Genetic Manipulation of Wild Human Gut Bacteroides

Abstract

Bacteroides is one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insights into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides species that does not require any alterations to the genetic background or a 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 included in the genomes of previously genetically tractable members of the human gut microbiome. IMPORTANCE Human gut Bacteroides species 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 medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.

Strain-level epidemiology of microbial communities and the human microbiome

“…Such an approach leads naturally to the subsequent biochemical characterization of these variants, either via isolation from primary samples [15,170] or by in silico retrieval of homologous sequences or related strains from databases or repositories (e.g., ATCC, BEI, DSMZ) [185]. Primary samples can be characterized as an entire community via gnotobiotics [186,187] or continuous culture [188,189], or individual isolate strains grown, characterized, or (when possible) genetically manipulated [15,190,191]. Such approaches dovetail nicely with "bottom up" approaches (analogous to reverse genetics) that identify and characterize healthrelevant strains by directly beginning with isolates and assessing their phenotypes in gnotobiotic mono-or combinatorial colonization [192][193][194][195][196][197] or, when possible, human feeding [198][199][200] or microbiota transplant clinical trials [201][202][203][204][205].…”

“…Such methods for capturing strains from the human microbiome go handin-hand with additional technologies for characterizing them at scale, including cheaper experimental systems such as gut-on-chip [211,212] or organoid variants [213,214] that sit in between single isolate culture and rich gnotobiotic models. Ultimately, understanding human microbiome biology will require not just the detection of specific microbial genetic variants in communities, but their introduction and manipulation, including the theoretical ability to genetically perturb any microbial strain either after or even before isolation from its host community [173,190].…”

A versatile genetic toolbox forPrevotella coprienables studying polysaccharide utilization systems

“…As targeted gene inactivation approaches enable gene function studies, they are frequently carried out in Bacteroides spp. by transferring a suicide plasmid from a donor strain into the recipient followed by selection of bacterial clones which underwent homologous recombination (Bencivenga-Barry et al, 2020; García-Bayona and Comstock, 2019; Koropatkin et al, 2008). To adapt the system for P. copri , we considered several key differences between Bacteroides spp.…”

“…(recipient) (Salyers et al, 1999), conjugation for Bacteroides spp. is routinely performed for at least 15 hours under aerobic conditions followed by transferring the cultures to anaerobic conditions permitting growth (Bencivenga-Barry et al, 2020; García-Bayona and Comstock, 2019). We initially tested aerotolerance of three P. copri strains, the type strain (DSM18205) and two strains (HDD04 and HDB01) from our lab collection containing recent isolates from healthy and diseased individuals (Figure 1A and Table S1).…”

“…In contrast to P. copri , members of the genus Bacteroides , as the best example, have been extensively studied via a variety of genetic tools (Bencivenga-Barry et al, 2020; García-Bayona and Comstock, 2019; Goodman et al, 2011; Koropatkin et al, 2008; Lim et al, 2017; Mimee et al, 2015). These studies have, for instance, identified genes required for various bacterial physiological functions and provide approaches to investigate bacteria-host interactions.…”