Simultaneous tracking of near-isogenic bacterial strains in syntheticArabidopsismicrobiota by chromosomally-integrated barcodes

Abstract

DNA amplicon-based microbiota profiling currently relies on polymorphisms in microbial marker genes to estimate species diversity and abundance. However, this technique cannot resolve genetic differences among microbial individuals of the same species. We report here the development of modular bacterial tags (MoBacTags) encoding DNA barcodes. These tags facilitate tracking of near-isogenic bacterial commensals in synthetic communities (SynComs), which allow assessment of the contributions of individual bacterial genes to root microbiota establishment inArabidopsis 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 wild-typePseudomonas capeferrumWCS358 and of pqqF and cyoB mutants with known defects in gluconic acid-mediated host immunosuppression and validated reduced root colonization of both mutants in a 15-member synthetic microbiota. We detected a reduced SynCom load on roots in the presence of the WCS358:pqqF mutant, but not WCS358:cyoB, revealing distinctpqqFandcyoBactivities in a community context. Using MoBacTags, we also show that WCS358 pqqF mutant-specific colonization and community establishment is nottrans-complemented by wild-type WCS358. Given that gluconic acid production inP. capeferrumis indirectly abolished in the pqqF mutant by disruption of pyrroloquinoline quinone (PQQ) biosynthesis, we propose that drastic changes in the root-associated community result from depletion of the cofactor PQQ, which might serve as a 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 the plant microbiota.