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Directed culturing of microorganisms using metatranscriptomics.

ABSTRACT: UNLABELLED:The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated- and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism's nutritional preferences and design a culture medium. IMPORTANCE:The number of prokaryotes on the planet has been estimated to exceed 10(30) cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.

SUBMITTER: Bomar L

PROVIDER: S-EPMC3069634 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Directed culturing of microorganisms using metatranscriptomics.

Bomar Lindsey L Maltz Michele M Colston Sophie S Graf Joerg J

mBio 20110405 2

<h4>Unlabelled</h4>The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo ver ...[more]

PMID: 21467263

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Project description:The microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor. IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.

Dataset Information

Directed culturing of microorganisms using metatranscriptomics.

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Directed culturing of microorganisms using metatranscriptomics.

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