Genetic manipulation of wild human gut Bacteroides.
Parole chiave
Astratto
Bacteroides are one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insight into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides that does not require any alterations to the genetic background or nutritionally defined culture medium. In this approach, dual antibacterial effectors normally delivered via Type VI secretion are targeted to the bacterial periplasm under the control of tightly regulated anhydrotetracycline (aTC)-inducible promoters. Introduction of aTC selects for recombination events producing the desired genetic modification, and the dual-effector design allows for broad applicability across strains that may have immunity to one counterselection effector. We demonstrate the utility of this approach across 21 human gut Bacteroides isolates representing diverse species, including strains isolated directly from human donors. We use this system to establish that antimicrobial peptide resistance in Bacteroides vulgatus is determined by the product of a gene that is not encoded in the genomes of previously genetically tractable members of the human gut microbiome.Importance Human gut Bacteroides exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined media formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of wildtype human gut Bacteroides from diverse species. This system enables genetic dissection of members of human and animal microbiomes outside of existing model organisms.