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 starvation resistance (SR). We first confirmed that 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 SR. A subsequent metagenome-wide association analysis predicted bacterial genes with potential influence on 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 SR in a way that was additive, but not interactive, with the microbiota. A direct role for bacterial methionine metabolism genes in SR was subsequently confirmed by analysis of flies that were reared individually with distinct methionine cycle mutants. The correlated responses of 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 SR is a microbiota-responsive trait, and specific bacterial genes underlie these influences. 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 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 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.

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.

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter, so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila. Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB, show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits.

19Organismal life history traits are ideally adapted to local environments when an organism has a 20 fitness advantage in one location relative to conspecifics from other geographies. Local 21 adaptation has been best studied across, for example, latitudinal gradients, where organisms 22 Significance statement 39Explanations of local adaptation have historically focused on how animal genotypes respond to 40 environmental selection. Although the impact of variation in host life histories on the composition 41 of the microbiota has been investigated for many associations, the scale and pattern of 42 microbial effects on host life history strategy are largely unknown. Here we demonstrate in the 43 fruit fly Drosophila melanogaster that microbiota effects on host life history strategy in the 44 laboratory are matched by patterns of microbiota composition in wild host populations. In 45 particular, microbiota composition varies with latitude and the effects of the microbiota on life 46 history traits are greater than host genetic adaptations. Together, these findings demonstrate 47

The ability of associated microorganisms (“microbiota”) to influence animal life history traits has been recognized and investigated, especially in the past 2 decades. For many microbial communities, there is not always a clear definition of whether the microbiota or its members are beneficial, pathogenic, or relatively neutral to their hosts’ fitness.