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Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics
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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. doi:10.1111/j.1462-2920.2008.01568.x ISSN 14622912.
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Official URL: http://dx.doi.org/10.1111/j.1462-2920.2008.01568.x
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 | ||||
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| Subjects: | Q Science > QP Physiology Q Science > QR Microbiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > 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 | ||||
| Official Date: | June 2008 | ||||
| Dates: |
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| Volume: | Vol.10 | ||||
| Number: | No.6 | ||||
| Number of Pages: | 10 | ||||
| Page Range: | pp. 1526-1535 | ||||
| DOI: | 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) |
Data sourced from Thomson Reuters' Web of Knowledge
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