Neugent, M. L.; Dalvi, H. H.; Saenz, C. N.; Gauch, J. L.; Koonjbearry, N. S.; Komarovsky, J.; Lim, K. C.; Spiro, E.; De Nisco, N. J.

The female urinary microbiome (FUM) has emerged a promising therapeutic target for recurrent urinary traction infection (rUTI). The FUM is dominated by a phylogenetically coherent group of Lactobacillus species whose metabolic relationship with the host remains poorly understood. Here, we demonstrate that fatty acid (FA) auxotrophy is a universal, conserved trait of FUM Lactobacillus species, mechanistically underpinned by complete loss or inactivation of the fab operon encoding Type II fatty acid synthesis (FASII). Stable isotope tracing with [U 13C] glucose confirms that L. crispatus and L. gasseri incorporate no glycolysis-derived carbon into cellular lipids, while generalist, non-FUM Lactobacillaceae species retain de novo FA biosynthesis. Gene synteny analysis across the Lactobacillaceae family reveals that fab operon loss is strongly associated with host-adapted lifestyles. Despite phenotypic FA auxotrophy among L. crispatus strains, a complete syntenic fab operon was identified in 31.9% of L. crispatus genomes. However, genomic inspection revealed a pervasive, inactivating frameshift mutation in fabH, the gene encoding the rate-limiting initiation condensing enzyme, in fab+ L. crispatus genomes highlighting two distinct evolutionary pathways of FA auxotrophy among this critically important FUM species. FA specificity assays establish L. crispatus and L. gasseri have an obligate requirement for monounsaturated FAs of C14-C20 chain length and that these species do not structurally modify supplemented FAs. Metabolic tracing further demonstrates that FUM lactobacilli directly scavenge and incorporate FAs from human bladder epithelial lipids, producing a bacterial membrane composition that mirrors the host. These findings establish FA auxotrophy as a defining adaptation to the female urogenital niche, with direct implications for development of microbiome-based therapies for rUTI.