Dumont, Marc G., Radajewski, Stefan M., Miguez, Carlos B., McDonald, Ian R. and Murrell, J. C. (J. Colin). (2006) Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. ENVIRONMENTAL MICROBIOLOGY, 8 (7). pp. 1240-1250. ISSN 1462-2912

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Abstract

Stable isotope probing (SIP) allows the isolation of nucleic acids from targeted metabolically active organisms in environmental samples. In previous studies, DNA-SIP has been performed with the one-carbon growth substrates methane and methanol to study methylotrophic organisms. The methylotrophs that incorporated the labelled substrate were identified with polymerase chain reaction and sequencing of 16S rRNA and 'functional genes' for methanotrophs (mxaF, pmoA, mmoX). In this study, a SIP experiment was performed using a forest soil sample incubated with (CH4)-C-13, and the C-13-DNA was purified and cloned into a bacterial artificial chromosome (BAC) plasmid. A library of 2300 clones was generated and most of the clones contained inserts between 10 and 30 kb. The library was probed for key methylotrophy genes and a 15.2 kb clone containing a pmoCAB operon, encoding particulate methane monooxygenase, was identified and sequenced. Analysis of the pmoA sequence suggested that the clone was most similar to that of a Methylocystis sp. previously detected in this forest soil. Twelve other open reading frames were identified on the clone, including the gene encoding beta-ribofuranosylaminobenzene 5'-phosphate synthase, which is involved in the biosynthesis of the 'archaeal' C-1-carrier, tetrahydromethanopterin, which is also found in methylotrophs. This study demonstrates that relatively large DNA fragments from uncultivated organisms can be readily isolated using DNA-SIP, and cloned into a vector for metagenomic analysis.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Journal or Publication Title:	ENVIRONMENTAL MICROBIOLOGY
Publisher:	BLACKWELL PUBLISHING
ISSN:	1462-2912
Date:	July 2006
Volume:	8
Number:	7
Number of Pages:	11
Page Range:	pp. 1240-1250
Identification Number:	10.1111/j.1462-2920.2006.01018.x
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/33478

Data sourced from Thomson Reuters' Web of Knowledge

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