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Bacteria often produce antimicrobial toxins to compete in microbial communities. Here we identify a family of broad-spectrum peptide toxins, named bacteroidetocins, produced by Bacteroidetes species. We study this toxin family using phenotypic, mutational, bioinformatic, and human metagenomic analyses. Bacteroidetocins are related to class IIa bacteriocins of Gram-positive bacteria and kill members of the Bacteroidetes phylum, including Bacteroides , Parabacteroides , and Prevotella gut species, as well as pathogenic Prevotella species. The bacteroidetocin biosynthesis genes are found in horizontally acquired mobile elements, which likely allow dissemination within the gut microbiota and may explain their wide distribution in human populations. Bacteroidetocins may have potential applications in microbiome engineering and as therapeutics for polymicrobial diseases such as bacterial vaginosis and periodontal disease. Bacteria often produce antimicrobial toxins to compete in microbial communities. Here, the authors identify a family of peptide toxins that are produced by, and target, Bacteroidetes species, some of which are widespread in the human gut microbiota.

We recently showed that human gut Bacteroidales species secrete antimicrobial proteins (BSAPs), and we characterized in vitro the first such BSAP produced by Bacteroides fragilis . In this study, we identified a second potent BSAP produced by the ubiquitous and abundant human gut species Bacteroides uniformis . The two BSAPs contain a membrane attack complex/perforin (MACPF) domain but share very little sequence similarity. We identified the target molecules of BSAP-sensitive cells and showed that each BSAP targets a different class of surface molecule: BSAP-1 targets an outer membrane protein of sensitive B. fragilis strains, and BSAP-2 targets the O-antigen glycan of lipopolysaccharide (LPS) of sensitive B. uniformis strains. Species-wide genomic and phenotypic analyses of B. fragilis and B. uniformis showed that BSAP-producing strains circumvent killing by synthesizing an orthologous nontargeted surface molecule. The BSAP genes are adjacent to the gene(s) encoding their target replacements, suggesting coacquisition. Using a gnotobiotic mouse competitive-colonization model, we found that the BSAP surface targets are important for colonization of the mammalian gut, thereby explaining why they are maintained in sensitive strains and why they were replaced rather than deleted in BSAP-producing strains. Using isogenic BSAP-producing, -sensitive, and -resistant strains, we show that a BSAP-producing strain outcompetes a sensitive strain but not a resistant strain in the mammalian gut. Human gut metagenomic datasets reveal that BSAP-1-sensitive strains do not cooccur with BSAP-1-producing strains in human gut microbiotas, further supporting the idea that BSAPs are important competitive factors with relevance to the strain-level composition of the human gut microbiota. IMPORTANCE We know relatively little about the ecology of the human intestinal microbiota and the combination of factors that dictate which strains and species occupy an individual’s gut microbial community. Interference competition, mediated by bacterial factors that directly harm other members, is beginning to be appreciated as important in contributing to species- and strain-level dynamics of abundant gut bacteria. Here, we show that gut Bacteroidales secrete antimicrobial proteins (BSAPs) that antagonize strains of the same species. We show that BSAPs target molecules of sensitive cells that are important for gut colonization and therefore are maintained in sensitive cells. In an experimental animal model of gut colonization, a BSAP-1-producing strain antagonized and outcompeted an isogenic sensitive strain. Furthermore, metagenomic analyses showed that BSAP-1-producing and -sensitive strains are not found together in human gut microbiotas. These data suggest that BSAPs are strong ecological drivers shaping the strain-level composition of gut communities. We know relatively little about the ecology of the human intestinal microbiota and the combination of factors that dictate which strains and species occupy an individual’s gut microbial community. Interference competition, mediated by bacterial factors that directly harm other members, is beginning to be appreciated as important in contributing to species- and strain-level dynamics of abundant gut bacteria. Here, we show that gut Bacteroidales secrete antimicrobial proteins (BSAPs) that antagonize strains of the same species. We show that BSAPs target molecules of sensitive cells that are important for gut colonization and therefore are maintained in sensitive cells. In an experimental animal model of gut colonization, a BSAP-1-producing strain antagonized and outcompeted an isogenic sensitive strain. Furthermore, metagenomic analyses showed that BSAP-1-producing and -sensitive strains are not found together in human gut microbiotas. These data suggest that BSAPs are strong ecological drivers shaping the strain-level composition of gut communities.

