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A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community
Wang, Yun1; Chen, Yin2; Zhou, Qian3,4; Huang, Shi3,4; Ning, Kang3,4; Xu, Jian3,4; Kalin, Robert M.5; Rolfe, Stephen6; Huang, Wei E.1
Source PublicationPLOS ONE

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 (13)C-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.

; 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 C-13-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 coexisted 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.
Subject Area功能基因组
WOS HeadingsScience & Technology
Indexed BySCI
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000311146900072
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Document Type期刊论文
Affiliation1.Univ Sheffield, Kroto Res Inst, Sheffield, S Yorkshire, England
2.Univ Warwick, Sch Life Sci, Coventry CV4 7AL, W Midlands, England
3.Chinese Acad Sci, Key Lab Biofuels, BioEnergy Genome Ctr, Qingdao, Peoples R China
4.Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, Shandong Key Lab Energy Genet, Qingdao, Peoples R China
5.Univ Strathclyde, David Livingstone Ctr Sustainabil, Glasgow, Lanark, Scotland
6.Univ Sheffield, Dept Anim & Plant Sci, Sheffield S10 2TN, S Yorkshire, England
Recommended Citation
GB/T 7714
Wang, Yun,Chen, Yin,Zhou, Qian,et al. A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community[J]. PLOS ONE,2012,7(10).
APA Wang, Yun.,Chen, Yin.,Zhou, Qian.,Huang, Shi.,Ning, Kang.,...&Huang, Wei E..(2012).A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community.PLOS ONE,7(10).
MLA Wang, Yun,et al."A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community".PLOS ONE 7.10(2012).
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