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Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

In prokaryotes, two-component signal transduction systems, consisting of a histidine kinase and a response regulator, play a critical role in regulating a range of cellular functions. A recent study suggests that XCC3315, a response regulator with a CheY-like receiver domain attached to an uncharacterized HD-related output domain (HDOD domain), plays a role in the general stress response of the Gram-negative bacterium Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in cruciferous plants. Here, we demonstrated genetically that XCC3315, designated as gsmR (general stress and motility regulator), is involved in the expression of genes responsible for flagellum synthesis, including rpoN2, flhF, flhB, and fliC. Site-directed mutagenesis revealed that Glu9 and Arg100 in the receiver domain and Gly205, Asp263, His287, Trp298 and His311 in the HDOD are critical amino acids for GsmR function in cell motility regulation. The gsmR transcription initiation site was mapped. Promoter analysis and gel retardation assay revealed that the expression of gsmR is positively controlled by the global transcriptional regulator Clp in a direct manner, and is subject to catabolite repression. Our findings not only extend the previous work on Clp regulation to show that it influences the expression of gsmR in Xcc, but are also the first to characterize the expression of this response regulator gene in this phytopathogen. Furthermore, GsmR is the first HDOD-containing protein of bacteria in which key amino acids have been experimentally identified and characterized., (© 2012 The Authors Journal compilation © 2012 FEBS.)

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

In Xanthomonas campestris pv. campestris (Xcc), which is the causative agent of black rot in crucifers, the virulence factor level is substantially decreased in the mutant deficient in RpfG, a phosphodiesterase that degrades the second messenger cyclic di-GMP. The rpfG mutant also grew in an aggregated state. It is indicated that expression of Pseudomonas GGDEF domain protein WspR (a diguanylate cyclase that synthesizes cyclic di-GMP) in wild-type Xcc can produce a phenocopy of the rpfG mutant. In this study, we showed that over-expression of GGDEF domain protein XCC2731 in wild-type Xcc caused (i) aggregation of cells, (ii) reduction in motility, and (iii) decrease in production of virulence factor extracellular enzymes and exopolysaccharides. Site-directed mutagenesis of the conserved G, G, and E residues of the GGDEF domain in XCC2731 abolished its function. The XCC2731 mutant has attenuated virulence. Furthermore, XCC2731 mutant was affected in surface attachment. Using the 5' RACE method, the XCC2731 transcription initiation site was mapped at nucleotide G, 15nt upstream of the XCC2731 start codon. Transcriptional fusion assay and gel retardation analysis indicated that Clp (cAMP receptor protein-like protein) positively regulates XCC2731 transcription in a direct manner. Reporter analysis also revealed that XCC2731 transcription is subject to catabolite repression, and reduced under conditions of oxygen limitation and high osmolarity. Our findings not only extend previous work on Clp regulation to show that it influences the expression of XCC2731 in Xcc, but also are the first to characterize the GGDEF domain protein gene expression in this phytopathogen., (Copyright © 2010 Elsevier GmbH. All rights reserved.)