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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
2012-10-17
发表期刊PLOS ONE
卷号7期号:10
摘要

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.
文章类型Article
学科领域功能基因组
WOS标题词Science & Technology
DOI10.1371/journal.pone.0047530
关键词[WOS]CATABOLIC NAG GENES ; SP STRAIN U2 ; RIBOSOMAL-RNA ; NAPHTHALENE-DEGRADATION ; SYSTEMS MICROBIOLOGY ; NUCLEOTIDE-SEQUENCE ; METAGENOMICS ; DNA ; CELL ; METABOLISM
收录类别SCI
语种英语
WOS研究方向Science & Technology - Other Topics
WOS类目Multidisciplinary Sciences
WOS记录号WOS:000311146900072
引用统计
文献类型期刊论文
条目标识符http://ir.qibebt.ac.cn/handle/337004/1418
专题单细胞中心组群
作者单位1.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
推荐引用方式
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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