Abstract
The social motility (S-motility) of Myxococcus xanthus is driven by the intricate interplay between type IVa pili (T4aP) and exopolysaccharide (EPS). Although T4aP retraction furnishes the mechanical force necessary for cellular movement, EPS serves as a matrix for T4aP anchoring and instigates pilus retraction. Despite this interdependence, the molecular determinants governing specific T4aP-EPS recognition and interaction have yet to be fully elucidated. Utilizing a comprehensive approach, our study identifies glucosamine as the key monosaccharide within EPS that interacts with the major pilin PilA, mediated by a tryptophan residue (W146). Significantly, mutagenesis of W146 to alanine (W146A) resulted in a markedly impaired EPS recognition and abrogation of S-motility, although the T4aP retained the capacity for extension and retraction. Saturation mutagenesis studies further emphasized the critical role of aromatic conservation at PilA position 146 in ensuring effective EPS recognition. Given the pivotal roles of T4aP and EPS in orchestrating bacterial multicellular behavior, gaining a profound understanding of their interaction will provide fundamental insights into the molecular basis of the collective behavior within bacterial communities.IMPORTANCEOur study reveals a key molecular mechanism that controls the interaction between T4aP and EPS, which is a critical process for bacterial social motility, biofilm formation, predation, and other collective behaviors. We identify a specific aromatic residue (W146) in the major pilin PilA that mediates direct binding to glucosamine-containing polysaccharides, thereby linking T4aP function and extracellular matrix recognition. This finding provides new insight into how bacteria use glycans to coordinate group behaviors within microbial communities. These results open potential strategies for controlling biofilm-related processes, such as disrupting infections or guiding beneficial microbial assemblies in environmental and industrial settings.