1887

Abstract

The methanotrophic bacterium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at low copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the operon is initiated from a promoter 5′ of . In this study the genes encoding () and a typical -dependent transcriptional activator () were cloned and sequenced. , a regulatory gene, and , a gene encoding a GroEL homologue, lie 5′ of the structural genes for the sMMO enzyme. Subsequent mutation of and by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of . An mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that plays a role in both nitrogen and carbon metabolism in OB3b. The data also indicate that is transcribed in a - and MmoR-independent manner. Marker-exchange mutagenesis of revealed that MmoG is necessary for gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR . The data presented allow the proposal of a more complete model for copper-mediated regulation of gene expression.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26060-0
2003-07-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/7/mic1491771.html?itemId=/content/journal/micro/10.1099/mic.0.26060-0&mimeType=html&fmt=ahah

References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic Local Alignment Search Tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  2. Arenghi, F. L. G., Pinti, M., Galli, E. & Barbieri, P. ( 1999; ). Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and its cognate promoter. Appl Environ Microbiol 65, 4057–4063.
    [Google Scholar]
  3. Barrios, H., Valderrama, B. & Morett, E. ( 1999; ). Compilation and analysis of σ 54-dependent promoter sequences. Nucleic Acids Res 27, 4305–4313.[CrossRef]
    [Google Scholar]
  4. Brusseau, G. A., Tsien, H.-C., Hanson, R. S. & Wackett, L. P. ( 1990; ). Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity. Biodegradation 1, 19–29.[CrossRef]
    [Google Scholar]
  5. Buck, M., Gallegos, M.-T., Studholme, D. J., Guo, Y. & Gralla, J. D. ( 2000; ). The bacterial enhancer-dependent σ 54 (σ N) transcription factor. J Bacteriol 182, 4129–4136.[CrossRef]
    [Google Scholar]
  6. Cardy, D. L. N., Laidler, V., Salmond, G. P. C. & Murrell, J. C. ( 1991; ). Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b. Mol Microbiol 5, 335–342.[CrossRef]
    [Google Scholar]
  7. Csáki, R., Bodrossy, L., Klem, J., Murrell, J. C. & Kovács, K. ( 2003; ). Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus Bath: cloning, sequencing and mutational analysis. Microbiology 149, 1785–1795.[CrossRef]
    [Google Scholar]
  8. Dennis, J. J. & Zylstra, G. J. ( 1998; ). Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64, 2710–2715.
    [Google Scholar]
  9. Feinberg, A. P. & Vogelstein, B. ( 1984; ). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137, 266–267.[CrossRef]
    [Google Scholar]
  10. Felsenstein, J. ( 1993; ). phylip (Phylogeny Interface Package) version 3.5c. Seattle: Department of Genetics, University of Washington.
  11. Fischer, H. M., Babst, M., Kaspar, T., Acuna, G., Arigoni, F. & Hennecke, H. ( 1993; ). One member of a groESL-like chaperonin multigene family of Bradyrhizobium japonicum is co-regulated with the symbiotic nitrogen fixation genes. EMBO J 12, 2901–2912.
    [Google Scholar]
  12. Garmendia, J., Devos, D., Valencia, A. & de Lorenzo, V. ( 2001; ). A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors. Mol Microbiol 42, 47–59.
    [Google Scholar]
  13. Gilbert, B., McDonald, I. R., Finch, R., Stafford, G. P., Nielsen, A. K. & Murrell, J. C. ( 2000; ). Molecular analysis of the pmo (particulate methane monooxygenase) operons from the two type II methanotrophs. Appl Environ Microbiol 66, 966–975.[CrossRef]
    [Google Scholar]
  14. Huala, E., Stigter, J. & Ausubel, F. M. ( 1992; ). The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol 174, 1428–1431.
    [Google Scholar]
  15. Kaneko, T., Nakamura, Y., Sato, S. & 21 other authors ( 2000; ). Complete genome sequence of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7, 331–338.[CrossRef]
    [Google Scholar]
  16. Koch, K. A., Pena, M. M. O. & Thiele, D. J. ( 1997; ). Copper-binding motifs in catalysis, transport, detoxification and signaling. Chem Biol 4, 549–560.[CrossRef]
    [Google Scholar]
  17. Krüger, N. & Steinbüchel, A. ( 1992; ). Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus HI6. J Bacteriol 174, 4391–4400.
    [Google Scholar]
  18. Kullik, I., Fritsche, S., Knobel, H., Sanjuan, J., Hennecke, H. & Fischer, H. M. ( 1991; ). Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN). J Bacteriol 173, 1125–1138.
