Identifying Genetic Determinants Needed to Establish a Human Gut Symbiont in Its Habitat

Abstract

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: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems.

Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut

“…cellulosilyticus WH2 was represented in the 15-member artificial community by a library of 93,458 isogenic mutants, with each mutant strain containing a single randomly inserted modified mariner transposon (Tn) (91.5% of predicted ORFs had insertions covering the first 80% of each gene with an average of 13.9 insertions per ORF). Because the modified Tn had engineered recognition sites for the type II restriction endonuclease MmeI at its ends, 16 bp of flanking chromosomal DNA could be excised together with the Tn after MmeI digestion of community DNA and sequenced (20). This makes it possible to use high throughput sequencing to define the precise location and abundance of each transposon mutant in the library (SI Methods).…”

“…This makes it possible to use high throughput sequencing to define the precise location and abundance of each transposon mutant in the library (SI Methods). Comparing the number of reads for each mutant in an "output" population after a given selection to the number of reads generated from an "input" population provides information about the effect each transposon insertion had on the fitness of the organism under the selection condition applied (20,21).…”

Exploring host–microbiota interactions in animal models and humans

“…The ASF consists of eight species: Five are from the genera Clostridium, Eubacterium, and Bacteroides; one is a spirochete from the Flexistipes group (Mucispirillum schaederli); and two are Lactobacillus species. The laboratory of Jeffrey Gordon (Goodman et al 2009;Faith et al 2011;McNulty et al 2011) has employed consortia of 10-15 human gut bacteria in several studies and used these communities to predict and test the response of the microbiota to diets and to unravel factors involved in model bacterial symbiont fitness in vivo (Goodman et al 2009;Faith et al 2011;McNulty et al 2011). The laboratory of Kenya Honda (Atarashi et al 2011) has used chloroform treatment of intestinal contents, which enriches for spore formers, as inocula into GF recipients as well as a cocktail of Clostridia strains.…”

“…While a pré cis of the discoveries stemming from monoassociation experiments of mice is beyond the scope of this review, a few technical innovations that are adding to the insight that can be gained from monoassociations are worth noting. Recently, Goodman et al (2009) employed insertion sequencing along with genome-wide transposon mutant libraries of B. thetaiotaomicron and used wildtype, mutant GF, and defined community mice to identify the genetic determinants of B. thetaiotaomicron fitness in the gut. Such approaches hold tremendous promise for unraveling the contributions of both host and microbiota genetics for coadaptation .…”

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

“…107 A similar massively parallel sequencing approach called INSeq (insertion sequencing) was developed independently to analyze transposon insertion sites in the gut commensal organism Bacteroides thetaiotaomicron. 108 The authors utilized this approach to analyze genes required for colonization of conventional and germ-free or mono-colonized gnotobiotic mice. The work revealed how colonization by Bacteroides is influenced by existing populations in the gut and competition for key nutrients in this environment.…”