DNA barcode MoBacTag NGS microbiota community profilling

Current DNA amplicon-based microbiota profiling relies on polymorphisms in microbial marker genes to estimate species diversity and abundance, but cannot resolve genetic differences among microbial individuals of the same species. We report the development of modular bacterial tags (MoBacTags) encoding DNA barcodes that facilitate tracking of near-isogenic bacterial commensals in natural or synthetic communities (SynComs) to assess the contributions of individual bacterial genes to root microbiota establishment in Arabidopsis thaliana. Chromosomally-integrated DNA barcodes are co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We generated MoBacTag-labeled strains of each wild-type Pseudomonas capeferrum WCS358 and pqqF and cyoB mutants with known defects in gluconic acid-mediated host immunosuppression and validated reduced root colonization of both mutants in a 14-member synthetic microbiota. We detected an unsuspected reduced SynCom load on roots in the presence of the WCS358:pqqF mutant, but not WCS358:cyoB, which cannot be trans-complemented by co-occurring wild-type P. capeferrum. Given that gluconic acid production in P. capeferrum is indirectly abolished in the pqqF mutant by disruption of PQQ biosynthesis, we propose that drastic changes in the root-associated community result from depletion of the cofactor PQQ, which might serve as common good during root microbiota establishment. Our proof-of-principle experiments illustrate how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in microbe-microbe and microbe-host interactions.