Molecular ecology of facultative methanotrophs

Abstract

Methylocella spp. are facultative methanotrophs, able to grow not only on one carbon molecules e.g., methane or methanol, but also on multi-carbon compounds e.g., acetate, succinate and malate. PCR-based molecular techniques were developed and validated to target the 16S rRNA genes of all the species of the genus Methylocella present in environmental samples. DNA extracted from a variety of environmental samples screened by PCR to determine the environmental distribution of Methylocella spp. Methylocella were found to be widely distributed, not only in acidic environments, but also in neutral and alkaline environments. New primers targeting the mmoX gene of Methylocella spp. that encoded the α-subunit of methane monooxygenases were designed. A SYBR® green-based real-time quantitative PCR assay was developed and validated using these Methylocella mmoX gene-targeting primers. The abundance of Methylocella spp. present in selected environmental samples was quantified using the newly developed real-time quantitative PCR assay A series of 13CH4 DNA-SIP experiments were carried either in the presence or absence of 12C acetate in microcosms containing Moor House peat soil to investigate the effect of acetate (0.5 mM) on the ability of Methylocella silvestris to oxidize methane. Methane oxidation data indicated that acetate repressed the ability of Methylocella silvestris to oxidize methane in peat soil microcosms. 13CH4 DNA-SIP experiments revealed that in presence of acetate, Methylocella silvestris did not utilize methane as a carbon source, suggesting that acetate might be the alternative source of carbon. However, when 13C-labelled acetate DNA-SIP experiments were carried out, Methylocella spp. were not found to be dominant acetate utilizers in the peat soil microcosms. Methylocella spp. seem to have been outcompeted by more efficient acetate utilizers such as Brevundimonas and Burkholderia. To identify genes that might be involved in the utilization of methane or acetate, Methylocella silvestris whole genome transcriptomics experiments were carried out. All the genes of the sMMO gene cluster were found to be highly upregulated during growth on methane. In addition to the sMMO gene cluster, a gene encoding Fur was also found to be highly upregulated during growth on methane. During growth on acetate, a gene cluster encoding glycine dehydrogenase was found to be highly upregulated. Microarray experiments carried out here provided potential candidate genes for further characterization by gene knockout based studies. Further work is also required to validate the microarray findings. A study was carried out on forest soils derived from Swedish islands that were at different successional stages. All islands were found to be positive for the presence of Methylocella spp., and identity of composition of other methanotrophs were determined using a pmoA-diagnostic microarray. All the islands were dominated by Methylococcus, Methylosinus and methanotrophs of the uncultivated RA14 clade. The diversity of methanotrophs was higher in late successional islands compared to mid and early successional islands. In addition the diversity of methanotrophs decreased as the soil depth increased