Increased Microbial Diversity and Decreased Prevalence of Common Pathogens in the Gut Microbiomes of Wild Turkeys Compared to Domestic Turkeys.

Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production, causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminate antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analyses of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella, and Escherichia coli compared to domestic turkeys. E. coli strains isolated from wild and domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII and kpsIII) or siderophore (iroN and fyuA) synthesis than were those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens. IMPORTANCE Due to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compare the microbiota of wild and domestic turkeys. The results suggest that free-ranging wild turkeys carry a distinct microbiome compared to farm-raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to that of farm-raised birds. The microbiomes of wild turkeys may be used to guide the development of new ways to control disease in large-scale poultry production.

Bacterial Metabolism and Transport Genes Are Associated with the Preference of Drosophila melanogaster for Dietary Yeast.

Many animal traits are influenced by their associated microorganisms ("microbiota"). To expand our understanding of the relationship between microbial genotype and host phenotype, we report an analysis of the influence of the microbiota on the dietary preference of the fruit fly Drosophila melanogaster. First, we confirmed through experiments on flies reared bacteria-free ("axenic") or in monoassociation with two different strains of bacteria that the microbiota significantly influences fruit fly dietary preference across a range of ratios of dietary yeast:dietary glucose. Then, focusing on microbiota-dependent changes in fly dietary preference for yeast (DPY), we performed a metagenome-wide association (MGWA) study to define microbial species specificity for this trait and to predict bacterial genes that influence it. In a subsequent mutant analysis, we confirmed that disrupting a subset of the MGWA-predicted genes influences fly DPY, including for genes involved in thiamine biosynthesis and glucose transport. Follow-up tests revealed that the bacterial influence on fly DPY did not depend on bacterial modification of the glucose or protein content of the fly diet, suggesting that the bacteria mediate their effects independent of the fly diet or through more specific dietary changes than broad ratios of protein and glucose. Together, these findings provide additional insight into bacterial determinants of host nutrition and behavior by revealing specific genetic disruptions that influence D. melanogaster DPY. IMPORTANCE Associated microorganisms ("microbiota") impact the physiology and behavior of their hosts, and defining the mechanisms underlying these interactions is a major gap in the field of host-microbe interactions. This study expands our understanding of how the microbiota can influence dietary preference for yeast (DPY) of a model host, Drosophila melanogaster. First, we show that fly preferences for a range of different dietary yeast:dietary glucose ratios vary significantly with the identity of the microbes that colonize the fruit flies. We then performed a metagenome-wide association study to identify candidate bacterial genes that contributed to some of these bacterial influences. We confirmed that disrupting some of the predicted genes, including genes involved in glucose transport and thiamine biosynthesis, resulted in changes to fly DPY and show that the influence of two of these genes is not through changes in dietary ratios of protein to glucose. Together, these efforts expand our understanding of the bacterial genetic influences on a feeding behavior of a model animal host.

Microbiome composition shapes rapid genomic adaptation of Drosophila melanogaster.

Population genomic data has revealed patterns of genetic variation associated with adaptation in many taxa. Yet understanding the adaptive process that drives such patterns is challenging; it requires disentangling the ecological agents of selection, determining the relevant timescales over which evolution occurs, and elucidating the genetic architecture of adaptation. Doing so for the adaptation of hosts to their microbiome is of particular interest with growing recognition of the importance and complexity of host-microbe interactions. Here, we track the pace and genomic architecture of adaptation to an experimental microbiome manipulation in replicate populations of Drosophila melanogaster in field mesocosms. Shifts in microbiome composition altered population dynamics and led to divergence between treatments in allele frequencies, with regions showing strong divergence found on all chromosomes. Moreover, at divergent loci previously associated with adaptation across natural populations, we found that the more common allele in fly populations experimentally enriched for a certain microbial group was also more common in natural populations with high relative abundance of that microbial group. These results suggest that microbiomes may be an agent of selection that shapes the pattern and process of adaptation and, more broadly, that variation in a single ecological factor within a complex environment can drive rapid, polygenic adaptation over short timescales.

Spatiotemporal variation in the fecal microbiota of mule deer is associated with proximate and future measures of host health.

