Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

Abstract

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FU nctional Metagenomics sequencing ( TFUM seq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo . Our approach uses metagenomic DNA to construct large‐scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUM seq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co‐evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo . image A platform for mining metagenomic DNA for genes contributing to fitness of commensal bacteria in vivo is presented. TFUM seq (Temporal FU nctional Metagenomics sequencing) uses shotgun libraries cloned into a recipient bacterial species, tracked over time in gnotobiotic mice by deep sequencing and computational methods. TFUM seq highlights the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo . Genes from a donor commensal bacterial species, Bacteroides thetaiotaomicron, confer fitness advantages to E. coli growing in the mouse gut. Analyses of population dynamics of E. coli clones harboring Bacteroides thetaiotaomicron genes reveals a galactokinase central to early colonization of the mouse gut, and subsequent dominance of a glycoside hydrolase enabling sucrose metabolism in E. coli . Co‐evolution of the donor plasmid library and the E. coli genome occurs, driving increased galactose utilization in E. coli .