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 <i>gyrB</i> and <i>recA</i>. However, certain bacterial genera, including <i>Xanthomonas</i> and <i>Pseudomonas</i>, present challenges in clear classification at the species or subspecies level using these single-marker genes. Here, we explored the potential of the <i>rfbA</i> gene as a novel marker for bacterial classification. The <i>rfbA</i> 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 <i>rfbA</i> has undergone host-specific adaptive evolution, distinguishing it from conserved housekeeping genes. Therefore, we targeted <i>Xanthomonas</i> and <i>Pseudomonas</i> species to demonstrate the utility of <i>rfbA</i> 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 <i>rfbA</i> variation and host specificity.