Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics.
In: Environmental Microbiology, Jg. 10 (2008-06-01), Heft 6, S. 1526-1535
Online
academicJournal
Zugriff:
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. [ABSTRACT FROM AUTHOR]
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Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics.
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Autor/in / Beteiligte Person: | Neufeld, Josh D. ; Chen, Yin ; Dumont, Marc G. ; Murrell, J. Colin |
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Zeitschrift: | Environmental Microbiology, Jg. 10 (2008-06-01), Heft 6, S. 1526-1535 |
Veröffentlichung: | 2008 |
Medientyp: | academicJournal |
ISSN: | 1462-2912 (print) |
DOI: | 10.1111/j.1462-2920.2008.01568.x |
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