BACKGROUND: Mule deer rely on fat and protein stored prior to the winter season as an energy source during the winter months when other food sources are sparse. Since associated microorganisms ('microbiota') play a significant role in nutrient metabolism of their hosts, we predicted that variation in the microbiota might be associated with nutrient storage and overwintering in mule deer populations. To test this hypothesis we performed a 16S rRNA marker gene survey of fecal samples from two deer populations in the western United States before and after onset of winter. RESULTS: PERMANOVA analysis revealed the deer microbiota varied interactively with geography and season. Further, using metadata collected at the time of sampling, we were able to identify different fecal bacterial taxa that could potentially act as bioindicators of mule deer health outcomes. First, we identified the abundance of Collinsella (family: Coriobacteriaceae) reads as a possible predictor of poor overwintering outcomes for deer herds in multiple locations. Second, we showed that reads assigned to the Bacteroides and Mollicutes Order RF39 were both positively correlated with deer protein levels, leading to the idea that these sequences might be useful in predicting mule deer protein storage. CONCLUSIONS: These analyses confirm that variation in the microbiota is associated with season-dependent health outcomes in mule deer, which may have useful implications for herd management strategies.

Genetic Influences of the Microbiota on the Life Span of Drosophila melanogaster.

To better understand how associated microorganisms ("microbiota") influence organismal aging, we focused on the model organism Drosophila melanogaster We conducted a metagenome-wide association (MGWA) as a screen to identify bacterial genes associated with variation in the D. melanogaster life span. The results of the MGWA predicted that bacterial cysteine and methionine metabolism genes influence fruit fly longevity. A mutant analysis, in which flies were inoculated with Escherichia coli strains bearing mutations in various methionine cycle genes, confirmed a role for some methionine cycle genes in extending or shortening fruit fly life span. Initially, we predicted these genes might influence longevity by mimicking or opposing methionine restriction, an established mechanism for life span extension in fruit flies. However, follow-up transcriptome sequencing (RNA-seq) and metabolomic experiments were generally inconsistent with this conclusion and instead implicated glucose and vitamin B6 metabolism in these influences. We then tested if bacteria could influence life span through methionine restriction using a different set of bacterial strains. Flies reared with a bacterial strain that ectopically expressed bacterial transsulfuration genes and lowered the methionine content of the fly diet also extended female D. melanogaster life span. Taken together, the microbial influences shown here overlap with established host genetic mechanisms for aging and therefore suggest overlapping roles for host and microbial metabolism genes in organismal aging.IMPORTANCE Associated microorganisms ("microbiota") are intimately connected to the behavior and physiology of their animal hosts, and defining the mechanisms of these interactions is an urgent imperative. This study focuses on how microorganisms influence the life span of a model host, the fruit fly Drosophila melanogaster First, we performed a screen that suggested a strong influence of bacterial methionine metabolism on host life span. Follow-up analyses of gene expression and metabolite abundance identified stronger roles for vitamin B6 and glucose than methionine metabolism among the tested mutants, possibly suggesting a more limited role for bacterial methionine metabolism genes in host life span effects. In a parallel set of experiments, we created a distinct bacterial strain that expressed life span-extending methionine metabolism genes and showed that this strain can extend fly life span. Therefore, this work identifies specific bacterial genes that influence host life span, including in ways that are consistent with the expectations of methionine restriction.

The microbiota influences the Drosophila melanogaster life history strategy.

Organisms are locally adapted when members of a population have a fitness advantage in one location relative to conspecifics in other geographies. For example, across latitudinal gradients, some organisms may trade off between traits that maximize fitness components in one, but not both, of somatic maintenance or reproductive output. Latitudinal gradients in life history strategies are traditionally attributed to environmental selection on an animal's genotype, without any consideration of the possible impact of associated microorganisms ("microbiota") on life history traits. Here, we show in Drosophila melanogaster, a key model for studying local adaptation and life history strategy, that excluding the microbiota from definitions of local adaptation is a major shortfall. First, we reveal that an isogenic fly line reared with different bacteria varies the investment in early reproduction versus somatic maintenance. Next, we show that in wild fruit flies, the abundance of these same bacteria was correlated with the latitude and life history strategy of the flies, suggesting geographic specificity of the microbiota composition. Variation in microbiota composition of locally adapted D. melanogaster could be attributed to both the wild environment and host genetic selection. Finally, by eliminating or manipulating the microbiota of fly lines collected across a latitudinal gradient, we reveal that host genotype contributes to latitude-specific life history traits independent of the microbiota and that variation in the microbiota can suppress or reverse the differences between locally adapted fly lines. Together, these findings establish the microbiota composition of a model animal as an essential consideration in local adaptation.

