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A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community.

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 (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.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3474725 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community.

Wang Yun Y Chen Yin Y Zhou Qian Q Huang Shi S Ning Kang K Xu Jian J Kalin Robert M RM Rolfe Stephen S Huang Wei E WE

PloS one 20121017 10

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) techn ...[more]

PMID: 23082176

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Project description:The aim of the present study was to assess the microecosystem of 13 homemade spontaneously fermented wheat sourdoughs from different regions of Greece, through the combined use of culture-dependent (classical approach; clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and identification by PCR species-specific for Lactiplantibacillus plantarum, and sequencing of the 16S-rRNA and 26S-rRNA gene, for Lactic Acid Bacteria (LAB) and yeasts, respectively) and independent approaches [DNA- and RNA-based PCR-Denaturing Gradient Gel Electrophoresis (DGGE)]. The pH and Total Titratable Acidity (TTA) values ranged from 3.64-5.05 and from 0.50-1.59% lactic acid, respectively. Yeast and lactic acid bacteria populations ranged within 4.60-6.32 and 6.28-9.20 log CFU/g, respectively. The yeast: LAB ratio varied from 1:23-1:10,000. A total of 207 bacterial and 195 yeast isolates were obtained and a culture-dependent assessment of their taxonomic affiliation revealed dominance of Lb. plantarum in three sourdoughs, Levilactobacillus brevis in four sourdoughs and co-dominance of these species in two sourdoughs. In addition, Companilactobacillusparalimentarius dominated in two sourdoughs and Fructilactobacillussanfranciscensis and Latilactobacillus sakei in one sourdough each. Lactococcus lactis, Lb. curvatus, Leuconostoc citreum, Ln. mesenteroides and Lb. zymae were also recovered from some samples. Regarding the yeast microbiota, it was dominated by Saccharomyces cerevisiae in 11 sourdoughs and Pichia membranifaciens and P. fermentans in one sourdough each. Wickerhamomyces anomalus and Kazachstania humilis were also recovered from one sample. RNA-based PCR-DGGE provided with nearly identical results with DNA-based one; in only one sample the latter provided an additional band. In general, the limitations of this approach, namely co-migration of amplicons from different species to the same electrophoretic position and multiband profile of specific isolates, greatly reduced resolution capacity, which resulted in only partial verification of the microbial ecology detected by culture-dependent approach in the majority of sourdough samples. Our knowledge regarding the microecosystem of spontaneously fermented Greek wheat-based sourdoughs was expanded, through the study of sourdoughs originating from regions of Greece that were not previously assessed.

Dataset Information

A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community.

Publications

A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community.

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