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...