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Summary The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach insertion-sequencing, or INSeq based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location.
We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals.
In vivo selection transforms this population, revealing functions necessary for survival in the gut: INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Introduction Our indigenous microbial communities play critical roles in shaping myriad features of our biology.
The distal gut hosts the majority of our microbes; these include representatives of all three domains of life, plus their viruses.
Most phylogenetic types phylotypes observed in the guts of humans and other mammals belong to just two bacterial divisions phyla - the Firmicutes and the Bacteroidetes Ley et al.
Microbial community microbiota exchange experiments indicate that gut community members are dynamically selected: Within the Firmicutes and Bacteroidetes, hundreds to thousands of phylotypes partition available niches professions to create a community able to maintain itself in this continuously perfused ecosystem despite shifts in host diet, regular ingestion of foreign bacteria, intense resource competition, high bacteriophage levels, and immune surveillance.
Comparisons of the sequenced genomes of cultured representatives of major gut phylogenetic lineages provide a means for identifying genomic features potentially important for colonization and competition in the gut. However, the recent surge in microbial genome sequencing projects has far outpaced development of broadly applicable tools for directly testing the role of genes in determining fitness in this habitat.
The paucity of tools is unfortunate, as fundamental questions connecting genome content to function remain unexplored.
For example, how are the determinants of fitness related to nutrient availability and how closely do the genes required for fitness in vivo mirror those required for maximizing growth rate in vitro?
Do the major recognized components of the host immune system play a dominant role in determining which genes are critical for symbiont fitness in vivo? To address these questions, we integrated a simple and broadly applicable genetic tool with second-generation DNA sequencers and gnotobiotic mouse models to identify fitness determinants in the genome of a human gut mutualist.
Mariner transposon mutagenesis is an attractive forward genetic strategy for connecting phenotype to gene because stable random insertions can be generated in a recipient genome without specific host factors: After alignment of the inverted repeat IR sequences that delimit mariner family transposons, we noted that a single G—T transversion at a nonconserved position would create a recognition sequence for the Type IIs restriction enzyme, MmeI.
When directed to this location, MmeI would cleave 16 bp outside of the transposon, capturing a genomic fragment that identifies the insertion site. Moreover, if the genome sequence of the recipient organism were known, the short genomic DNA sequences captured by this MmeI digestion would be sufficient to uniquely map transposon location.
We reasoned that in a mixed population of transposon mutants produced in a given recipient bacterial species, the relative abundance of each MmeI-liberated genomic fragment, identified after limited PCR amplification and massively parallel sequencing, would in principle mirror the abundance of each mutant in the population.
Putting this population under potentially selective conditions e.Sep 17, · The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition.
To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location.
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