The rfbA Gene as a High-Resolution Marker for Pathovar-Level Classification of Xanthomonas citri and Pseudomonas syringae

Bacterial identification and classification are commonly performed based on the sequence of the 16S rRNA and several gold-standard marker genes, such as gyrB and recA. However, certain bacterial genera, including Xanthomonas and Pseudomonas, present challenges in clear classification at the species or subspecies level using these single-marker genes. Here, we explored the potential of the rfbA gene as a novel marker for bacterial classification. The rfbA gene, involved in lipopolysaccharide synthesis, is a conserved gene present in most gram-negative bacteria and is sufficiently short (approximately 850 bp). In this study, we identified that rfbA has undergone host-specific adaptive evolution, distinguishing it from conserved housekeeping genes. Therefore, we targeted Xanthomonas and Pseudomonas species to demonstrate the utility of rfbA in resolving taxonomic challenges at the pathovar level. These differences were strongly associated with the hosts of the respective strains, suggesting a potential evolutionary link between rfbA variation and host specificity.