The evolutionary dynamics and functional role of the REPIN-RAYT system in the hut locus of Pseudomonas fluorescens SBW25

Mobile genetic elements (MGEs) shape bacterial genomes but often degenerate or vanish. In contrast, the REPIN-RAYT system, composed of repetitive extragenic palindromic doublets forming hairpins (REPINs) and REP-associated tyrosine transposases (RAYTs), is widespread and persists. REPINs can form hairpin structures and affect gene regulation, while RAYTs resemble housekeeping genes. The system is thought to provide a fitness benefit, though mechanisms remain unclear.

In Chapter 3, I examined the evolutionary dynamics of RAYTs in Pseudomonas fluorescens SBW25 and related strains, focusing on the histidine utilization (hut) locus. I found that horizontal transfer of RAYTs occurs more often than assumed and that RAYTs can associate with multiple REPIN groups. Six REPIN sites in the hut locus were conserved across strains, though sequences varied depending on the RAYT present.

In Chapter 4, I tested REPIN function using deletion mutants. Loss of REPIN RH, located between hutH2 and hutTh, caused reduced fitness early in competition but increased fitness later. RT-qPCR indicated that REPIN RH downregulates these genes, explaining the dynamic phenotype. Deletion of RAYTs had no detectable effect.

In Chapter 5, I explored the role of hutD, a gene of unclear function. ΔhutD populations evolved for ~200 generations showed increased fitness. Genome sequencing revealed adaptive changes mainly in motility and biofilm genes, suggesting environmental adaptation rather than compensation for hutD loss.

Overall, my work shows that REPINs can modulate gene expression and bacterial fitness, and that the REPIN-RAYT system is more dynamic than previously recognized. These findings highlight REPINs as regulatory elements and advance understanding of the hut locus.

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