Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1999 Aug;152(4):1439–1447. doi: 10.1093/genetics/152.4.1439

Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase.

W L Gardner 1, W B Whitman 1
PMCID: PMC1460687  PMID: 10430574

Abstract

A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.

Full Text

The Full Text of this article is available as a PDF (181.2 KB).

Selected References

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

  1. Beneke S., Bestgen H., Klein A. Use of the Escherichia coli uidA gene as a reporter in Methanococcus voltae for the analysis of the regulatory function of the intergenic region between the operons encoding selenium-free hydrogenases. Mol Gen Genet. 1995 Jul 28;248(2):225–228. doi: 10.1007/BF02190804. [DOI] [PubMed] [Google Scholar]
  2. Bock A. K., Schönheit P. Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation. J Bacteriol. 1995 Apr;177(8):2002–2007. doi: 10.1128/jb.177.8.2002-2007.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowen T. L., Union J., Tumbula D. L., Whitman W. B. Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus. Gene. 1997 Mar 25;188(1):77–84. doi: 10.1016/s0378-1119(96)00779-2. [DOI] [PubMed] [Google Scholar]
  4. Cohen-Kupiec R., Blank C., Leigh J. A. Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1316–1320. doi: 10.1073/pnas.94.4.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gernhardt P., Possot O., Foglino M., Sibold L., Klein A. Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol Gen Genet. 1990 Apr;221(2):273–279. doi: 10.1007/BF00261731. [DOI] [PubMed] [Google Scholar]
  6. Jones W. J., Whitman W. B., Fields R. D., Wolfe R. S. Growth and plating efficiency of methanococci on agar media. Appl Environ Microbiol. 1983 Jul;46(1):220–226. doi: 10.1128/aem.46.1.220-226.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Park H. S., Xing R., Whitman W. B. Nonenzymatic acetolactate oxidation to diacetyl by flavin, nicotinamide and quinone coenzymes. Biochim Biophys Acta. 1995 Dec 14;1245(3):366–370. doi: 10.1016/0304-4165(95)00103-4. [DOI] [PubMed] [Google Scholar]
  9. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  10. Shieh J. S., Whitman W. B. Pathway of acetate assimilation in autotrophic and heterotrophic methanococci. J Bacteriol. 1987 Nov;169(11):5327–5329. doi: 10.1128/jb.169.11.5327-5329.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Shieh J., Mesbah M., Whitman W. B. Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine. J Bacteriol. 1988 Sep;170(9):4091–4096. doi: 10.1128/jb.170.9.4091-4096.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tumbula D. L., Bowen T. L., Whitman W. B. Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector. J Bacteriol. 1997 May;179(9):2976–2986. doi: 10.1128/jb.179.9.2976-2986.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Whitman W. B., Sohn S., Kuk S., Xing R. Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp. Appl Environ Microbiol. 1987 Oct;53(10):2373–2378. doi: 10.1128/aem.53.10.2373-2378.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Xing R. Y., Whitman W. B. Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp. J Bacteriol. 1987 Oct;169(10):4486–4492. doi: 10.1128/jb.169.10.4486-4492.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Xing R., Whitman W. B. Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus. J Bacteriol. 1994 Mar;176(5):1207–1213. doi: 10.1128/jb.176.5.1207-1213.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES