Predation-resistant Pseudomonas bacteria engage in symbiont-like behavior with the social amoeba Dictyostelium discoideum.

The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.

The Gut Microbiota Protects Bees from Invasion by a Bacterial Pathogen.

Commensal microbes in animal guts often help to exclude bacterial pathogens. In honey bees, perturbing or depleting the gut microbiota increases host mortality rates upon challenge with the opportunistic pathogen Serratia marcescens, suggesting antagonism between S. marcescens and one or more members of the bee gut microbiota. In laboratory culture, S. marcescens uses a type VI secretion system (T6SS) to kill bacterial competitors, but the role of this T6SS within hosts is unknown. Using infection assays, we determined how the microbiota impacts the abundance and persistence of S. marcescens in the gut and visualized colocalization of S. marcescens with specific community members in situ. Using T6SS-deficient S. marcescens strains, we measured T6SS-dependent killing of gut isolates in vitro and compared the persistence of mutant and wild-type strains in the gut. We found that S. marcescens is rapidly eliminated in the presence of the microbiota but persists in microbiota-free guts. Protection is reduced in monocolonized and antibiotic-treated bees, possibly because different symbionts occupy distinct niches. Serratia marcescens uses a T6SS to antagonize Escherichia coli and other S. marcescens strains but shows limited ability to kill bee symbionts. Furthermore, wild-type and T6SS-deficient S. marcescens strains achieved similar abundance and persistence in bee guts. Thus, an intact gut microbiota offers robust protection against this common pathogen, whose T6SSs do not confer the ability to compete with commensal species. IMPORTANCE Bacteria living within guts of animals can provide protection against infection by pathogens. Some pathogens have been shown to use a molecular weapon known as a T6SS to kill beneficial bacteria during invasion of the mouse gut. In this study, we examined how bacteria native to the honey bee gut work together to exclude the opportunistic pathogen Serratia marcescens. Although S. marcescens has a T6SS that can kill bacteria, bee gut bacteria seem resistant to its effects. This limitation may partially explain why ingestion of S. marcescens is rarely lethal to insects with healthy gut communities.

Evolution of Interbacterial Antagonism in Bee Gut Microbiota Reflects Host and Symbiont Diversification.

Gram-negative bacteria frequently possess type VI secretion systems (T6SSs), protein complexes that are able to inject toxic proteins into nearby cells. Many aspects of T6SS structure and function have been characterized for model species, but less is known about the evolutionary processes that shape T6SS and effector (toxin) diversity in host-associated microbial communities. The bee gut microbiota is a simple community that has codiversified with bees for >80 million years. This study investigated how complements of T6SSs and effectors within the bee microbiota changed as bacteria and their hosts diversified into isolated species. We used protein homology to survey 198 isolate genomes of 9 Gram-negative species for genes encoding T6SS structural components; Rhs toxins, which are common T6SS effectors; and VgrG proteins, which are structural components associated with specific toxins. T6SS loci were present in 5 species clusters found only in bees, namely Apibacter spp., Gilliamella spp., Frischella perrara, "Candidatus Schmidhempelia bombi," and Snodgrassella alvi The distribution of T6SS loci suggests that at least 3 were present in the microbiota of the common ancestor of social bees and that loss of these genes in some bacterial lineages was linked to both host and bacterial speciation. Isolates differed enormously in repertoires of Rhs and VgrG proteins. We found that bacterial species employ different mechanisms for toxin acquisition and diversification and that species and strains sometimes lose the T6SS entirely, likely causing shifts in competitive dynamics within these communities.IMPORTANCE Antagonistic interactions between bacteria affect diversity and dynamics of host-associated communities, including gut communities that are linked to host health. In many bacterial communities, including human and honey bee gut microbiotas, antagonism is mediated by type VI secretion systems (T6SSs) that deliver lethal toxins to competing strains. In this study, we explored how T6SSs and associated toxins have evolved in the simple, host-specific gut microbiota of honey bees and bumble bees. Using comparative genomics, we explored the conservation, recombination, horizontal transfer, and loss of T6SSs and effectors during 80 million years of evolution of this bee-associated community. We find that that patterns of T6SS loss and retention are linked to differences in biology across host species, while trends in effector diversification are mostly specific to bacterial lineages.

