Wang, Yun, Chen, Yin, Zhou, Qian, Huang, Shi, Ning, Kang, Xu, Jian, Kalin, Robert M., Rolfe, Stephen A. and Huang, Wei E. (2012) A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community. PLoS One, Vol.7 (No.10). e47530. doi:10.1371/journal.pone.0047530 ISSN 1932-6203.

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Abstract

Most microorganisms in nature are uncultured with unknown functionality. Sequence-based metagenomics alone answers ‘who/what are there?’ but not ‘what are they doing and who is doing it and how?’. Function-based metagenomics reveals gene function but is usually limited by the specificity and sensitivity of screening strategies, especially the identification of clones whose functional gene expression has no distinguishable activity or phenotypes. A ‘biosensor-based genetic transducer’ (BGT) technique, which employs a whole-cell biosensor to quantitatively detect expression of inserted genes encoding designated functions, is able to screen for functionality of unknown genes from uncultured microorganisms. In this study, BGT was integrated with Stable isotope probing (SIP)-enabled Metagenomics to form a culture-independent SMB toolbox. The utility of this approach was demonstrated in the discovery of a novel functional gene cluster in naphthalene contaminated groundwater. Specifically, metagenomic sequencing of the 13C-DNA fraction obtained by SIP indicated that an uncultured Acidovorax sp. was the dominant key naphthalene degrader in-situ, although three culturable Pseudomonas sp. degraders were also present in the same groundwater. BGT verified the functionality of a new nag2 operon which co-existed with two other nag and two nah operons for naphthalene biodegradation in the same microbial community. Pyrosequencing analysis showed that the nag2 operon was the key functional operon in naphthalene degradation in-situ, and shared homology with both nag operons in Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2. The SMB toolbox will be useful in providing deep insights into uncultured microorganisms and unravelling their ecological roles in natural environments.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Metagenomics, Microorganisms -- Genetics, Biosensors
Journal or Publication Title:	PLoS One
Publisher:	Public Library of Science
ISSN:	1932-6203
Official Date:	17 October 2012
Dates:	Date Event 17 October 2012 Published
Volume:	Vol.7
Number:	No.10
Page Range:	e47530
DOI:	10.1371/journal.pone.0047530
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)
Date of first compliant deposit:	23 December 2015
Date of first compliant Open Access:	23 December 2015
Funder:	China Scholarship Council (CSC), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)
Grant number:	BRM19108 (BBSRC), BRM19109 (BBSRC)

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