Human gut Bacteroides species produce different types of toxins that antagonize closely related members of the gut microbiota. Some are toxic effectors delivered by type VI secretion systems, and others are non-contact-dependent secreted antimicrobial proteins. Many strains of Bacteroides fragilis secrete antimicrobial molecules, but only one of these toxins has been described to date ( Bacteroidales secreted antimicrobial protein 1 [BSAP-1]). In this study, we describe a novel secreted protein produced by B. fragilis strain 638R that mediated intraspecies antagonism. Using transposon mutagenesis and deletion mutation, we identified a gene encoding a eukaryotic-like ubiquitin protein (BfUbb) necessary for toxin activity against a subset of B. fragilis strains. The addition of ubb into a heterologous background strain conferred toxic activity on that strain. We found this gene to be one of the most highly expressed in the B. fragilis genome. The mature protein is 84% similar to human ubiquitin but has an N-terminal signal peptidase I (SpI) signal sequence and is secreted extracellularly. We found that the mature 76-amino-acid synthetic protein has very potent activity, confirming that BfUbb mediates the activity. Analyses of human gut metagenomic data sets revealed that ubb is present in 12% of the metagenomes that have evidence of B. fragilis . As 638R produces both BSAP-1 and BfUbb, we performed a comprehensive analysis of the toxin activity of BSAP-1 and BfUbb against a set of 40 B. fragilis strains, revealing that 75% of B. fragilis strains are targeted by one or the other of these two secreted proteins of strain 638R. IMPORTANCE We are just beginning to understand some of the important interactions that occur between microbes of the human gut microbiota that dictate the composition and abundance of its constituent members. The ability of one member to produce molecules that directly kill a coresident member has been shown among minor gut species and is just starting to be studied in the abundant Bacteroides species. Here, we show that some strains of Bacteroides fragilis have acquired a gene encoding a secreted eukaryotic-like ubiquitin protein with potent inhibitory activity against other B. fragilis stains. This is the first bacterially encoded ubiquitin-like molecule shown to function like a bacterial toxin. This molecule is an example of a gut symbiont acquiring and adapting a eukaryotic molecule likely to increase its competitiveness in the mammalian gut. Understanding antagonistic factors produced by abundant gut symbionts is an important prerequisite to properly engineer strains to colonize the gut for health benefits. We are just beginning to understand some of the important interactions that occur between microbes of the human gut microbiota that dictate the composition and abundance of its constituent members. The ability of one member to produce molecules that directly kill a coresident member has been shown among minor gut species and is just starting to be studied in the abundant Bacteroides species. Here, we show that some strains of Bacteroides fragilis have acquired a gene encoding a secreted eukaryotic-like ubiquitin protein with potent inhibitory activity against other B. fragilis stains. This is the first bacterially encoded ubiquitin-like molecule shown to function like a bacterial toxin. This molecule is an example of a gut symbiont acquiring and adapting a eukaryotic molecule likely to increase its competitiveness in the mammalian gut. Understanding antagonistic factors produced by abundant gut symbionts is an important prerequisite to properly engineer strains to colonize the gut for health benefits.