MAGNAMWAR: an R package for genome-wide association studies of bacterial orthologs.

Summary: Here we report on an R package for genome-wide association studies of orthologous genes in bacteria. Before using the software, orthologs from bacterial genomes or metagenomes are defined using local or online implementations of OrthoMCL. These presence-absence patterns are statistically associated with variation in user-collected phenotypes using the Mono-Associated GNotobiotic Animals Metagenome-Wide Association R package (MAGNAMWAR). Genotype-phenotype associations can be performed with several different statistical tests based on the type and distribution of the data. Availability and implementation: MAGNAMWAR is available on CRAN. Contact: john_chaston@byu.edu.

Bacterial Methionine Metabolism Genes Influence Drosophila melanogaster Starvation Resistance.

Animal-associated microorganisms (microbiota) dramatically influence the nutritional and physiological traits of their hosts. To expand our understanding of such influences, we predicted bacterial genes that influence a quantitative animal trait by a comparative genomic approach, and we extended these predictions via mutant analysis. We focused on Drosophila melanogaster starvation resistance (SR). We first confirmed that D. melanogaster SR responds to the microbiota by demonstrating that bacterium-free flies have greater SR than flies bearing a standard 5-species microbial community, and we extended this analysis by revealing the species-specific influences of 38 genome-sequenced bacterial species on D. melanogaster SR. A subsequent metagenome-wide association analysis predicted bacterial genes with potential influence on D. melanogaster SR, among which were significant enrichments in bacterial genes for the metabolism of sulfur-containing amino acids and B vitamins. Dietary supplementation experiments established that the addition of methionine, but not B vitamins, to the diets significantly lowered D. melanogaster SR in a way that was additive, but not interactive, with the microbiota. A direct role for bacterial methionine metabolism genes in D. melanogaster SR was subsequently confirmed by analysis of flies that were reared individually with distinct methionine cycle Escherichia coli mutants. The correlated responses of D. melanogaster SR to bacterial methionine metabolism mutants and dietary modification are consistent with the established finding that bacteria can influence fly phenotypes through dietary modification, although we do not provide explicit evidence of this conclusion. Taken together, this work reveals that D. melanogaster SR is a microbiota-responsive trait, and specific bacterial genes underlie these influences.IMPORTANCE Extending descriptive studies of animal-associated microorganisms (microbiota) to define causal mechanistic bases for their influence on animal traits is an emerging imperative. In this study, we reveal that D. melanogaster starvation resistance (SR), a model quantitative trait in animal genetics, responds to the presence and identity of the microbiota. Using a predictive analysis, we reveal that the amino acid methionine has a key influence on D. melanogaster SR and show that bacterial methionine metabolism mutants alter normal patterns of SR in flies bearing the bacteria. Our data further suggest that these effects are additive, and we propose the untested hypothesis that, similar to bacterial effects on fruit fly triacylglyceride deposition, the bacterial influence may be through dietary modification. Together, these findings expand our understanding of the bacterial genetic basis for influence on a nutritionally relevant trait of a model animal host.

A genomic investigation of ecological differentiation between free-living and Drosophila-associated bacteria.

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome-scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.

The Drosophila transcriptional network is structured by microbiota.

BACKGROUND: Resident microorganisms (microbiota) have far-reaching effects on the biology of their animal hosts, with major consequences for the host's health and fitness. A full understanding of microbiota-dependent gene regulation requires analysis of the overall architecture of the host transcriptome, by identifying suites of genes that are expressed synchronously. In this study, we investigated the impact of the microbiota on gene coexpression in Drosophila. RESULTS: Our transcriptomic analysis, of 17 lines representative of the global genetic diversity of Drosophila, yielded a total of 11 transcriptional modules of co-expressed genes. For seven of these modules, the strength of the transcriptional network (defined as gene-gene coexpression) differed significantly between flies bearing a defined gut microbiota (gnotobiotic flies) and flies reared under microbiologically sterile conditions (axenic flies). Furthermore, gene coexpression was uniformly stronger in these microbiota-dependent modules than in both the microbiota-independent modules in gnotobiotic flies and all modules in axenic flies, indicating that the presence of the microbiota directs gene regulation in a subset of the transcriptome. The genes constituting the microbiota-dependent transcriptional modules include regulators of growth, metabolism and neurophysiology, previously implicated in mediating phenotypic effects of microbiota on Drosophila phenotype. Together these results provide the first evidence that the microbiota enhances the coexpression of specific and functionally-related genes relative to the animal's intrinsic baseline level of coexpression. CONCLUSIONS: Our system-wide analysis demonstrates that the presence of microbiota enhances gene coexpression, thereby structuring the transcriptional network in the animal host. This finding has potentially major implications for understanding of the mechanisms by which microbiota affect host health and fitness, and the ways in which hosts and their resident microbiota coevolve.

Host Genetic Control of the Microbiota Mediates the Drosophila Nutritional Phenotype.

A wealth of studies has demonstrated that resident microorganisms (microbiota) influence the pattern of nutrient allocation to animal protein and energy stores, but it is unclear how the effects of the microbiota interact with other determinants of animal nutrition, including animal genetic factors and diet. Here, we demonstrate that members of the gut microbiota in Drosophila melanogaster mediate the effect of certain animal genetic determinants on an important nutritional trait, triglyceride (lipid) content. Parallel analysis of the taxonomic composition of the associated bacterial community and host nutritional indices (glucose, glycogen, triglyceride, and protein contents) in multiple Drosophila genotypes revealed significant associations between the abundance of certain microbial taxa, especially Acetobacteraceae and Xanthamonadaceae, and host nutritional phenotype. By a genome-wide association study of Drosophila lines colonized with a defined microbiota, multiple host genes were statistically associated with the abundance of one bacterium, Acetobacter tropicalis. Experiments using mutant Drosophila validated the genetic association evidence and reveal that host genetic control of microbiota abundance affects the nutritional status of the flies. These data indicate that the abundance of the resident microbiota is influenced by host genotype, with consequent effects on nutrient allocation patterns, demonstrating that host genetic control of the microbiome contributes to the genotype-phenotype relationship of the animal host.

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes.

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.

The bacterial communities in plant phloem-sap-feeding insects.

The resident microbiota of animals represents an important contribution to the global microbial diversity, but it is poorly known in many animals. This study investigated the bacterial diversity in plant phloem-sap-feeding whiteflies, aphids and psyllids by pyrosequencing bacterial 16S rRNA gene amplicons. After correction for sequencing error, just 3-7 bacterial operational taxonomic units were recovered from each insect sample sequenced to sufficient depth for saturation of rarefaction curves. Most samples were dominated by primary and secondary symbionts, which are localized to insect cells or the body cavity, indicative of a dearth of bacterial colonists of the gut lumen. Diversity indices of the bacterial communities (Shannon's index: 0.40-1.46, Simpson's index: 0.15-0.74) did not differ significantly between laboratory and field samples of the phloem-feeding insects, but were significantly lower than in drosophilid flies quantified by the same methods. Both the low bacterial content of the phloem sap diet and biological processes in the insect may contribute to the apparently low bacterial diversity in these phloem-feeding insects.

Previously unrecognized stages of species-specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes.

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species-specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species-specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal-intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species-specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.

Humidity determines penetrance of a latitudinal gradient in genetic selection on the microbiota by Drosophila melanogaster.

The fruit fly Drosophila melanogaster is a model for understanding how hosts and their microbial partners interact as the host adapts to wild environments. These interactions are readily interrogated because of the low taxonomic and numeric complexity of the flies' bacterial communities. Previous work has established that host genotype, the environment, diet, and interspecies microbial interactions can all influence host fitness and microbiota composition, but the specific processes and characters mediating these processes are incompletely understood. Here, we compared the variation in microbiota composition between wild-derived fly populations when flies could choose between the microorganisms in their diets and when flies were reared under environmental perturbation (different humidities). We also compared the colonization of the resident and transient microorganisms. We show that the ability to choose between microorganisms in the diet and the environmental condition of the flies can influence the relative abundance of the microbiota. There were also key differences in the abundances of the resident and transient microbiota. However, the microbiota only differed between populations when the flies were reared at humidities at or above 50% relative humidity. We also show that elevated humidity determined the penetrance of a gradient in host genetic selection on the microbiota that is associated with the latitude the flies were collected from. Finally, we show that the treatment-dependent variation in microbiota composition is associated with variation in host stress survival. Together, these findings emphasize that host genetic selection on the microbiota composition of a model animal host can be patterned with the source geography, and that such variation has the potential to influence their survival in the wild. Importance: The fruit fly Drosophila melanogaster is a model for understanding how hosts and their microbial partners interact as hosts adapt in wild environments. Our understanding of what causes geographic variation in the fruit fly microbiota remains incomplete. Previous work has shown that the D. melanogaster microbiota has relatively low numerical and taxonomic complexity. Variation in the fly microbiota composition can be attributed to environmental characters and host genetic variation, and variation in microbiota composition can be patterned with the source location of the flies. In this work we explored three possible causes of patterned variation in microbiota composition. We show that host feeding choices, the host niche colonized by the bacteria, and a single environmental character can all contribute to variation in microbiota composition. We also show that penetrance of latitudinally-patterned host genetic selection is only observed at elevated humidities. Together, these results identify several factors that influence microbiota composition in wild fly genotypes and emphasize the interplay between environmental and host genetic factors in determining the microbiota composition of these model hosts.

