Yulin Song

Antagonistic interactions often shape microbial communities, as co-residents compete for resources. One system of interbacterial antagonism involves tailocins: phage tail-like particles produced by some bacteria to kill close relatives. Their roles in natural communities are poorly studied. Here, we identified putative tailocin loci ubiquitously present in genomes of Snodgrassella species, core members of the bee gut microbiota. We purified tailocins from culture supernatants of different Snodgrassella strains and found that these inhibited growth of multiple Snodgrassella strains but not of strains of other species that cooccur in bee guts. Proteomic analyses corroborated the production of tailocins, and cryogenic electron microscopy imaging revealed their morphology and killing activity via membrane disruption. We generated single- and double-gene knockout mutants of a predator Snodgrassella strain that naturally possesses both tailocin and a type VI secretion system (T6SS) that was previously shown to function in intraspecies competition in bee guts. These mutants were used in tests for antagonism against a prey Snodgrassella strain in vivo. Co-inoculations within gnotobiotic honeybees including visualization within guts showed that both tailocin and T6SS are important for Snodgrassella in intraspecies competition. Each of these two antagonism mechanisms inhibits the establishment of susceptible strains within the ileum. Outcomes depend on the timing of colonization by competing strains, with both tailocin and T6SS giving a stronger advantage when the producing strain colonizes first. Thus, tailocin production can underlie priority effects that have been widely observed in the bee gut community and other host-associated bacterial communities. Significance Statement: Interbacterial competition plays a pivotal role in determining bacterial colonization success in natural habitats, including guts of animals. Tailocins are phage tail-like bacteriocins that are widely encoded in bacterial genomes and that function in intraspecies killing. They have been little studied in natural communities, including those in gut environments. Here, we show that bee gut bacteria produce tailocins that kill close relatives within the gut. Tailocins function as phage-derived antagonism mechanisms together with T6SS, which are also phage-derived. The possession of both weapons provides a double safeguard, supporting that intense competition happens in bee gut environments. Our findings bring new insights into roles of antagonistic weapons in natural communities.

Colibactin is a bacterial genotoxin that damages DNA and is linked to colorectal cancer. It is implicated in interbacterial competition but its role in natural microbiomes is unclear. Here we show that Frischella perrara, a colibactin-producing symbiont restricted to honeybee guts, lowers bee lifespan but increases survivorship following challenge with the opportunistic bee pathogen Serratia marcescens. F. perrara mutants lacking colibactin production fail to improve survivorship following challenge. The symbiont reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. Colibactin is toxic to the pathogen under anoxic conditions, reflecting the bee gut. clbS, a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but absent from opportunistic colonizers, including pathogens. Indeed, F. perrara colonization representing colibactin pressure had limited impact on the bee gut microbiome where strains encode clbS. Our findings suggest that colibactin functions in interbacterial competition and protects against pathogens while permitting co-evolved symbionts to persist.

Symbiotic bacteria in the gut have an important physiological impact on their hosts, but the mechanisms that underlie the exchange of molecular signals remain poorly understood. Membrane vesicles have been suggested to mediate the direct exchange of cytoplasmic content like nucleic acids (NAs), but their study is complicated by conflicting and imprecise reports of their type and composition. Here, we show that honeybee gut symbionts produce non-lytic membrane vesicles (MVs) enriched in NA, potentially explaining the RNAi activity of engineered Snodgrassella alvi in honeybees. Using cryogenic electron microscopy (cryo-EM), we developed a method to distinguish lytic from non-lytic MV production in Gram-negative bacteria and to differentiate outer membrane vesicles (OMVs) from outer-inner membrane vesicles (OIMVs) based on membrane ultrastructure. Among the strains studied, S. alvi, Gilliamella apicola, and Gilliamella apis exhibit clear OMV and OIMV budding, while Escherichia coli and Salmonella enterica show membrane debris and self-assembled vesicles, indicating lytic release. MVs from the symbionts carry significantly more NAs than non-symbionts. Assays on DNA and RNA contents confirm the cytoplasmic origin of MV cargo in S. alvi, suggesting a role in mediating NA delivery to the insect host. These findings enhance our understanding of symbiotic vesiculation and highlight the potential for engineering symbionts to boost honeybee immunity and deliver NA-based therapeutics via vesicular transport.

Colibactin is a bacterial genotoxin that is linked to colorectal cancer and implicated in interbacterial competition; however, its role in natural communities remains unknown. Frischella perrara , a symbiont living only in honeybee guts, produces colibactin and causes DNA damage. Here, we found that F. perrara mono-colonization reduces bee lifespan but increases survivorship following challenge with an opportunistic pathogen. F. perrara reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. clbS , a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but is absent from opportunistic colonizers including bee pathogens. Our findings provide evidence that colibactin functions in interbacterial competition, exerts selective pressure on community members, and confers protection against pathogens, while permitting co-evolved gut symbionts to persist.

Spirochetes are widely distributed in various anoxic environments and commonly form molecular hydrogen as a major fermentation product. Here, we show that two closely related members of the family Treponemataceae differ strongly in their sensitivity to high hydrogen partial pressure, and we explain the metabolic mechanisms that cause these differences by comparative genome analysis.

The intestinal tracts of termites are abundantly colonized by a diverse assemblage of spirochetes. Most of them belong to ‘termite cluster I’, a monophyletic group within the radiation of the genus Treponema that occurs exclusively in termite guts. Phylogenomic analysis revealed that members of the genus Treponema are extremely diverse and represent two separate, family-level lineages: the Treponemataceae sensu stricto, which comprise the majority of the validly described Treponema species, and a second lineage that comprises the remaining members of the genus Treponema , including all members of ‘termite cluster I’ from termites and the recently isolated Breznakiella homolactica from cockroaches. Here, we present the formal description of Breznakiellaceae fam. nov. and of the new genera required to accommodate the misplaced Treponema species in the new family as new combinations ( Leadbettera azotonutricia , Gracilinema caldarium , Helmutkoenigia isoptericolens and Zuelzera stenostrepta). To avoid paraphyly of Treponemataceae , we propose Rectinemataceae fam. nov. to include the genus Rectinema .

Summary: Spirochetes of the genus Treponema are surprisingly abundant in termite guts, where they play an important role in reductive acetogenesis. Although they occur in all termites investigated, their evolutionary origin is obscure. Here, we isolated the first representative of ‘termite gut treponemes’ from cockroaches, the closest relatives of termites. Phylogenomic analysis revealed that Breznakiella homolactica gen. nov. sp. nov. represents the most basal lineage of the highly diverse ‘termite cluster I', a deep‐branching sister group of Treponemataceae (fam. ‘ Termitinemataceae ’) that was present already in the cockroach ancestor of termites and subsequently coevolved with its host. Breznakiella homolactica is obligately anaerobic and catalyses the homolactic fermentation of both hexoses and pentoses. Resting cells produced acetate in the presence of oxygen. Genome analysis revealed the presence of pyruvate oxidase and catalase, and a cryptic potential for the formation of acetate, ethanol, formate, CO ₂ and H ₂ ‐ the fermentation products of termite gut isolates. Genes encoding key enzymes of reductive acetogenesis, however, are absent, confirming the hypothesis that the ancestral metabolism of the cluster was fermentative, and that the capacity for acetogenesis from H ₂ plus CO ₂ ‐ the most intriguing property among termite gut treponemes ‐ was acquired by lateral gene transfer.