Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Cloning, sequencing and mutational analysis

Róbert Csáki, Levente Bodrossy, József Klem, J. Colin Murrell, K. Kovács

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus (Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative transcriptional start sites, containing a σ70- and a σN-dependent motif, were identified in the 5′ region of mmoX. The promoter region of mmoX was mapped using truncated 5′ end regions fused to a promoterless green fluorescent protein gene. A 9.5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (3′) from the last gene of the operon, mmoC, four ORFs were found, mmoG, mmoQ, mmoS and mmoR. mmoG shows significant identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation, two genes, mmoQ and mmoS, showed significant identity to two-component sensor-regulator system genes. Next to mmoS, a gene encoding a putative σN-dependent transcriptional activator, mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate that mmoG and mmoR are essential for the expression of sMMO in Mc. capsulatus (Bath).

Original languageEnglish
Pages (from-to)1785-1795
Number of pages11
JournalMicrobiology
Volume149
Issue number7
Publication statusPublished - Jul 1 2003

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methane monooxygenase
Methylococcus capsulatus
Baths
Organism Cloning
Copper
Genes
Methane
Chaperonins
Bacterial Genes
Enzymes
Regulator Genes
Operon
Multigene Family
Green Fluorescent Proteins
Genetic Promoter Regions
Mutagenesis
Open Reading Frames
Methanol

ASJC Scopus subject areas

  • Microbiology

Cite this

Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath) : Cloning, sequencing and mutational analysis. / Csáki, Róbert; Bodrossy, Levente; Klem, József; Murrell, J. Colin; Kovács, K.

In: Microbiology, Vol. 149, No. 7, 01.07.2003, p. 1785-1795.

Research output: Contribution to journalArticle

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abstract = "The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus (Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative transcriptional start sites, containing a σ70- and a σN-dependent motif, were identified in the 5′ region of mmoX. The promoter region of mmoX was mapped using truncated 5′ end regions fused to a promoterless green fluorescent protein gene. A 9.5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (3′) from the last gene of the operon, mmoC, four ORFs were found, mmoG, mmoQ, mmoS and mmoR. mmoG shows significant identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation, two genes, mmoQ and mmoS, showed significant identity to two-component sensor-regulator system genes. Next to mmoS, a gene encoding a putative σN-dependent transcriptional activator, mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate that mmoG and mmoR are essential for the expression of sMMO in Mc. capsulatus (Bath).",
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