3). Thus, XrvB may regulate not only hrp gene

expression but also the expression of genes involved in bacterial growth. H-NS protein binds preferentially to curved DNA, which is commonly associated with promoters, via its conserved C-terminal domain (Tendeng & Bertin, 2003; Dorman 2004). Generally, the binding of H-NS leads to repression of gene expression, while its release leads to gene activation. We found that XrvB has an amino acid sequence similar to the ABT 888 core motif in the C-terminal domain of H-NS (Fig. S1). We, therefore, investigated whether XrvB has DNA-binding activity and whether the protein binds to the promoter region of hrpG. The XrvB protein tagged with six histidine residues at its C-terminus was extracted and purified from E. coli transformed with pETXrvB harboring the coding region of xrvB (Fig. 4a),

and then incubated with SspI-, PvuII- and BamHI-digested pBSHrpG-Pro, in which the putative promoter region of hrpG (−686 to +56) is contained. Electrophoresis, followed by staining with ethidium bromide revealed that, like other H-NS proteins reported previously (Zuber et al., 1994; Tendeng et al., 2003), XrvB bound to a 500-bp SspI–PvuII fragment containing the bla promoter Selleck AZD0530 with a curved structure, along with the 1900-bp fragment derived from the vector sequence (Fig. 4b). The electrophoretic mobility of the fragment was completely retarded at a protein concentration of 1.8 μM. Under the same conditions, the 740-bp BamHI fragment containing the hrpG promoter was not retarded as much as the 500- and 1900-bp fragments. When the predicted promoter region of hrpG was examined using the bend-it computer program (http://www.icgeb.org/dna/bend_it.html), the

possibility that it possesses curved regions Flavopiridol (Alvocidib) was low (data not shown). The results suggest that XrvB possesses DNA-binding activity, but that it does not bind to the hrpG promoter. It is likely that the regulation of hrpG expression by XrvB is indirect and that some unknown gene(s)/protein(s) mediate the regulation. Although many researchers have contributed to identifying various hrp regulatory genes in Xoo and other Xanthomonas spp., the entire scheme of the complicated hrp-regulatory cascade remains unclear (Tsuge et al., 2006; Lee et al., 2008; Zhang et al., 2008; Feng et al., 2009; Huang et al., 2009). Here, our study suggests that the H-NS-like, DNA-binding protein XrvB is involved in the negative regulation of hrp gene expression in Xoo by repressing the expression of a key hrp regulatory gene hrpG. Besides the regulation of hrp gene expression, XrvB is likely to be involved in the regulation of various genes because the growth of the mutant decreased under the culture conditions. Moreover, virulence of the XrvB mutant on rice decreased compared with the wild type (data not shown).