    [Google Scholar]
  19. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  20. Lee, W. T., Terlesky, K. C. & Tabita, F. R. ( 1997; ). Cloning and characterisation of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon. J Bacteriol 179, 487–495.
    [Google Scholar]
  21. Lemos, J. A. C., Chen, Y. M. & Burne, R. A. ( 2001; ). Genetic and physiological analysis of the groE operon and role of the HrcA repressor in stress regulation and acid tolerance in Streptococcus mutans. J Bacteriol 183, 6074–6084.[CrossRef]
    [Google Scholar]
  22. Lipscomb, J. D. ( 1994; ). Biochemistry of soluble methane monooxygenase. Annu Rev Microbiol 48, 371–399.[CrossRef]
    [Google Scholar]
  23. Lund, P. ( 2001; ). Microbial molecular chaperones. Adv Microbial Physiology 44, 93–140.
    [Google Scholar]
  24. Martin, H. & Murrell, J. C. ( 1995; ). Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis. FEMS Microbiol Lett 127, 243–248.[CrossRef]
    [Google Scholar]
  25. McDonald, I. R., Uchiyama, H., Kambe, S., Yagi, O. & Murrell, J. C. ( 1997; ). The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M. Appl Environ Microbiol 63, 1898–1904.
    [Google Scholar]
  26. Meijer, W. & Tabita, F. ( 1992; ). Isolation and characterisation of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides. J Bacteriol 174, 3855–3566.
    [Google Scholar]
  27. Merkx, M. & Lippard, S. J. ( 2002; ). Why OrfY? Characterization of mmoD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 277, 5858–5865.[CrossRef]
    [Google Scholar]
  28. Merrick, M. J. ( 1993; ). In a class of its own – the RNA polymerase sigma factor σ 54 (σ N). Mol Microbiol 10, 903–909.[CrossRef]
    [Google Scholar]
  29. Merrick, M. J. & Edwards, R. A. ( 1995; ). Nitrogen control in bacteria. Microbiol Rev 59, 604–622.
    [Google Scholar]
  30. Michiels, J., Van Soom, T., D'Hooghe, I., Dombrecht, B., Benhassine, T., De Wilde, P. & Vanderleyden, J. ( 1998; ). The Rhizobium etli rpoN locus: sequence analysis and phenotypical characterisation of rpoN, ptsN, and ptsA mutants. J Bacteriol 180, 1729–1740.
    [Google Scholar]
  31. Morrett, E. & Segovia, L. ( 1993; ). The σ 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol 175, 6067–6074.
    [Google Scholar]
  32. Murrell, J. C. & Dalton, H. ( 1983a; ). Nitrogen fixation in obligate methanotrophs. J Gen Microbiol 129, 3481–3496.
    [Google Scholar]
  33. Murrell, J. C. & Dalton, H. ( 1983b; ). Ammonia assimilation in Methylococcus capsulatus (Bath) and other obligate methanotrophs. J Gen Microbiol 129, 1197–1206.
    [Google Scholar]
  34. Nguyen, H.-H. T., Shiemke, A. K., Jacobs, S. J., Hales, B. J., Lidstrom, M. E. & Chan, S. I. ( 1994; ). The nature of the copper ions in the membranes containing the particulate methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 269, 14995–15005.
    [Google Scholar]
  35. Nielsen, A. K., Gerdes, K., Degn, H. & Murrell, J. C. ( 1996; ). Regulation of bacterial methane oxidation: transcription of the soluble methane monooxygenase operon of Methylococcus capsulatus (Bath) is repressed by copper ions. Microbiology 142, 1289–1296.[CrossRef]
    [Google Scholar]
  36. Nielsen, A. K., Gerdes, K. & Murrell, J. C. ( 1997; ). Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium. Mol Microbiol 25, 399–409.[CrossRef]
    [Google Scholar]
  37. Oakley, C. J. & Murrell, J. C. ( 1988; ). nifH genes in obligate methane oxidizing bacteria. FEMS Microbiol Lett 49, 53–57.[CrossRef]
    [Google Scholar]
  38. Parkhill, J., Achtman, M., James, K. D. & 25 other authors ( 2000; ). Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404, 502–506.[CrossRef]
    [Google Scholar]
  39. Powell, B. S., Court, D. L., Inada, T. & 7 other authors ( 1995; ). Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. J Biol Chem 270, 4822–4839.[CrossRef]
    [Google Scholar]
  40. Reitzer, L. & Schneider, B. L. ( 2001; ). Metabolic context and possible physiological themes of σ 54 dependent genes in Escherichia coli. Microbiol Mol Biol Rev 65, 422–444.[CrossRef]
    [Google Scholar]
  41. Ronson, C. W., Nixon, B. T., Albright, L. M. & Ausubel, F. M. ( 1987; ). Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol 169, 2424–2431.
