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

Abstract

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...

Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2

“…In addition, three monocistronic pmoC paralogs were identified, with one present on plasmid pBSC2-2 [50]. As expected for an alphaproteobacterial methanotroph, we could identify the genes involved in the serine pathway of formaldehyde assimilation, but not those involved in the RuMP pathway.…”

“…The genome of strain SC2 totals 4,146,594 bp and consists of three replicons: a circular chromosome of 3,773,444 bp (Figure 1) and two plasmids of 229,614 (pBSC2-1) and 143,536 bp (pBSC2-2), with an average GC content of 63, 61 and 60%, respectively [20], [50].…”

“…Additional toxin-antitoxin systems encoded on the chromosome include two mazEF systems (BN69_0515, BN69_0516; and BN69_2525, BN69_2526) and one yoeB – yefM system (BN69_3397, BN69_3398). A relBE toxin-antitoxin system was identified in the plasmid pBSC2-1 [50]. All toxin-antitoxin systems encode toxins that target diverse cellular functions like DNA replication, mRNA stability, protein synthesis, cell wall biosynthesis, and ATP synthesis [54].…”

Complete Genome Sequence of Methylocystis sp. Strain SC2, an Aerobic Methanotroph with High-Affinity Methane Oxidation Potential

“…While HAO oxidizes hydroxylamine to nitrite, HCP detoxifies hydroxylamine by reducing it to ammonia. Genes encoding nitric oxide reductase and nitrous oxide reductase are present in the plasmids (5). The genome sequence of Methylocystis sp.…”

Spatial Distribution of Microbial Communities in the Alluvial Aquifer along the Oyodo River, Miyakonojo Basin, Japan

“…Methylocystis sp. SC2 could involve in expression of two particulate methane monooxygenases [17], but also encode the hydroxylamine oxidoreductase, hydroxylamine reductase, nitric oxide reductase and nitrous oxide reductase [18]. S. hydrogenivorans sk43H could oxidize thiosulfate, elemental sulfur or hydrogen as energy sources for autotrophic growth, or assimilate acetate, benzoate, pyruvate or succinate for heterotrophic growth utilizing nitrate as an electron acceptor [19].…”