Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome

ABSTRACT Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DN...

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Autores principales:	Kathy N. Lam, Eric C. Martens, Trevor C. Charles
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Bacteroides thetaiotaomicron anaerobic sulfatase maturating enzyme chondroitin sulfate utilization fosmid library functional metagenomics functional screening Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0aa5845cdd2c4fda90d1e92b24b1fe2b
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spelling	oai:doaj.org-article:0aa5845cdd2c4fda90d1e92b24b1fe2b2021-12-02T19:46:17ZDeveloping a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome10.1128/mSystems.00195-172379-5077https://doaj.org/article/0aa5845cdd2c4fda90d1e92b24b1fe2b2018-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00195-17https://doaj.org/toc/2379-5077ABSTRACT Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be introduced into the gut microbe Bacteroides thetaiotaomicron to identify genes based on activity screening. Our results support the continuing development of genetically tractable systems to obtain information about gene function.Kathy N. LamEric C. MartensTrevor C. CharlesAmerican Society for MicrobiologyarticleBacteroides thetaiotaomicronanaerobic sulfatase maturating enzymechondroitin sulfate utilizationfosmid libraryfunctional metagenomicsfunctional screeningMicrobiologyQR1-502ENmSystems, Vol 3, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Bacteroides thetaiotaomicron anaerobic sulfatase maturating enzyme chondroitin sulfate utilization fosmid library functional metagenomics functional screening Microbiology QR1-502
spellingShingle	Bacteroides thetaiotaomicron anaerobic sulfatase maturating enzyme chondroitin sulfate utilization fosmid library functional metagenomics functional screening Microbiology QR1-502 Kathy N. Lam Eric C. Martens Trevor C. Charles Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
description	ABSTRACT Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be introduced into the gut microbe Bacteroides thetaiotaomicron to identify genes based on activity screening. Our results support the continuing development of genetically tractable systems to obtain information about gene function.
format	article
author	Kathy N. Lam Eric C. Martens Trevor C. Charles
author_facet	Kathy N. Lam Eric C. Martens Trevor C. Charles
author_sort	Kathy N. Lam
title	Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
title_short	Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
title_full	Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
title_fullStr	Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
title_full_unstemmed	Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome
title_sort	developing a <italic toggle="yes">bacteroides</italic> system for function-based screening of dna from the human gut microbiome
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/0aa5845cdd2c4fda90d1e92b24b1fe2b
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Developing a <italic toggle="yes">Bacteroides</italic> System for Function-Based Screening of DNA from the Human Gut Microbiome

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