Abstract
Summary The concentrations of one‐carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally‐relevant substrate concentrations in stable‐isotope probing (SIP) incubations to yield sufficient DNA for large‐insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface‐water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled 13 C‐DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high‐molecular‐weight DNA (≤ 40 kb) and cloned to produce a fosmid library of > 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga ‐like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9‐kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure–function studies of microbial communities to achieve the long‐desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.