A syntenic pangenome of Gardnerella reveals novel plasmids and phage, taxonomic boundaries, and species-level stratification of metabolic and virulence potential.

Gardnerella species are key drivers of bacterial vaginosis (BV), a prevalent condition affecting nearly one in three women of reproductive age and associated with adverse reproductive outcomes. Despite decades of study, progress in defining Gardnerella diversity has been hindered by inconsistent taxonomy and poor-quality genomic resources. Here we sequenced 392 Gardnerella isolates spanning asymptomatic and BV-associated microbiota and integrated this collection with all publicly available genomes to create a curated, high-quality reference set of 312 genomes. We resolved 21 genomic lineages encompassing 11 species and 15 subspecies using phylogenomics, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and assigned each a provisional taxonomic name. Long-read assemblies enabled construction of a syntenic Gardnerella pangenome, revealing lineage-specific repertoires of virulence, metabolic, and defense, including variable sialidases (NanH), vaginolysin, and amino-acid biosynthetic pathways alongside conserved genomic organization. Comparative methylome profiling uncovered restriction-modification system diversity suggesting barriers to genetic exchange. Finally, we identified native cryptic plasmids in Gardnerella, overturning the assumption that the genus lacks plasmids. Together, these results establish a complete genomic and functional framework for Gardnerella, providing a reproducible foundation for mechanistic and translational studies of BV and a model for resolving taxonomy and functional stratification in other urogenital-associated bacteria.