1887

Abstract

The growth of AM1 on C compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn transposon mutagenesis procedure was performed to identify genes involved in the growth of AM1 on succinate and pyruvate. Of the 15 000 insertion colonies screened, 71 mutants were found that grew on methanol but either grew slowly or were unable to grow on one or both of the multicarbon substrates. For each of these mutants, the chromosomal region adjacent to the insertion site was sequenced, and 55 different genes were identified and assigned putative functions. These genes fell into a number of predicted categories, including central carbon metabolism, carbohydrate metabolism, regulation, transport and non-essential housekeeping functions. This study focused on genes predicted to encode enzymes of central heterotrophic metabolism: 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase and NADH : ubiquinone oxidoreductase. In each case, the mutants showed normal growth on methanol and impaired growth on pyruvate and succinate, consistent with a role specific to heterotrophic metabolism. For the first two cases, no detectable activity of the corresponding enzyme was found in the mutant, verifying the predictions. The results of this study were used to reconstruct multicarbon metabolism of AM1 during growth on methanol, succinate and pyruvate.

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2003-03-01
2024-04-20
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References

  1. Anthony C. 1982 The Biochemistry of Methylotrophs London: Associated Press;
    [Google Scholar]
  2. Arps P. J, Fulton G. F, Minnich E. C., Lidstrom M. E. 1993; Genetics of serine pathway enzymes in Methylobacterium extorquens AM1: phospho enol pyruvate carboxylase and malyl coenzyme A lyase. J Bacteriol 175:3776–3783
    [Google Scholar]
  3. Attwood M. M., Harder W. 1972; A rapid and specific enrichment procedure for Hyphomicrobium spp. Antonie Van Leeuwenhoek 38:369–377
    [Google Scholar]
  4. Bolbot J. A., Anthony C. 1980; The metabolism of 1,2-propanediol by the facultative methylotroph Pseudomonas AM1. J Gen Microbiol 120:245–254
    [Google Scholar]
  5. Chistoserdov A. Y, Chistoserdova L. V, McIntire W. S., Lidstrom M. E. 1994; The genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176:4052–4065
    [Google Scholar]
  6. Chistoserdova L. 1996; Metabolism of formaldehyde in M. extorquens AM1: molecular genetic analysis and mutant characterization. In Microbial Growth on C1 Compounds pp  16–24 Edited by Lidstrom M. E., Tabita F. R. Dordrecht: Kluwer Academic;
    [Google Scholar]
  7. Chistoserdova L., Lidstrom M. E. 1997; Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1. J Bacteriol 179:4946–4948
    [Google Scholar]
  8. Chun K. T, Edenberg H. J, Kelley M. R., Goebl M. G. 1997; Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast 13:233–240
    [Google Scholar]
  9. D'Argenio D. A, Gallagher L. A, Berg C. A., Manoil C. 2001; Drosophila as a model host for Pseudomonas aeruginosa infection. J Bacteriol 183:1466–1471
    [Google Scholar]
  10. Dupuis A, Darrouzet E, Duborjal H, Pierrard B, Chevallet M, van Belzen R, Albracht S. P., Lunardi J. 1998; Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH-ubiquinone oxidoreductase. Mol Microbiol 28:531–541
    [Google Scholar]
  11. Flint D. H. 1994; Initial kinetic and mechanistic characterization of Escherichia coli fumarase A. Arch Biochem Biophys 311:509–516
    [Google Scholar]
  12. Green L. S, Li Y, Emerich D. W, Bergersen F. J., Day D. A. 2000; Catabolism of α-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum : evidence for an alternative tricarboxylic acid cycle. J Bacteriol 182:2838–2844
    [Google Scholar]
  13. Korotkova N, Chistoserdova L, Kuksa V., Lidstrom M. E. 2002; Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1. J Bacteriol 184:1750–1758
    [Google Scholar]
  14. Lidstrom M. E. 1992; The genetics and molecular biology of methanol-utilizing bacteria. In Methane and Methanol Utilizers pp  183–206 Edited by Murrell J. C., Dalton H. New York: Plenum;
    [Google Scholar]
  15. Marx C. J., Lidstrom M. E. 2001; Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147:2065–2075
    [Google Scholar]
  16. Marx C. J, O'Brien B. N, Breezee J., Lidstrom M. E. 2003; Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase. J Bacteriol 185:669–673
    [Google Scholar]
  17. Miller V. L., Mekalanos J. J. 1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR . J Bacteriol 170:2575–2583
    [Google Scholar]
  18. Saito H., Miura K.-I. 1963; Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
    [Google Scholar]
  19. Salem A. R, Wagner C, Hacking A. J., Quayle J. R. 1973; The metabolism of lactate and pyruvate by Pseudomonas AM1. J Gen Microbiol 76:375–388
    [Google Scholar]
  20. Sambrook J, Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Schreyer R., Bock A. 1980; Phosphoglucose isomerase from Escherichia coli K10: purification, properties and formation under aerobic and anaerobic condition. Arch Microbiol 127:289–298
    [Google Scholar]
  22. Simon R, Priefer U., Puhler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791
    [Google Scholar]
  23. Taylor I. J., Anthony C. 1976; A biochemical basis for obligate methylotrophy: properties of a mutant of Pseudomonas AM1 lacking 2-oxoglutarate dehydrogenase. J Gen Microbiol 93:259–265
    [Google Scholar]
  24. Thissen J, Desai S, McCartney P., Komuniecki R. 1986; Improved purification of the pyruvate dehydrogenase complex from Ascaris suum body wall muscle and characterization of PDHa kinase activity. Mol Biochem Parasitol 21:129–138
    [Google Scholar]
  25. van der Oost J, van Walraven H. S, Bogerd J, Smit A. B, Ewart G. D., Smith G. D. 1989; Nucleotide sequence of the gene proposed to encode the small subunit of the soluble hydrogenase of the thermophilic unicellular cyanobacterium Synechococcus PCC 6716. Nucleic Acids Res 17:10098
    [Google Scholar]
  26. Van Dien S. J., Lidstrom M. E. 2002; Stoichiometric model for evaluating the metabolic capabilities of the facultative methylotroph Methylobacterium extorquens AM1, with application to reconstruction of C3 and C4 metabolism. Biotechnol Bioeng 78:296–312
    [Google Scholar]
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