Neufeld, Josh D., Chen, Yin, Dumont, Marc G. and Murrell, J. C. (J. Colin). (2008) Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environmental Microbiology, Vol.10 (No.6). pp. 1526-1535. ISSN 14622912

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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 C-13-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.

Item Type:	Journal Article
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)
Library of Congress Subject Headings (LCSH):	Methylotrophic microorganisms, Marine microbiology, Stable isotope tracers, Metagenomics, Gene amplification, Methylotrophic bacteria, Polymerase chain reaction
Journal or Publication Title:	Environmental Microbiology
Publisher:	Wiley-Blackwell Publishing Ltd.
ISSN:	14622912
Date:	June 2008
Volume:	Vol.10
Number:	No.6
Number of Pages:	10
Page Range:	pp. 1526-1535
Identification Number:	10.1111/j.1462-2920.2008.01568.x
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Natural Environment Research Council (Great Britain) (NERC), Natural Sciences and Engineering Research Council Canada (NSERC)
Grant number:	NE/C001 923/1 (NERC)
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URI:	http://wrap.warwick.ac.uk/id/eprint/30078

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