A Mobile Glycosylation Locus Modulates Cell Wall Architecture in Lactobacillus crispatus

Lactobacillus crispatus dominance in the vaginal microbiome is associated with beneficial health outcomes, yet strain-level variation and its implications remain poorly understood. Here, we resolve the genomic context of three glycosyltransferase gene fragments (GT1-3) previously linked with dysbiotic states. Long-read resequencing revealed that GT1-3 are part of a ~18.7 kb Wzx/Wzy-dependent cell wall polysaccharide (CWPS) locus, containing several IS256-family transposases. Serial propagation in vitro produced isolates with 4.1 kb excised via a composite transposon encompassing the GT3, UDP-galactopyranose mutase, flippase, and hypothetical protein, demonstrating structural plasticity. Transmission electron microscopy showed a ~20%-25% thinner peptidoglycan layer in the derived strains, while FT-IR and monosaccharide analysis indicated no gross changes. Molecular dynamics simulations suggest that GT3 contributes to the structural stability of the glycosyltransferase complex, without compromising catalytic function. Together, these findings establish the CWPS locus as a mobile, structurally plastic element that directly influences cell wall architecture in L. crispatus.