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 with, 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 varying levels, independent of dietary replenishment. We then repeated the analysis with an expanded panel of bacterial strains and performed a metagenome wide association (MGWA) to identify distinct bacterial genes that are significantly correlated with the colonization level of persistent bacterial strains. Based on the MGWA, we tested if 44 bacterial transposon insertion mutants from 6 gene categories affect bacterial persistence with the flies. We identified that transposon insertions in four flagellar genes, one urea carboxylase gene, one phosphatidyl inositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly influenced the colonization of an Acetobacter fabarum strain with D. melanogaster. Follow-up experiments revealed that each flagellar mutant was non-motile, even though the wild-type strain was motile. Taken together, these results reveal 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. IMPORTANCEDespite 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 colonization persistence phenotype of the microbiota in Drosophila melanogaster, which reveals specific bacterial factors that influence establishment of the microbiota with its host. 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 further developing the Drosophila microbiota as a model for microbiome research.