Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1997 Aug;146(4):1211–1220. doi: 10.1093/genetics/146.4.1211

Megaplasmid and Chromosomal Loci for the Phb Degradation Pathway in Rhizobium (Sinorhizobium) Meliloti

T C Charles 1, C Cai 1, P Aneja 1
PMCID: PMC1208069  PMID: 9258668

Abstract

Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutyrate (PHB) degradation pathway in Rhizobium meliloti were identified. A clone that restores the ability of certain R. meliloti mutants with defined deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or acetoacetate as the sole carbon source was isolated from a cosmid library of R. meliloti genomic DNA. Tn5 insertion mutagenesis, followed by merodiploid complementation analysis, demonstrated that the locus consists of at least four transcriptional units, bhbA-D. We also identified loci involved in 3-hydroxybutyrate and/or acetoacetate utilization by screening for mutants that had lost the ability to use 3-hydroxybutyrate as the sole carbon source while retaining the ability to use acetate (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway). These mutants fell into four classes, as determined by replicon mobilization experiments and genetic linkage in phage transduction; one class corresponded to the bhb locus on pRmeSU47b, two classes mapped to different regions on the chromosome and the fourth, bdhA, represented by a single mutant, mapped to another pRmeSU47b locus, near bacA. The bdhA mutant is deficient in 3-hydroxybutrate dehydrogenase activity.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Anderson A. J., Dawes E. A. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990 Dec;54(4):450–472. doi: 10.1128/mr.54.4.450-472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bardin S., Dan S., Osteras M., Finan T. M. A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti. J Bacteriol. 1996 Aug;178(15):4540–4547. doi: 10.1128/jb.178.15.4540-4547.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergmeyer H. U., Gawehn K., Klotzsch H., Krebs H. A., Williamson D. H. Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Biochem J. 1967 Feb;102(2):423–431. doi: 10.1042/bj1020423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Briese B. H., Jendrossek D., Schlegel H. G. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by aerobic sewage sludge. FEMS Microbiol Lett. 1994 Mar 15;117(1):107–111. doi: 10.1111/j.1574-6968.1994.tb06750.x. [DOI] [PubMed] [Google Scholar]
  6. Cevallos M. A., Encarnación S., Leija A., Mora Y., Mora J. Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. J Bacteriol. 1996 Mar;178(6):1646–1654. doi: 10.1128/jb.178.6.1646-1654.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charles T. C., Finan T. M. Analysis of a 1600-kilobase Rhizobium meliloti megaplasmid using defined deletions generated in vivo. Genetics. 1991 Jan;127(1):5–20. doi: 10.1093/genetics/127.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charles T. C., Finan T. M. Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b. J Bacteriol. 1990 May;172(5):2469–2476. doi: 10.1128/jb.172.5.2469-2476.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charles T. C., Newcomb W., Finan T. M. ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium meliloti, is required for normal nodule development. J Bacteriol. 1991 Jul;173(13):3981–3992. doi: 10.1128/jb.173.13.3981-3992.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Churchill P., Hempel J., Romovacek H., Zhang W. W., Brennan M., Churchill S. Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase. Biochemistry. 1992 Apr 21;31(15):3793–3799. doi: 10.1021/bi00130a009. [DOI] [PubMed] [Google Scholar]
  11. Engelke T., Jording D., Kapp D., Pühler A. Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier. J Bacteriol. 1989 Oct;171(10):5551–5560. doi: 10.1128/jb.171.10.5551-5560.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finan T. M. Genetic and physical analyses of group E exo- mutants of Rhizobium meliloti. J Bacteriol. 1988 Jan;170(1):474–477. doi: 10.1128/jb.170.1.474-477.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finan T. M., Hartweig E., LeMieux K., Bergman K., Walker G. C., Signer E. R. General transduction in Rhizobium meliloti. J Bacteriol. 1984 Jul;159(1):120–124. doi: 10.1128/jb.159.1.120-124.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gage D. J., Bobo T., Long S. R. Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa). J Bacteriol. 1996 Dec;178(24):7159–7166. doi: 10.1128/jb.178.24.7159-7166.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirsch A. M., Bang M., Ausubel F. M. Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):367–380. doi: 10.1128/jb.155.1.367-380.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirsch A. M., Long S. R., Bang M., Haskins N., Ausubel F. M. Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meliloti. J Bacteriol. 1982 Jul;151(1):411–419. doi: 10.1128/jb.151.1.411-419.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huisman G. W., Wonink E., Meima R., Kazemier B., Terpstra P., Witholt B. Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J Biol Chem. 1991 Feb 5;266(4):2191–2198. [PubMed] [Google Scholar]
  20. Jenkins L. S., Nunn W. D. Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system. J Bacteriol. 1987 Jan;169(1):42–52. doi: 10.1128/jb.169.1.42-52.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pauli G., Overath P. ato Operon: a highly inducible system for acetoacetate and butyrate degradation in Escherichia coli. Eur J Biochem. 1972 Sep 25;29(3):553–562. doi: 10.1111/j.1432-1033.1972.tb02021.x. [DOI] [PubMed] [Google Scholar]
  22. Peoples O. P., Masamune S., Walsh C. T., Sinskey A. J. Biosynthetic thiolase from Zoogloea ramigera. III. Isolation and characterization of the structural gene. J Biol Chem. 1987 Jan 5;262(1):97–102. [PubMed] [Google Scholar]
  23. Peoples O. P., Sinskey A. J. Fine structural analysis of the Zoogloea ramigera phbA-phbB locus encoding beta-ketothiolase and acetoacetyl-CoA reductase: nucleotide sequence of phbB. Mol Microbiol. 1989 Mar;3(3):349–357. doi: 10.1111/j.1365-2958.1989.tb00180.x. [DOI] [PubMed] [Google Scholar]
  24. Peoples O. P., Sinskey A. J. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J Biol Chem. 1989 Sep 15;264(26):15298–15303. [PubMed] [Google Scholar]
  25. Peoples O. P., Sinskey A. J. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem. 1989 Sep 15;264(26):15293–15297. [PubMed] [Google Scholar]
  26. Schubert P., Steinbüchel A., Schlegel H. G. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol. 1988 Dec;170(12):5837–5847. doi: 10.1128/jb.170.12.5837-5847.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Selander R. K., Caugant D. A., Ochman H., Musser J. M., Gilmour M. N., Whittam T. S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol. 1986 May;51(5):873–884. doi: 10.1128/aem.51.5.873-884.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Senior P. J., Dawes E. A. The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J. 1973 May;134(1):225–238. doi: 10.1042/bj1340225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Slater S. C., Voige W. H., Dennis D. E. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. J Bacteriol. 1988 Oct;170(10):4431–4436. doi: 10.1128/jb.170.10.4431-4436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tombolini R., Povolo S., Buson A., Squartini A., Nuti M. P. Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41. Microbiology. 1995 Oct;141(Pt 10):2553–2559. doi: 10.1099/13500872-141-10-2553. [DOI] [PubMed] [Google Scholar]
  31. Watson R. J., Chan Y. K., Wheatcroft R., Yang A. F., Han S. H. Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid. J Bacteriol. 1988 Feb;170(2):927–934. doi: 10.1128/jb.170.2.927-934.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES