Functional characterization, transcriptome and metabolome analyses reveal that pacR possesses multifaceted...

Xanthomonas campestris pathovar campestris (Xcc) is the pathogen responsible for causing black rot in cruciferous plants. In this study, we show that mutation of AAW18_RS04175 (pacR, encodes a hypothetical protein containing a domain of unknown function, DUF1631) of Xcc strain Xc17 had decreased bacterial attachment, exopolysaccharide production, hypersensitive response and virulence. Furthermore, the pacR mutant exhibited reduced cell membrane integrity and outer membrane vesicle production. Transcriptomic analysis indicated that 225 genes were differentially expressed following pacR mutation. These genes can be classified into various functional categories, such as the type three secretion system and membrane component. Among them, genes associated with attachment, exopolysaccharide synthesis, the type three secretion system, and nucleotide metabolism were further verified by quantitative RT-PCR. Metabolomic analysis showed that 81 and 132 metabolites in positive and negative modes, respectively, were altered after pacR mutation. Among the identified metabolites, some are known to belong to different pathways, such as biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and nucleotide and purine metabolism, while others have not been previously documented in microbial systems. Additionally, the transcription initiation point of pacR was mapped, and promoter analysis indicated that pacR expression is influenced by different conditions. Taken together, our findings advance the understanding of PacR function and expression in Xcc and offer new insights into the role of the DUF1631-containing hypothetical protein in bacterial physiology.