Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis

Lidstrom, Mary E.; Wopat, Ann E.

doi:10.1007/bf00409767

Cited by 30 publications

(20 citation statements)

References 17 publications

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“…A comprehensive phylogenetic analysis of sMMO and other soluble di-iron monooxygenases (reviewed by Leahy et al, 2003) suggests that these enzymes have been largely spread through horizontal gene transfer, and are not maintained permanently in a bacterial lineage. The identification of a second lone copy of mmoX in Ms. sporium, and the isolation of plasmids from this organism in previous studies (Lidstrom & Wopat, 1984), suggest that sMMO genes in Ms. sporium may have been acquired through horizontal gene transfer. Functional redundancy is common among bacterial genes, and in this case, the identification of a duplicate copy of mmoX may indicate a competitive advantage for Ms. sporium in adapting to local environments, such as peat-bogs, where copper is likely to be limiting for growth.…”

Section: Discussionmentioning

confidence: 74%

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

et al. 2006

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The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a σ 54 promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the α subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO− mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO− mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.

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Section: Discussionmentioning

confidence: 74%

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

et al. 2006

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“…This raised the possibility that one set of genes might be plasmid encoded. The only plasmid in a methanotroph that has been characterized is a 65-kb plasmid that has been isolated from Methylobacter albus BG8 and for which a restriction map has been determined (24). To determine whether one of the hybridizing fragments was part of this Methylobacter albus BG8 plasmid, the plasmid was isolated, digested with HindIII, BstYI, and BamHI, and separated on gels.…”

Section: Purificationmentioning

confidence: 99%

Particulate methane monooxygenase genes in methanotrophs

et al. 1995

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A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45-and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.Methanotrophs are a group of gram-negative bacteria that utilize methane as their sole source of carbon and energy. The initial transformation involves the conversion of methane into methanol by methane monooxygenase (MMO):

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“…Methanotrophs have been shown to contain plasmids of considerable sizes (24), but no function has been ascribed to any of them yet. Earlier surveys have reported plasmids to be present in both type I and type II methanotrophs, ranging in size from 8 to 186 kb (23,24,40). However, no detectable homology was found among the plasmids isolated from different methanotrophs by using DNA-DNA hybridization and restriction pattern analysis (24).…”

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confidence: 99%

Complete Sequence Analysis of Two Methanotroph-Specific repABC -Containing Plasmids from Methylocystis sp. Strain SC2

Dam

Kube

Dam

et al. 2012

Appl Environ Microbiol

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bThe complete nucleotide sequences of two large, low-copy-number plasmids of 229.6 kb (pBSC2-1) and 143.5 kb (pBSC2-2) were determined during assembly of the whole-genome shotgun sequences of the methane-oxidizing bacterium Methylocystis sp. strain SC2. The physical existence of the two plasmids in strain SC2 was confirmed by pulsed-field gel electrophoresis followed by Southern hybridization. Both plasmids have a conserved replication module of the repABC system and carry genes involved in their faithful maintenance and conjugation. In addition, they contain genes that might be involved in essential metabolic processes. These include several heavy metal resistance genes and copper transport genes in pBSC2-1 and a complete nitrous oxide reductase operon and a pmoC singleton in pBSC2-2, the latter encoding the PmoC subunit of particulate methane monooxygenase. Methanotrophic bacteria, or methanotrophs, are able to oxidize the greenhouse gas methane via the enzyme methane monooxygenase (MMO), which is present in the particulate form (pMMO) in most of them (29). Methanotrophs have been shown to contain plasmids of considerable sizes (24), but no function has been ascribed to any of them yet. Earlier surveys have reported plasmids to be present in both type I and type II methanotrophs, ranging in size from 8 to 186 kb (23,24,40). However, no detectable homology was found among the plasmids isolated from different methanotrophs by using DNA-DNA hybridization and restriction pattern analysis (24). Given a genome size of approximately 4 Mb for methanotrophic bacteria, the collective size of the plasmids reported to be present in a single organism would account for approximately 5 to 10% of their total gene content. This calls for sequencing these large plasmids, in order to characterize the genes located on them and eventually understand their putative function in methanotrophs. For a long time, Methylococcus capsulatus Bath was the only methanotrophic bacterium whose complete genome sequence was available (39). However, it was known not to contain any plasmid (24). With the availability of highthroughput sequencing facilities, an increasing number of methanotroph genomes are being sequenced. This includes the type I methanotrophs Methylobacter tundripaludum SV96 (36), Methylomonas methanica MC09 (5), and Methylomicrobium alcaliphilum strain 20Z (38), the type II methanotrophs Methylosinus trichosporium OB3b (33) and Methylocystis sp. strain Rockwell (ATCC 49242) (31), the facultative Methylocella silvestris BL2 (12), and the acidophilic "Candidatus Methylacidiphilum infernorum" V4 (22). However, apart from the very recently announced genome of the type I methanotroph Mm. alcaliphilum strain 20Z, no plasmid sequence has been reported for any of these methanotrophic bacteria.The present study reports the complete sequences of two novel, large plasmids, pBSC2-1 and pBSC2-2, identified in Methylocystis sp. strain SC2. Basic plasmid-related features and the putative functions of genes present in the two plasmids, including...

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Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis

Cited by 30 publications

References 17 publications

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

Particulate methane monooxygenase genes in methanotrophs

Complete Sequence Analysis of Two Methanotroph-Specific repABC -Containing Plasmids from Methylocystis sp. Strain SC2

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