffelman, R.J.H. Okker, and B. J.J. Lugtenberg, EMBO J. 8:2811-2818, 1989; B. Scheres, C. van de Wiel, A. Zalensky, B. Horvath, H. Spaink, H. van Eck, F. Zwartkruis, A.M. Wolters, T. Gloudemans, A. van Kammen, and T. Bisseling, Cell 60:281-294, 1990). The nodFE region of Rhizobium leguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive beta-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled beta-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with beta-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.

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Selected References

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

  1. Anderson M. S., Raetz C. R. Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. J Biol Chem. 1987 Apr 15;262(11):5159–5169. [PubMed] [Google Scholar]
  2. Bibb M. J., Biró S., Motamedi H., Collins J. F., Hutchinson C. R. Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis. EMBO J. 1989 Sep;8(9):2727–2736. doi: 10.1002/j.1460-2075.1989.tb08414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brody S., Mikolajczyk S. Neurospora mitochondria contain an acyl-carrier protein. Eur J Biochem. 1988 Apr 15;173(2):353–359. doi: 10.1111/j.1432-1033.1988.tb14005.x. [DOI] [PubMed] [Google Scholar]
  4. Cooper C. L., Hsu L., Jackowski S., Rock C. O. 2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein. J Biol Chem. 1989 May 5;264(13):7384–7389. [PubMed] [Google Scholar]
  5. Cronan J. E., Jr Beta-alanine synthesis in Escherichia coli. J Bacteriol. 1980 Mar;141(3):1291–1297. doi: 10.1128/jb.141.3.1291-1297.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Downie J. A. The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE. Mol Microbiol. 1989 Nov;3(11):1649–1651. doi: 10.1111/j.1365-2958.1989.tb00150.x. [DOI] [PubMed] [Google Scholar]
  9. Feng D. F., Doolittle R. F. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. doi: 10.1007/BF02603120. [DOI] [PubMed] [Google Scholar]
  10. Fischl A. S., Kennedy E. P. Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli. J Bacteriol. 1990 Sep;172(9):5445–5449. doi: 10.1128/jb.172.9.5445-5449.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goethals K., Gao M., Tomekpe K., Van Montagu M., Holsters M. Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: nucleotide sequence and plant-inducible expression. Mol Gen Genet. 1989 Oct;219(1-2):289–298. doi: 10.1007/BF00261190. [DOI] [PubMed] [Google Scholar]
  12. Halverson L. J., Stacey G. Signal exchange in plant-microbe interactions. Microbiol Rev. 1986 Jun;50(2):193–225. doi: 10.1128/mr.50.2.193-225.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hancock W. S., Marshall G. R., Vagelos P. R. Acyl carrier protein. XX. Chemical synthesis and characterization of analogues of acyl carrier protein. J Biol Chem. 1973 Apr 10;248(7):2424–2434. [PubMed] [Google Scholar]
  14. Jackowski S., Rock C. O. Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants. J Bacteriol. 1987 Apr;169(4):1469–1473. doi: 10.1128/jb.169.4.1469-1473.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackowski S., Rock C. O. Ratio of active to inactive forms of acyl carrier protein in Escherichia coli. J Biol Chem. 1983 Dec 25;258(24):15186–15191. [PubMed] [Google Scholar]
  16. Jackowski S., Rock C. O. Regulation of coenzyme A biosynthesis. J Bacteriol. 1981 Dec;148(3):926–932. doi: 10.1128/jb.148.3.926-932.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lakin-Thomas P. L., Brody S. Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein. J Biol Chem. 1986 Apr 15;261(11):4785–4788. [PubMed] [Google Scholar]
  18. Lerouge P., Roche P., Faucher C., Maillet F., Truchet G., Promé J. C., Dénarié J. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature. 1990 Apr 19;344(6268):781–784. doi: 10.1038/344781a0. [DOI] [PubMed] [Google Scholar]
  19. Long S. R. Rhizobium-legume nodulation: life together in the underground. Cell. 1989 Jan 27;56(2):203–214. doi: 10.1016/0092-8674(89)90893-3. [DOI] [PubMed] [Google Scholar]
  20. MAJERUS P. W., ALBERTS A. W., VAGELOS P. R. ACYL CARRIER PROTEIN. IV. THE IDENTIFICATION OF 4'-PHOSPHOPANTETHEINE AS THE PROSTHETIC GROUP OF THE ACYL CARRIER PROTEIN. Proc Natl Acad Sci U S A. 1965 Feb;53:410–417. doi: 10.1073/pnas.53.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  23. Miller K. J., Gore R. S., Benesi A. J. Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol. J Bacteriol. 1988 Oct;170(10):4569–4575. doi: 10.1128/jb.170.10.4569-4575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Platt M. W., Miller K. J., Lane W. S., Kennedy E. P. Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti. J Bacteriol. 1990 Sep;172(9):5440–5444. doi: 10.1128/jb.172.9.5440-5444.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  26. Rosenfeld I. S., D'Agnolo G., Vagelos P. R. Identification and quantitation of acyl thioesters by thin-layer chromatography of hydroxamic acid derivatives. Anal Biochem. 1975 Mar;64(1):221–228. doi: 10.1016/0003-2697(75)90422-4. [DOI] [PubMed] [Google Scholar]
  27. Scheres B., Van De Wiel C., Zalensky A., Horvath B., Spaink H., Van Eck H., Zwartkruis F., Wolters A. M., Gloudemans T., Van Kammen A. The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction. Cell. 1990 Jan 26;60(2):281–294. doi: 10.1016/0092-8674(90)90743-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Sherman D. H., Malpartida F., Bibb M. J., Kieser H. M., Bibb M. J., Hopwood D. A. Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22. EMBO J. 1989 Sep;8(9):2717–2725. doi: 10.1002/j.1460-2075.1989.tb08413.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spaink H. P., Weinman J., Djordjevic M. A., Wijffelman C. A., Okker R. J., Lugtenberg B. J. Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein. EMBO J. 1989 Oct;8(10):2811–2818. doi: 10.1002/j.1460-2075.1989.tb08427.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Surin B. P., Watson J. M., Hamilton W. D., Economou A., Downie J. A. Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum. Mol Microbiol. 1990 Feb;4(2):245–252. doi: 10.1111/j.1365-2958.1990.tb00591.x. [DOI] [PubMed] [Google Scholar]
  34. Therisod H., Kennedy E. P. The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8235–8238. doi: 10.1073/pnas.84.23.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Therisod H., Weissborn A. C., Kennedy E. P. An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7236–7240. doi: 10.1073/pnas.83.19.7236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vanaman T. C., Wakil S. J., Hill R. L. The complete amino acid sequence of the acyl carrier protein of Escherichia coli. J Biol Chem. 1968 Dec 25;243(24):6420–6431. [PubMed] [Google Scholar]
  37. de Maagd R. A., Wijfjes A. H., Spaink H. P., Ruiz-Sainz J. E., Wijffelman C. A., Okker R. J., Lugtenberg B. J. nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein. J Bacteriol. 1989 Dec;171(12):6764–6770. doi: 10.1128/jb.171.12.6764-6770.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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