title: Isolation of Genetically Tractable Most-Wanted Bacteria by Metaparental Mating.
abstract: 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.
authors: CSIRO Preventative Health Flagship Research Program, Queensland, Australia.|The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Queensland, Australia.
authors: Páraic Ó
authors: PÓ
authors: Cuív
authors: CSIRO Preventative Health Flagship Research Program, Queensland, Australia.
authors: Wendy J
authors: WJ
authors: Smith
authors: The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Queensland, Australia.
authors: Sian
authors: S
authors: Pottenger
authors: The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Queensland, Australia.
authors: Sriti
authors: S
authors: Burman
authors: The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Queensland, Australia.|Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Queensland, Australia.
authors: Erin R
authors: ER
authors: Shanahan
authors: CSIRO Preventative Health Flagship Research Program, Queensland, Australia.|The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Queensland, Australia.
authors: Mark
authors: M
authors: Morrison
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D044822:Biodiversity; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification*; D003227:Conjugation, Genetic; D005456:Fluorescent Dyes / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D056186:Metagenomics / Q000379:methods*; D008856:Microscopy, Fluorescence; D010802:Phylogeny; D016133:Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes