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Metagenomics has rapidly advanced our inventory and appreciation of the genetic potential inherent to the gut microbiome. However it is widely accepted that two key constraints to further genetic dissection of the gut microbiota and host-microbe interactions have been our inability to recover new isolates from the human gut, and the paucity of genetically tractable gut microbes. To address this challenge we developed a modular RP4 mobilisable recombinant vector system and an approach termed metaparental mating to support the rapid and directed isolation of genetically tractable fastidious gut bacteria. Using this approach we isolated transconjugants affiliated with  Clostridium  cluster IV ( Faecalibacterium  and  Oscillibacter  spp.),  Clostridium  cluster XI ( Anaerococcus ) and  Clostridium  XIVa ( Blautia  spp.) and group 2 ruminococci amongst others, and demonstrated that the recombinant vectors were stably maintained in their recipient hosts. By a similar approach we constructed fluorescently labelled bacterial transconjugants affiliated with  Clostridium  cluster IV (including  Flavonifractor  and  Pseudoflavonifractor  spp.),  Clostridium  XIVa ( Blautia  spp.) and  Clostridium  cluster XVIII ( Clostridium ramosum ) that expressed a flavin mononucleotide-based reporter gene ( evoglow-C-Bs2 ). Our approach will advance the integration of bacterial genetics with metagenomics and realize new directions to support a more mechanistic dissection of host-microbe associations relevant to human health and disease.

Isolation of Genetically Tractable Most-Wanted Bacteria by Metaparental Mating