Bacteroides species are the most abundant Gram-negative bacteria of the human colonic microbiota. These endogenous organisms are unique in that they synthesize an extensive number of phase-variable surface polysaccharides. Pathogenic bacteria phase vary expression of surface molecules for immune evasion, but the importance of the synthesis of multiple phase-variable polysaccharides to these commensal bacteria is unknown. We previously showed that a Bacteroides fragilis mutant unable to synthesize 4 of the 8 capsular polysaccharides and unable to glycosylate proteins properly is rapidly outcompeted by the wild-type strain for colonization of the gnotobiotic mouse intestine. In the present study, we constructed mutants defective only in capsule polysaccharide synthesis to define better the importance of these surface molecules to intestinal colonization. We discovered a key enzymatic activity required for synthesis of 7 of the 8 capsular polysaccharides. Deletion of its gene resulted in the first B. fragilis mutant able to synthesize only one phase-variable polysaccharide, and further mutation resulted in a stable acapsular mutant. We show that the acapsular mutant is rapidly outcompeted, but synthesis of a single polysaccharide is sufficient for the organism to colonize the gnotobiotic intestine competitively. These data demonstrate that initial colonization of the gnotobiotic mouse intestine by B. fragilis requires that the organism synthesize only a single polysaccharide and suggest that the synthesis of multiple phase-variable polysaccharides is important for the bacteria's long-term maintenance in the normally complex and competitive ecosystem.

Bacteroides is an abundant genus of bacteria of the human intestinal microbiota. Bacteroides species synthesize a large number of capsular polysaccharides (PS), a biological property not shared with closely related oral species, suggesting importance for intestinal survival. Bacteroides fragilis, for example, synthesizes eight capsular polysaccharides per strain, each of which phase varies via inversion of the promoters located upstream of seven of the eight polysaccharide biosynthesis operons. In a single cell, many of these polysaccharide loci promoters can be simultaneously oriented on for transcription of the downstream biosynthesis operons. Here, we demonstrate that despite the promoter orientations, concomitant transcription of multiple polysaccharide loci within a cell is inhibited. The proteins encoded by the second gene of each of these eight loci, collectively designated the UpxZ proteins, inhibit the synthesis of heterologous polysaccharides. These unique proteins interfere with the ability of UpxY proteins encoded by other polysaccharide loci to function in transcriptional antitermination of their respective operon. The eight UpxZs have different inhibitory spectra, thus establishing a hierarchical regulatory network for polysaccharide synthesis. Limitation of concurrent polysaccharide synthesis strongly suggests that these bacteria evolved this property as an evasion-type mechanism to avoid killing by polysaccharide-targeting factors in the ecosystem.

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.

The ability to antagonize competing strains and species is often important for bacterial fitness in microbial communities. The extent to which intra-species antagonism drives phenotypic diversity of bacterial species is rarely examined in a comprehensive manner at both the genetic and phenotypic levels. Here we show that for nine abundant human gut Bacteroides species examined, there are only a few LPS glycan genetic types. We show that for a given Bacteroides species, there is a predominant lipopolysaccharide (LPS) glycan locus present in the majority of strains. However, other strains have replacements of glycosyltransferase-encoding genes, in most cases, adjacent to a membrane attack/perforin (MACPF) domain-encoding gene not present in the predominant type. We show that the MACPF genes present in LPS glycan biosynthesis loci of four Bacteroides species encode antimicrobial proteins and in Bacteroides vulgatus and Bacteroides dorei , we show the MACPF toxin targets the LPS of strains with the predominant LPS glycan locus. By a combination of gene deletion and replacement, we converted a MACPF toxin-producing strain into a sensitive strain. Genetic diversity of LPS glycan biosynthesis regions in Bacteroides is similar to phage serotype conversion whereby the receptor is altered to render the strain immune to infection/toxicity, and is a rare example in bacteria of toxin immunity conferred to the toxin-producing strain by replacement of genetic material to modify the receptor rather than by a cognate immunity protein.

DNA transfer is ubiquitous in the gut microbiota, especially among species of Bacteroidales. analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. Here, we show that acquisition of a ubiquitous integrative and conjugative element encoding an antagonistic system shuts down the native contact-dependent antagonistic system of . Despite inactivating the native antagonism system, mobile element acquisition increases fitness of the transconjugant over its progenitor by arming it with a new weapon. This DNA transfer causes the strain to change allegiances so that it no longer targets ecosystem members containing the same element yet is armed for communal defense.