XCC2731, a GGDEF domain protein in Xanthomonas campestris, is involved in bacterial attachment and is positively regulated by Clp.
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.
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan, ROC. ymhsiao@ctust.edu.tw
Yi-Min
YM
Hsiao
Yu-Fan
YF
Liu
Mei-Chiung
MC
Fang
Wan-Ling
WL
Song
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000378:metabolism*; D001937:Brassica / Q000382:microbiology; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008124:Locomotion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010935:Plant Diseases / Q000382:microbiology; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*; D024363:Transcription Initiation Site; D037521:Virulence Factors / Q000096:biosynthesis*; D016959:Xanthomonas campestris / Q000235:genetics* / Q000472:pathogenicity*
D001426:Bacterial Proteins; C436751:Clp protein, Xanthomonas campestris; D011135:Polysaccharides, Bacterial; D014157:Transcription Factors; D037521:Virulence Factors