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
2020-03-28
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 Biol215:403–410
    [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 Microbiol65:4057–4063
    [Google Scholar]
  3. Barrios H., Valderrama B., Morett E.. 1999; Compilation and analysis of σ 54-dependent promoter sequences. Nucleic Acids Res27:4305–4313
    [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. Biodegradation1:19–29
    [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 Bacteriol182:4129–4136
    [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 Microbiol5:335–342
    [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. Microbiology149:1785–1795
    [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 Microbiol64:2710–2715
    [Google Scholar]
  9. Feinberg A. P., Vogelstein B.. 1984; A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem137:266–267
    [Google Scholar]
  10. Felsenstein J.. 1993; phylip (Phylogeny Interface Package), version 3.5c. Seattle: Department of Genetics, University of Washington;
    [Google Scholar]
  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 J12:2901–2912
    [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 Microbiol42:47–59
    [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 Microbiol66:966–975
    [Google Scholar]
  14. Huala E., Stigter J., Ausubel F. M.. 1992; The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol174:1428–1431
    [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 Res7:331–338
    [Google Scholar]
  16. Koch K. A., Pena M. M. O., Thiele D. J.. 1997; Copper-binding motifs in catalysis, transport, detoxification and signaling. Chem Biol4:549–560
    [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 Bacteriol174:4391–4400
    [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 Bacteriol173:1125–1138
    [Google Scholar]
  19. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685
    [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 Bacteriol179:487–495
    [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 Bacteriol183:6074–6084
    [Google Scholar]
  22. Lipscomb J. D.. 1994; Biochemistry of soluble methane monooxygenase. Annu Rev Microbiol48:371–399
    [Google Scholar]
  23. Lund P.. 2001; Microbial molecular chaperones. Adv Microbial Physiology44:93–140
    [Google Scholar]
  24. Martin H., Murrell J. C.. 1995; Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis. FEMS Microbiol Lett127:243–248
    [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 Microbiol63:1898–1904
    [Google Scholar]
  26. Meijer W., Tabita F.. 1992; Isolation and characterisation of the nifUSVW - rpoN gene cluster from Rhodobacter sphaeroides . J Bacteriol174:3855–3566
    [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 Chem277:5858–5865
    [Google Scholar]
  28. Merrick M. J.. 1993; In a class of its own – the RNA polymerase sigma factor σ 54 ( σ N). Mol Microbiol10:903–909
    [Google Scholar]
  29. Merrick M. J., Edwards R. A.. 1995; Nitrogen control in bacteria. Microbiol Rev59:604–622
    [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 Bacteriol180:1729–1740
    [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 Bacteriol175:6067–6074
    [Google Scholar]
  32. Murrell J. C., Dalton H.. 1983a; Nitrogen fixation in obligate methanotrophs. J Gen Microbiol129:3481–3496
    [Google Scholar]
  33. Murrell J. C., Dalton H.. 1983b; Ammonia assimilation in Methylococcus capsulatus (Bath) and other obligate methanotrophs. J Gen Microbiol129:1197–1206
    [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 Chem269:14995–15005
    [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. Microbiology142:1289–1296
    [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 Microbiol25:399–409
    [Google Scholar]
  37. Oakley C. J., Murrell J. C.. 1988; nifH genes in obligate methane oxidizing bacteria. FEMS Microbiol Lett49:53–57
    [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. Nature404:502–506
    [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 Chem270:4822–4839
    [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 Rev65:422–444
    [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 Bacteriol169:2424–2431
    [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;
    [Google Scholar]
  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 . Gene145:69–73
    [Google Scholar]
  44. Segal G., Ron E. Z.. 1996; Regulation and organization of the groE and dnaK operons in eubacteria. FEMS Microbiol Lett138:1–10
    [Google Scholar]
  45. Semrau J. D., Chistoserdov A., Lebron J.. 7 other authors 1995; Particulate methane monooxygenase genes in methanotrophs. J Bacteriol177:3071–3079
    [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 Microbiol65:5198–5206
    [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 Lett1:784–791
    [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 Bacteriol182:3008–3016
    [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). Gene91:27–34
    [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 Lett5:487–492
    [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. Microbiology145:1235–1244
    [Google Scholar]
  52. Stolyar S., Franke M., Lidstrom M. E.. 2001; Expression of individual copies of Methylococcus capsulatus Bath particulate methane monooxygenase genes. J Bacteriol183:1810–1812
    [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 Chem274:11170–11177
    [Google Scholar]
  54. Studholme D. J., Buck M.. 2000; The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiol Lett186:1–9
    [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 Chem137:161–168
    [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 Acta1343:335–348
    [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 Microbiol158:107–114
    [Google Scholar]
  58. Whittenbury R., Phillips K. C., Wilkinson J. F.. 1970; Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol61:205–218
    [Google Scholar]
  59. Zahn J. A., Dispirito A. A.. 1996; Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J Bacteriol178:1018–1029
    [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 Lett55:181–186
    [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...

Most cited this month

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