Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism

The metabolism of one-carbon (C-1) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C-1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in C-13-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from C-13-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C-1 compounds in the coastal marine environment.