A Syntenic Pangenome of Gardnerella Reveals Novel Plasmids and Phage, Taxonomic Boundaries, and Species-Level Stratification of Metabolic and Virulence Potential

Abstract

Gardnerella species are central to bacterial vaginosis (BV), a condition affecting nearly one in three women of reproductive age and associated with preterm birth and increased susceptibility to sexually transmitted infections. Despite decades of study, progress in defining Gardnerella diversity has been hindered by inconsistent taxonomy and poor-quality genomic resources. Here, we sequenced 392 new Gardnerella isolates from asymptomatic and BV-associated microbiota and integrated this collection with all publicly available genomes. After stringent curation, we generated a high-quality reference set of 313 distinct genomes that underpins a comprehensive taxonomic framework. Using average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and phylogenomics, we resolved 21 genomic lineages encompassing 11 species and 15 subspecies, each assigned a provisional formal name. Integration of complete long-read assemblies enabled construction of the first syntenic Gardnerella pangenome, revealing lineage-specific repertoires of virulence, metabolic, and defense systems - including variable sialidases (NanH), vaginolysin, and amino-acid biosynthetic pathways - and defining conserved genomic architecture across species. Comparative methylome profiling further highlighted restriction-modification system diversity that may influence genetic exchange. Finally, we identified the first native cryptic plasmids in Gardnerella, overturning the assumption that the genus lacks plasmids, and demonstrated their use in generating a replicative E. coli-Gardnerella shuttle vector. 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.