Genetic Manipulation of Wild Human Gut Bacteroides

ABSTRACT Bacteroides is one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insights into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides species that does not require any alterations to the genetic background or a nutritionally defined culture medium. In this approach, dual antibacterial effectors normally delivered via type VI secretion are targeted to the bacterial periplasm under the control of tightly regulated anhydrotetracycline (aTC)-inducible promoters. Introduction of aTC selects for recombination events producing the desired genetic modification, and the dual effector design allows for broad applicability across strains that may have immunity to one counterselection effector. We demonstrate the utility of this approach across 21 human gut Bacteroides isolates representing diverse species, including strains isolated directly from human donors. We use this system to establish that antimicrobial peptide resistance in Bacteroides vulgatus is determined by the product of a gene that is not included in the genomes of previously genetically tractable members of the human gut microbiome. IMPORTANCE Human gut Bacteroides species exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.

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Complete Genome Sequence of Phascolarctobacterium faecium JCM 30894, a Succinate-Utilizing Bacterium Isolated from Human Feces

Microbiology Resource Announcements ◽

10.1128/mra.01487-18 ◽

2019 ◽

Vol 8 (3) ◽

Cited By ~ 7

Author(s):

Yusuke Ogata ◽

Wataru Suda ◽

Nao Ikeyama ◽

Masahira Hattori ◽

Moriya Ohkuma ◽

...

Keyword(s):

Genome Sequence ◽

Genome Analysis ◽

Gut Microbiome ◽

Complete Genome Sequence ◽

Complete Genome ◽

Human Feces ◽

Human Gut ◽

Content Type ◽

Human Gut Microbiome

Phascolarctobacterium faecium is an anaerobic microbe known as a member of the human gut microbiome. Here, we report the complete genome sequence of Phascolarctobacterium faecium JCM 30894 and the elucidation of the mechanism for utilization of succinate by this bacterium based on the genome analysis.

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Genome Sequence of Christensenella minuta DSM 22607T

Genome Announcements ◽

10.1128/genomea.01451-16 ◽

2017 ◽

Vol 5 (2) ◽

Cited By ~ 7

Author(s):

Bruce A. Rosa ◽

Kymberlie Hallsworth-Pepin ◽

John Martin ◽

Aye Wollam ◽

Makedonka Mitreva

Keyword(s):

Genome Sequence ◽

Gut Microbiome ◽

Bacterial Species ◽

Biological Mechanism ◽

Valuable Resource ◽

Human Gut ◽

Content Type ◽

Human Gut Microbiome

ABSTRACT Obesity influences and is influenced by the human gut microbiome. Here, we present the genome of Christensenella minuta, a highly heritable bacterial species which has been found to be strongly associated with obesity through an unknown biological mechanism. This novel genome provides a valuable resource for future obesity therapeutic studies.

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Developing a Bacteroides System for Function-Based Screening of DNA from the Human Gut Microbiome

mSystems ◽

10.1128/msystems.00195-17 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 3

Author(s):

Kathy N. Lam ◽

Eric C. Martens ◽

Trevor C. Charles

Keyword(s):

Gene Function ◽

Gut Microbiome ◽

Environmental Dna ◽

Functional Metagenomics ◽

Human Gut ◽

Metagenomic Dna ◽

Content Type ◽

Human Gut Microbiome ◽

Surrogate Host ◽

Functional Screens

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.

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Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains

Applied and Environmental Microbiology ◽

10.1128/aem.02117-19 ◽

2019 ◽

Vol 86 (3) ◽

Cited By ~ 2

Author(s):

Nina Kirmiz ◽

Kadir Galindo ◽

Karissa L. Cross ◽

Estefani Luna ◽

Nicholas Rhoades ◽

...

Keyword(s):

Gut Microbiome ◽

Species Level ◽

Vitamin B ◽

Human Gut ◽

Content Type ◽

Human Host ◽

Akkermansia Muciniphila ◽

Human Gut Microbiome ◽

Physiological Diversity ◽

Bacterial Lineage

ABSTRACT Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a “next-generation probiotic.” However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome. IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila. However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12. The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.

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