A ten gene-containing genomic island determines flagellin glycosylation: implication for its regulatory role in motility and virulence of Xanthomonas oryzae pv. oryzae

Abstract
    Flagellin glycosylation plays a crucial role in flagellar assembly, motility and virulence in several pathogenic bacteria. However, little is known about the genetic determinants and biological functions of flagellin glycosylation in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Here, the structure, regulation and functions of a ten-gene cluster gigX (glycosylation island genes of Xoo), which was embedded in a flagellar regulon, were characterized. gigX1 to gigX10 encoded putative enzymes or proteins involved in glycan biosynthesis and transfer, including a nucleotide sugar transaminase, an acyl-carrier protein (ACP), a 3-oxoacyl-ACP synthase, a 3-oxoacyl-ACP reductase, a dehydrogenase, an acetyltransferase, a ring hydroxylating dioxygenase, a hypothetical protein, a methyltransferanse and a glycosyltransferase, respectively. The gigX genes were co-transcribed in an operon and up-regulated by the upstream r54 factor RpoN2 and transcriptional activator FleQ. In-frame deletion of each gigX gene affected flagellin glycosylation modification, meaning that the unglycosylated flagellin of the mutants was smaller than the glycosylated flagellin of the wild-type. No significant changes in flagellar filament and motility were observed in the DgigX mutants, among which only DgigX6 displayed increased swimming ability. Importantly, all mutants, except DgigX9, showed significantly increased virulence and bacterial growth in the susceptible rice cultivar IR24, and DgigX1 and DgigX10 showed enhanced type III secretion system (T3SS)-related gene expression. Moreover, the glycosylated flagellin of the wild-type induced higher H2O2 levels in rice leaves than did the unglycosylated flagellins of DgigX1 or DgigX10. Taken together, this study reveals that the gigX cluster determines flagellin glycosylation, and implicates the regulatory role of post-translational modification with the glycosylation, acetylation and methylation of flagellin in the regulation of motility and virulence of Xoo.