Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

A pervasive large conjugative plasmid mediates multispecies biofilm formation in the intestinal microbiota increasing resilience to perturbations.

Although horizontal gene transfer is pervasive in the intestinal microbiota, we understand only superficially the roles of most exchanged genes and how the mobile repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying the ability of a community to recover after a perturbation. Here, we identified and functionally characterized a large conjugative plasmid that is one of the most frequently transferred elements among Bacteroidales species and is ubiquitous in diverse human populations. This plasmid encodes both an extracellular polysaccharide and fimbriae, which promote the formation of multispecies biofilms in the mammalian gut. We use a hybridization-based approach to visualize biofilms in clarified whole colon tissue with unprecedented 3D spatial resolution. These biofilms increase bacterial survival to common stressors encountered in the gut, increasing strain resiliency, and providing a rationale for the plasmid's recent spread and high worldwide prevalence.

Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species and strain level diversity and evolution.

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome with diverse impacts on human health. While Bacteroidales strains and species are genomically and functionally diverse, order-wide comparative analyses are lacking. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, mobile gene, and metabolomic analyses. Families, genera, and species could be grouped based on many distinctive features. However, we also show extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-specific diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses can serve as a resource to facilitate informed selection of strains for microbiome reconstitution.

Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria.

Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations.