title: Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics.
abstract: 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 (<or= 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.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Josh D
authors.initials: JD
authors.lastname: Neufeld
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: Marc G
authors.initials: MG
authors.lastname: Dumont
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D001419:Bacteria / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D023281:Genomics; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)