Honey bees as models for gut microbiota research.

The gut microbiota of the honey bee (Apis mellifera) offers several advantages as an experimental system for addressing how gut communities affect their hosts and for exploring the processes that determine gut community composition and dynamics. A small number of bacterial species dominate the honey bee gut community. These species are restricted to bee guts and can be grown axenically and genetically manipulated. Large numbers of microbiota-free hosts can be economically reared and then inoculated with single isolates or defined communities to examine colonization patterns and effects on host phenotypes. Honey bees have been studied extensively, due to their importance as agricultural pollinators and as models for sociality. Because of this history of bee research, the physiology, development, and behavior of honey bees is relatively well understood, and established behavioral and phenotypic assays are available. To date, studies on the honey bee gut microbiota show that it affects host nutrition, weight gain, endocrine signaling, immune function, and pathogen resistance, while perturbation of the microbiota can lead to reduced host fitness. As in humans, the microbiota is concentrated in the distal part of the gut, where it contributes to digestion and fermentation of plant cell wall components. Much like the human gut microbiota, many bee gut bacteria are specific to the bee gut and can be directly transmitted between individuals through social interaction. Although simpler than the human gut microbiota, the bee gut community presents opportunities to understand the processes that govern the assembly of specialized gut communities as well as the routes through which gut communities impact host biology.

Genome Sequences of Apibacter spp., Gut Symbionts of Asian Honey Bees.

Honey bees have distinct gut microbiomes consisting almost entirely of several host-specific bacterial species. We present the genomes of three strains of Apibacter spp., bacteria of the Bacteroidetes phylum that are endemic to Asian honey bee species (Apis dorsata and Apis cerana). The Apibacter strains have similar metabolic abilities to each other and to Apibacter mensalis, a species isolated from a bumble bee. They use microaerobic respiration and fermentation to catabolize a limited set of monosaccharides and dicarboxylic acids. All strains are capable of gliding motility and encode a type IX secretion system. Two strains and A. mensalis have type VI secretion systems, and all strains encode Rhs or VgrG proteins used in intercellular interactions. The characteristics of Apibacter spp. are consistent with adaptions to life in a gut environment; however, the factors responsible for host-specificity and mutualistic interactions remain to be uncovered.

Diversification of Type VI Secretion System Toxins Reveals Ancient Antagonism among Bee Gut Microbes.

Microbial communities are shaped by interactions among their constituent members. Some Gram-negative bacteria employ type VI secretion systems (T6SSs) to inject protein toxins into neighboring cells. These interactions have been theorized to affect the composition of host-associated microbiomes, but the role of T6SSs in the evolution of gut communities is not well understood. We report the discovery of two T6SSs and numerous T6SS-associated Rhs toxins within the gut bacteria of honey bees and bumble bees. We sequenced the genomes of 28 strains of Snodgrassella alvi, a characteristic bee gut microbe, and found tremendous variability in their Rhs toxin complements: altogether, these strains appear to encode hundreds of unique toxins. Some toxins are shared with Gilliamella apicola, a coresident gut symbiont, implicating horizontal gene transfer as a source of toxin diversity in the bee gut. We use data from a transposon mutagenesis screen to identify toxins with antibacterial function in the bee gut and validate the function and specificity of a subset of these toxin and immunity genes in Escherichia coli Using transcriptome sequencing, we demonstrate that S. alvi T6SSs and associated toxins are upregulated in the gut environment. We find that S. alvi Rhs loci have a conserved architecture, consistent with the C-terminal displacement model of toxin diversification, with Rhs toxins, toxin fragments, and cognate immunity genes that are expressed and confer strong fitness effects in vivo Our findings of T6SS activity and Rhs toxin diversity suggest that T6SS-mediated competition may be an important driver of coevolution within the bee gut microbiota.IMPORTANCE The structure and composition of host-associated bacterial communities are of broad interest, because these communities affect host health. Bees have a simple, conserved gut microbiota, which provides an opportunity to explore interactions between species that have coevolved within their host over millions of years. This study examined the role of type VI secretion systems (T6SSs)-protein complexes used to deliver toxic proteins into bacterial competitors-within the bee gut microbiota. We identified two T6SSs and diverse T6SS-associated toxins in bacterial strains from bees. Expression of these genes is increased in bacteria in the bee gut, and toxin and immunity genes demonstrate antibacterial and protective functions, respectively, when expressed in Escherichia coli Our results suggest that coevolution among bacterial species in the bee gut has favored toxin diversification and maintenance of T6SS machinery, and demonstrate the importance of antagonistic interactions within host-associated microbial communities.

Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling.

Social bees harbor a simple and specialized microbiota that is spatially organized into different gut compartments. Recent results on the potential involvement of bee gut communities in pathogen protection and nutritional function have drawn attention to the impact of the microbiota on bee health. However, the contributions of gut microbiota to host physiology have yet to be investigated. Here we show that the gut microbiota promotes weight gain of both whole body and the gut in individual honey bees. This effect is likely mediated by changes in host vitellogenin, insulin signaling, and gustatory response. We found that microbial metabolism markedly reduces gut pH and redox potential through the production of short-chain fatty acids and that the bacteria adjacent to the gut wall form an oxygen gradient within the intestine. The short-chain fatty acid profile contributed by dominant gut species was confirmed in vitro. Furthermore, metabolomic analyses revealed that the gut community has striking impacts on the metabolic profiles of the gut compartments and the hemolymph, suggesting that gut bacteria degrade plant polymers from pollen and that the resulting metabolites contribute to host nutrition. Our results demonstrate how microbial metabolism affects bee growth, hormonal signaling, behavior, and gut physicochemical conditions. These findings indicate that the bee gut microbiota has basic roles similar to those found in some other animals and thus provides a model in studies of host-microbe interactions.

Metabolism of Toxic Sugars by Strains of the Bee Gut Symbiont Gilliamella apicola.

Social bees collect carbohydrate-rich food to support their colonies, and yet, certain carbohydrates present in their diet or produced through the breakdown of pollen are toxic to bees. The gut microbiota of social bees is dominated by a few core bacterial species, including the Gram-negative species Gilliamella apicola We isolated 42 strains of G. apicola from guts of honey bees and bumble bees and sequenced their genomes. All of the G. apicola strains share high 16S rRNA gene similarity, but they vary extensively in gene repertoires related to carbohydrate metabolism. Predicted abilities to utilize different sugars were verified experimentally. Some strains can utilize mannose, arabinose, xylose, or rhamnose (monosaccharides that can cause toxicity in bees) as their sole carbon and energy source. All of the G. apicola strains possess a manO-associated mannose family phosphotransferase system; phylogenetic analyses suggest that this was acquired from Firmicutes through horizontal gene transfer. The metabolism of mannose is specifically dependent on the presence of mannose-6-phosphate isomerase (MPI). Neither growth rates nor the utilization of glucose and fructose are affected in the presence of mannose when the gene encoding MPI is absent from the genome, suggesting that mannose is not taken up by G. apicola strains which harbor the phosphotransferase system but do not encode the MPI. Given their ability to simultaneously utilize glucose, fructose, and mannose, as well as the ability of many strains to break down other potentially toxic carbohydrates, G. apicola bacteria may have key roles in improving dietary tolerances and maintaining the health of their bee hosts. IMPORTANCE: Bees are important pollinators of agricultural plants. Our study documents the ability of Gilliamella apicola, a dominant gut bacterium in honey bees and bumble bees, to utilize several sugars that are harmful to bee hosts. Using genome sequencing and growth assays, we found that the ability to metabolize certain toxic carbohydrates is directly correlated with the presence of their respective degradation pathways, indicating that metabolic potential can be accurately predicted from genomic data in these gut symbionts. Strains vary considerably in their range of utilizable carbohydrates, which likely reflects historical horizontal gene transfer and gene deletion events. Unlike their bee hosts, G. apicola bacteria are not detrimentally affected by growth on mannose-containing medium, even in strains that cannot metabolize this sugar. These results suggest that G. apicola may be an important player in modulating nutrition in the bee gut, with ultimate effects on host health.