Pseudovibriamides from Pseudovibrio marine sponge bacteria promote swarming motility via transcriptional modulation

Pseudovibrio a-Proteobacteria have been repeatedly isolated from marine sponges and proposed to be beneficial to the host. Bacterial motility is known to contribute to host colonization. We have previously identified pseudovibriamides A and B, produced in culture by Pseudovibrio brasiliensis Ab134, and shown that pseudovibriamide A promotes flagellar motility. Pseudovibriamides are encoded in a hybrid nonribosomal peptide synthetase-polyketide synthase gene cluster that also includes several accessory genes. Pseudovibriamide A is a linear heptapeptide and pseudovibriamide B is a nonadepsipeptide derived from pseudovibriamide A. Here we define the borders of the pseudovibriamides gene cluster, assign function to biosynthetic genes using reverse genetics and test the hypothesis that pseudovibriamides impact motility by modulating gene transcription. RNA-seq transcriptomic analyses of strains having different compositions of pseudovibriamides suggested that both pseudovibriamides A and B affect genes potentially involved in motility, and that a compensatory mechanism is at play in mutants that produce only pseudovibriamide A, resulting in comparable swarming motility as the wild type. The data gathered suggest that pseudovibriamides A and B have opposite roles in modulating a subset of genes, with pseudovibriamide B having a primary effect in gene activation, and pseudovibriamide A on inhibition. Finally, we observed many differentially expressed genes (up to 29% of the total gene number) indicating that pseudovibriamides have a global effect on transcription that goes beyond motility. Overall design: To investiagte the impact of different compositions of pseudovibriamides on gene transcription, we extracted RNA from 24-h triplicate liquid cultures of the wild type (all pseudovibriamides are present), the ?pppA mutant (only pseudovibriamide C is present), the ?pppD mutant (only pseudovibriamide A is present) and the ?pppE mutant (no pseudovibriamides are present). We then calculated gene expression levels using raw reads obtained from RNA-seq of each strain. Finally, we performed pairwise differential expression analysis between each mutant and the wild type, and between the ?pppA mutant and the ?pppD mutant using average TPM values.