Diversity, classification, and evolution of myxobacterial PilY1 proteins

Type IVa pili (T4aP) mediate one of the most widespread forms of bacterial surface motility through coordinated cycles of extension, attachment, and retraction that generate pulling forces to propel cells forward. This process is well characterized in diverse Gram-negative bacteria such as Pseudomonas, Myxococcus, and Neisseria, where T4aP filaments are composed of thousands of major pilin subunits and a tip complex formed by minor pilins and the PilY1 adhesin proteins. PilY1 is a multifunctional protein localized at the T4aP machine and pilus tip, playing critical roles in pilus priming, surface adhesion, motility, and virulence. Myxococcus xanthus possesses three distinct PilY1 adhesins with conserved C-terminal but different N-terminal, where each is encoded within separate minor pilin/pilY1 gene clusters, suggesting functional specialization. This study investigates the extent of PilY1 diversity and domain architecture conservation across the phylum Myxococcota using genomic, phylogenetic, and structural approaches, suggesting a remarkable evolutionary strategy for tailoring T4aP tip complexes to diverse environmental and physiological demands. Our analysis of sixty-seven representative genomes reveals that PilY1 proteins are widely distributed and typically occur in multiple copies, with an average of two homologs per genome. Phylogenetic reconstruction identifies several well-supported clades supported by myxobacterial taxonomy, domain architecture, protein length, and cysteine content. Notably, M. xanthus paralogs PilY1.1 and PilY1.2 form a conserved lineage characterized by a DUF4114 domain and appear to have evolved primarily through vertical inheritance, whereas PilY1.3 clusters with homologs from diverse bacterial phyla, suggesting acquisition via horizontal gene transfer. We reconfirmed that pilY1 genes frequently occur in conserved operons with minor pilins (pilX, pilW, pilV, and fimU), supporting their role in forming priming complexes initiating pilus assembly. Structural modeling predicts conserved interaction patterns within minor pilins and PilY1 via β-strand complementation between PilX and PilY1, highlighting a potentially conserved structural feature of T4aP tip complexes. Together, our findings reveal extensive diversification of PilY1 proteins within Myxococcota and suggest that variation in their N-terminal domains contributes to functional specialization of T4aP systems. Future experimental studies will be essential to determine how this diversity shapes mechanosensing, adhesion, and environmental adaptation in myxobacteria and other bacteria.