    [Google Scholar]
  42. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  43. Schäfer, A., Tauch, A., Jäger, W., Lalinowski, J., Thierbach, G. & Pühler, A. ( 1994; ). Small mobilisable multi-purpose cloning vector derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69–73.[CrossRef]
    [Google Scholar]
  44. Segal, G. & Ron, E. Z. ( 1996; ). Regulation and organization of the groE and dnaK operons in eubacteria. FEMS Microbiol Lett 138, 1–10.[CrossRef]
    [Google Scholar]
  45. Semrau, J. D., Chistoserdov, A., Lebron, J. & 7 other authors (1995; ). Particulate methane monooxygenase genes in methanotrophs. J Bacteriol 177, 3071–3079.
    [Google Scholar]
  46. Shigematsu, T., Handa, S., Eguchi, M., Kamagata, Y., Kanagawa, T. & Kurane, R. ( 1999; ). Soluble methane monooxygenase gene clusters from trichloroethylene-degrading Methylomonas sp. strains and detection of methanotrophs during in situ bioremediation. Appl Environ Microbiol 65, 5198–5206.
    [Google Scholar]
  47. Simon, R., Priefer, U. & Pühler, A. ( 1983; ). A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis on Gram-negative bacteria. Biotechnol Lett 1, 784–791.[CrossRef]
    [Google Scholar]
  48. Skärfstad, E., O'Neill, E., Garmendia, J. & Shingler, V. ( 2000; ). Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling. J Bacteriol 182, 3008–3016.[CrossRef]
    [Google Scholar]
  49. Stainthorpe, A. C., Lees, V., Salmond, G. P. C., Dalton, H. & Murrell, J. C. ( 1990; ). The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath). Gene 91, 27–34.[CrossRef]
    [Google Scholar]
  50. Stanley, S. H., Prior, S. D., Leak, D. J. & Dalton, H. ( 1983; ). Copper stress underlies the fundamental change in intracellular location of methane monooxygenase in methane-oxidizing organisms: studies in batch and continuous cultures. Biotechnol Lett 5, 487–492.[CrossRef]
    [Google Scholar]
  51. Stolyar, S., Costello, A. M., Peeples, T. L. & Lidstrom, M. E. ( 1999; ). Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiology 145, 1235–1244.[CrossRef]
    [Google Scholar]
  52. Stolyar, S., Franke, M. & Lidstrom, M. E. ( 2001; ). Expression of individual copies of Methylococcus capsulatus Bath particulate methane monooxygenase genes. J Bacteriol 183, 1810–1812.[CrossRef]
    [Google Scholar]
  53. Strausak, D., La Fontaine, S., Hill, J., Firth, S. D., Lockhart, P. J. & Mercer, J. F. B. ( 1999; ). The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein. J Biol Chem 274, 11170–11177.[CrossRef]
    [Google Scholar]
  54. Studholme, D. J. & Buck, M. ( 2000; ). The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiol Lett 186, 1–9.[CrossRef]
    [Google Scholar]
  55. Takeguchi, M., Miyakawa, K. & Okura, I. ( 1999; ). The role of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b. J Mol Catal A Chem 137, 161–168.[CrossRef]
    [Google Scholar]
  56. Tanaka, N., Hiyama, T. & Nakamoto, H. ( 1997; ). Cloning, characterization and functional analysis of groESL operon from thermophilic cyanobacterium Synechococcus vulcanus. Biochim Biophys Acta 1343, 335–348.[CrossRef]
    [Google Scholar]
  57. Warrelmann, J., Eitinger, M., Schwartz, E., Rommermann, D. & Friedrich, B. ( 1992; ). Nucleotide sequence of the rpoN (hno) gene region of Alcaligenes eutrophus: evidence for a conserved gene cluster. Arch Microbiol 158, 107–114.[CrossRef]
    [Google Scholar]
  58. Whittenbury, R., Phillips, K. C. & Wilkinson, J. F. ( 1970; ). Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61, 205–218.[CrossRef]
    [Google Scholar]
  59. Zahn, J. A. & Dispirito, A. A. ( 1996; ). Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J Bacteriol 178, 1018–1029.
    [Google Scholar]
  60. Zharkikh, A. & Li, W.-H. ( 1992; ). Statistical properties of bootstrap estimation of phylogenetic variability from nucleotide sequences. I. Four taxa with a molecular clock. FEMS Microbiol Lett 55, 181–186.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26060-0
Loading
/content/journal/micro/10.1099/mic.0.26060-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error