Elucidating Functions of FleQ in Xanthomonas oryzae pv. oryzae by Comparative Proteomic and Phenotypic Analyses

To acclimate to different environments, gene expression has to be controlled using diverse transcriptional activators. FleQ activates σ⁵⁴-dependent transcription initiation and regulates flagellar biosynthesis and other mechanisms in several bacteria. Xanthomonas oryzae pv. oryzae (Xoo), which is a causal agent of bacterial leaf blight on rice, lacking FleQ loses swimming motility and virulence is not altered. However, other biological mechanisms related with FleQ in Xoo are unknown. In this study, we generated the FleQ-overexpressing strain, Xoo(FleQ), and knockout mutant, XooΔfleQ. To predict the mechanisms affected by FleQ, label-free shotgun comparative proteomics was carried out. Based on proteomic results, we performed diverse phenotypic assays. Xoo(FleQ) had reduced ability to elicit disease symptoms and exopolysaccharide production. Additionally, the ability of XooΔfleQ(EV) (empty vector) and Xoo(FleQ) to form biofilm was decreased. Swarming motility of XooΔfleQ(EV) was abolished, but was only reduced for Xoo(FleQ). Additionally, abnormal twitching motility was observed in both strains. Siderophore production of Xoo(FleQ) was enhanced in iron-rich conditions. The proteomic and phenotypic analyses revealed that FleQ is involved in flagellar-dependent motility and other mechanisms, including symptom development, twitching motility, exopolysaccharide production, biofilm formation, and siderophore production. Thus, this study provides fundamental information about a σ⁵⁴-dependent transcription activator in Xoo.