Flagellar Genes Are Associated with the Colonization Persistence Phenotype of the Drosophila melanogaster Microbiota.

In this work, we use Drosophila melanogaster as a model to identify bacterial genes necessary for bacteria to colonize their hosts independent of the bulk flow of diet. Early work on this model system established that dietary replenishment drives the composition of the D. melanogaster gut microbiota, and subsequent research has shown that some bacterial strains can stably colonize, or persist within, the fly independent of dietary replenishment. Here, we reveal transposon insertions in specific bacterial genes that influence the bacterial colonization persistence phenotype by using a gene association approach. We initially established that different bacterial strains persist at various levels, independent of dietary replenishment. We then repeated the analysis with an expanded panel of bacterial strains and performed a metagenome-wide association (MGWA) study to identify distinct bacterial genes that are significantly correlated with the level of colonization by persistent bacterial strains. Based on the MGWA study, we tested if 44 bacterial transposon insertion mutants from 6 gene categories affect bacterial persistence within the flies. We identified that transposon insertions in four flagellar genes, one urea carboxylase gene, one phosphatidylinositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly influenced the colonization of D. melanogaster by an Acetobacter fabarum strain. Follow-up experiments revealed that each flagellar mutant was nonmotile, even though the wild-type strain was motile. Taken together, these results reveal that transposon insertions in specific bacterial genes, including motility genes, are necessary for at least one member of the fly microbiota to persistently colonize the fly. IMPORTANCE Despite the growing body of research on the microbiota, the mechanisms by which the microbiota colonizes a host can still be further elucidated. This study identifies bacterial genes that are associated with the colonization persistence phenotype of the microbiota in Drosophila melanogaster, which reveals specific bacterial factors that influence the establishment of the microbiota within its host. The identification of specific genes that affect persistence can help inform how the microbiota colonizes a host. Furthermore, a deeper understanding of the genetic mechanisms of the establishment of the microbiota could aid in the further development of the Drosophila microbiota as a model for microbiome research.

Mutational analyses reveal overall topology and functional regions of NilB, a bacterial outer membrane protein required for host association in a model of animal-microbe mutualism.

The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon noncognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.

Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes.

Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.

Glacial Legacies: Microbial Communities of Antarctic Refugia.

In the cold deserts of the McMurdo Dry Valleys (MDV) the suitability of soil for microbial life is determined by both contemporary processes and legacy effects. Climatic changes and accompanying glacial activity have caused local extinctions and lasting geochemical changes to parts of these soil ecosystems over several million years, while areas of refugia may have escaped these disturbances and existed under relatively stable conditions. This study describes the impact of historical glacial and lacustrine disturbance events on microbial communities across the MDV to investigate how this divergent disturbance history influenced the structuring of microbial communities across this otherwise very stable ecosystem. Soil bacterial communities from 17 sites representing either putative refugia or sites disturbed during the Last Glacial Maximum (LGM) (22-17 kya) were characterized using 16 S metabarcoding. Regardless of geographic distance, several putative refugia sites at elevations above 600 m displayed highly similar microbial communities. At a regional scale, community composition was found to be influenced by elevation and geographic proximity more so than soil geochemical properties. These results suggest that despite the extreme conditions, diverse microbial communities exist in these putative refugia that have presumably remained undisturbed at least through the LGM. We suggest that similarities in microbial communities can be interpreted as evidence for historical climate legacies on an ecosystem-wide scale.