Isolation of a Methylocystis strain containing a novel pmoA-like gene

Abstract

A type II methanotrophic bacterium (Methylocystis strain SC2) was isolated from a polluted aquifer and identified based on morphology and on 16S rRNA gene phylogeny. Primers targeting the particulate methane monooxygenase subunit A gene (pmoA) were used to obtain a PCR product from DNA extract of strain SC2. Denaturing gradient gel electrophoresis of this PCR product demonstrated that strain SC2 contained two very different pmoA-like genes. One gene (pmoA1) had very high sequence homology to pmoA genes of other type II methanotrophic bacteria (identical amino acid sequence to pmoA of some other Methylocystis strains). The second gene (pmoA2) possessed only 73% identity with the first gene at the nucleotide level and 68.5% identity (83% similarity) at the amino acid level. The presence of both pmoA-like genes was verified by developing specific oligonucleotide probes for each and using these in Southern hybridisation of genomic DNA. Purity of the culture was exhaustively verified with a variety of methods to ensure that both genes were present in a single genospecies. These included microscopic examination, plating on various media, denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene (universal to bacteria) and of the methanol dehydrogenase alpha-subunit gene mxaF (universal to methylotrophic bacteria), and whole-cell hybridisation with fluorescently labelled 16S rRNA-targeted oligonucleotide probes specific for the genera Methylosinus and Methylocystis, or specific for strain SC2. Reverse transcription PCR of extracted RNA suggested that the novel pmoA2 gene was not expressed during growth under standard conditions used for the cultivation of these bacteria. The presence of multiple, diverse pmoA-like genes in a single genospecies of methanotrophic bacteria implies that pmoA must be cautiously applied as a phylogenetic marker in cultivation-independent molecular ecology studies.

Genomic and Transcriptomic Analyses of the Facultative Methanotroph Methylocystis sp. Strain SB2 Grown on Methane or Ethanol

“…strain SC2, and Methylocystis sp. strain Rockwell (ATCC 49242) (45)(46)(47), using the suite of bioinformatic tools of the MicroScope online platform (48), indicates that the TCA cycle and EMC pathway are complete in all of these strains (data not shown). We also investigated the complement of genes that were common to Methylocystis sp.…”

Microbial CH4 and N2O Consumption in Acidic Wetlands

“…Methylocystis species that were frequently detected in acidic wetlands are closely related to the solely known methanotroph strains that are capable of consumption of atmospheric CH 4 , i.e., strains LR1, SC2, and DWT (Dunfield et al, 1999, 2002; Kolb et al, 2005; Baani and Liesack, 2008; Dedysh, 2009; Wieczorek et al, 2011). Nonetheless, pmoA2 genes (that encode for the hydroxylase subunit of a high affinity CH 4 mono-oxygenase) that would indicate the capability of consumption of atmospheric CH 4 (Dunfield et al, 2002; Yimga et al, 2003; Ricke et al, 2004; Baani and Liesack, 2008) have only been detected at low relative abundances (<5%; Wieczorek et al, 2011). To warrant consumption of atmospheric CH 4 in wetlands a small fraction (about 10 7 cells per gram dry weight in forest soils that consume atmospheric CH 4 ; Kolb et al, 2005; Degelmann et al, 2010) of the total cell number of present methanotrophs (about 10 8 cells per gram dry weight) need presumably be active to allow for consumption of atmospheric CH 4 at rates that were observed in acidic wetland sites.…”

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

“…Although the motility of Ms. trichosporium OB3b is well known [104], all Methylocystis spp. studied so far, including strain SC2, are reported to be non-motile [1], [105], [106], [107], [108], [109]. This is due to the absence of flagella and, as expected, no flagella biosynthesis-related genes were detected in strain SC2 (Figure 8).…”