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.