Amplification of Marine Methanotrophic Enrichment DNA with 16S rDNA PCR Primers for Type Ⅱ α Proteobacteria Methanotrophs

摘要

Type Ⅱ α proteobacteria methanotrophs are capable of a wide range of cometa-bolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type Ⅰ γ proteobacteria which do not have this activity to the extent of Type Ⅱ methanotrophs. In previous work in our laboratory, determining the presence of Type Ⅱ α proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type Ⅱ methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type Ⅰ methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type Ⅱ methanotrophs and a high nitrogen/high copper enrichment to select for Type Ⅰ methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type Ⅱ methanotrophs, suggesting the presence of higher numbers of Type Ⅱ methanotrophs. These results provide further evidence for the existence of Type Ⅱ marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.