Evolution of flagellar assembly regulation in host-associated bacteria: a case study on plant commensal Acidovorax delafieldii isolates

In host-associated bacteria, benefits of traits common in their free-living counterparts, such as flagellar motility, can be offset by energetic costs and the risk of triggering immune responses. While the conserved and immunogenic flagellin gene is often lost, we expect other genes of the flagellar assembly pathway to undergo diversifying selection, in particular the master regulator flhDC. FlhDC has been shown to integrate environmental signals and regulate processes other than flagellar assembly, including virulence of pathogenic bacteria. We were also interested in how loss of flhC would affect the transcriptional landscape of two closely related flhC+ and flhC- Acidovorax delafieldii strains, isolated from Lotus japonicus.

Firstly, we mined 1839 reference Proteobacteria genomes for elements of the flagellar assembly pathway using hidden Markov models. Quantity and quality of each sequence were analyzed in a phylogenetic framework, comparing statistics across environmental and host-associated strains. We constructed knock-out and revertant mutants for the flhC+ isolate and tested their motility. We cultivated the subjects in media spiked with crude extracts from Lotus japonicus, axenic or infected by Rhizophagus irregularis, an arbuscular mycorrhizal fungus. After sequencing the transcriptome, we analyzed differential expression and regulation of ecologically relevant genes and studied differences in transcriptional networks topology.

We detected lower prevalence, number of copies and scores for most flagellar assembly genes of host-associated strains. However, flhDC shows similar prevalence in both groups, but lower scores in host-associated strains. This suggests that mutation might be more beneficial than loss. As a case study, flhC- co-expression networks show widespread deregulation, with effects on several plant-associated traits and an altered transcriptional balance between type II and VI secretion systems.

Our study provides insights into evolutionary dynamics of the flagella and examines a loss of regulation scenario, showing how effects on the phenotype can extend beyond motility, indirectly affecting host-microbe-microbe relationships.