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Adenosine kinase (ADK) is a purine salvage enzyme and a typical housekeeping enzyme in eukaryotes which catalyzes the phosphorylation of adenosine to form AMP. Since prokaryotes synthesize purines de novo and no endogenous ADK activity is detectable in Escherichia coli, ADK has long been considered to be rare in bacteria. To date, only two prokaryotes, both of which are gram-positive bacteria, have been reported to contain ADK. Here we report that the gram-negative bacterium Xanthomonas campestris pathovar campestris, the causal agent of black rot of crucifers, possesses a gene (designated [adk.sub.Xcc]) encoding an ADK (named [ADK.sub.Xcc]), and we demonstrate genetically that the [ADK.sub.Xcc] is involved in extracellular polysaccharide (EPS) production, cell motility, and pathogenicity of X. campestris pv. campestris, [adk.sub.Xcc] was overexpressed as a [His.sub.6]-tagged protein in E. coli, and the purified [His.sub.6]-tagged protein exhibited ADK activity. Mutation of [adk.sub.Xcc] did not affect bacterial growth in rich and minimal media but led to an accumulation of intracellular adenosine and diminutions of intracellular ADK activity and ATP level, as well as EPS. The [adk.sub.Xcc] mutant displayed significant reductions in bacterial growth and virulence in the host plant.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glucose catabolism, converting glyceraldehyde 3-phosphates to 1,3-bisphosphoglycerates. Open reading frame (ORF) XC_0972 in the genome of Xanthomonas campestris pv. campestris (Xcc) strain 8004 is the only ORF in this strain annotated to encode a GAPDH. In this work, we have demonstrated genetically that this ORF encodes a unique GAPDH in Xcc strain 8004, which seems to be constitutively expressed. A GAPDH-deficient mutant could still grow in medium with glucose or other sugars as the sole carbon source, and no phosphofructokinase activity was detectable in strain 8004. These facts suggest that Xcc may employ the Entner--Doudoroff pathway, but not glycolysis, to utilize glucose. The mutant could not utilize pyruvate as sole carbon source, whereas the wild-type could, implying that the GAPDH of Xcc is involved in gluconeogenesis. Furthermore, inactivation of the Xcc GAPDH resulted in impairment of bacterial growth and virulence in the host plant, and reduction of intracellular ATP and extracellular polysaccharide (EPS). This reveals that GAPDH is required for EPS production and full pathogenicity of Xcc.

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant: both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed [avrAC.sub.Xcc8004], functions as an avirulence gene whose product seems to be recognized in vascular tissues.

The genome of the Xanthomonas campestris pathovar campestris (Xcc) strain 8004 encodes three uncharacterized proteins, XC1166, XC1223 and XC1976, annotated as glucose kinase (Glk) by bioinformatic studies. Here we have investigated the biochemical characteristics and physiological roles of these proteins with particular reference to the synthesis of extracellular polysaccharide (EPS). XC1166, XC1223 and XC1976 were overexpressed as fusion proteins with a [His.sub.6] affinity tag and purified by nickel affinity chromatography. The standard GIk activity assay revealed that all three proteins possessed apparent Glk activity, with XC1976-[His.sub.6] being the most active; the specific activity values were 1.16 x [10.sup.6] U [mg.sup.-1] for XC1166-[His.sup.6], 4.36x[10.sup.7] U [mg.sup.-1] for XC1223-[His.sub.6] and 2.63 x [10.sup.8] U [mg.sup.-1] for XC1976-[His.sub.6]. TLC analysis showed, however, that only XC1976-[His.sub.6] could phosphorylate glucose. Insertional mutants of XC1166, XC1223 and XC1976 were generated using the suicide plasmid pK18mob. Although mutant strains with insertions in XC1166 or XC1223 had GIk activity similar to that of the wild-type strain, the XC1976 mutant had only about 6% of the wild-type activity. Mutation in XC1976 had complex effects on EPS production. In media containing arabinose, glucose, galactose, sucrose or maltose, the XC1976 mutant produced about 40-75% of the wild-type level of EPS, whereas in medium containing fructose, the mutant showed a 30% increase in EPS production compared to the wild-type strain. The X01976 mutant also showed attenuated virulence on the host plant Chinese radish (Raphanus sativus). The results indicate that XC1976 has the most significant role for the parameters tested.

The MarR family of transcriptional regulators of bacteria are involved in the regulation of many cellular processes, including pathogenesis. In this work, we have demonstrated genetically that hpaR (hpa, hrp associated), which encodes a putative MarR family regulator, is involved in the hypersensitive response (HR), pathogenicity, and extracellular protease production of the phytopathogenic bacterium Xanthomonas campestris pathovar campestris. A mutation in hpaR resulted in complete loss of virulence in the host plant cabbage, a delayed and weakened HR in the nonhost plant pepper ECW-10R, and an increase in extracellular protease production. Detection of the [beta]-glucuronidase activity of a plasmid-driven hpaR promoter-gusA reporter revealed that the expression of hpaR is positively controlled by HrpG and HrpX and is suppressed in rich medium while being strongly induced in minimal and hrp-inducing media and inside the host. These findings indicate that hpaR belongs to the hrpG and hrpX regulon and that HrpX regulates the extracellular protease production via hpaR in X. campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. The extracellular polysaccharide (EPS) produced by Xcc is an important pathogenicity factor and also has a range of industrial uses. In preliminary work a number of transposon-mediated insertion mutants in Xcc with defects in EPS production were identified. Here, one of these mutated loci was investigated in detail. Six ORFs within the locus (ORFs XC3811-3816) were disrupted by plasmid integration. Mutation of XC3813, XC3814 or XC3815 resulted in significantly reduced EPS production and significantly reduced virulence on the host plant Chinese radish (Raphanus sativus). The EPS production and virulence of XC3813, XC3814 and XC3815 mutants could be restored by intact XC3813, XC3814 and XC3815 genes, respectively, when provided in trans. Although bioinformatic analysis suggested a role for XC3814 and XC3815 in lipopolysaccharide biosynthesis, the lipopolysaccharides produced by the mutants were indistinguishable from those of the wild-type, as judged by electrophoretic mobility in SDS-polyacrylamide gels. These results reveal that XC3813, XC3814 and XC3815 comprise a novel gene cluster involved in EPS production and virulence of Xcc.

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.

Abstract Background One of the major tasks of the post-genomic era is "reading" genomic sequences in order to extract all the biological information contained in them. Although a wide variety of techniques is used to solve the gene finding problem and a number of prokaryotic gene-finding software are available, gene recognition in bacteria is far from being always straightforward. Results This study reported a thorough search for new CDS in the two published Xcc genomes. In the first, putative CDSs encoded in the two genomes were re-predicted using three gene finders, resulting in the identification of 2850 putative new CDSs. In the second, similarity searching was conducted and 278 CDSs were found to have homologs in other bacterial species. In the third, oligonucleotide microarray and RT-PCR analysis identified 147 CDSs with detectable mRNA transcripts. Finally, in-frame deletion and subsequent phenotype analysis of confirmed that Xcc_CDS002 encoding a novel SIR2-like domain protein is involved in virulence and Xcc_CDS1553 encoding a ArsR family transcription factor is involved in arsenate resistance. Conclusions Despite sophisticated approaches available for genome annotation, many cellular transcripts have remained unidentified so far in Xcc genomes. Through a combined strategy involving bioinformatic, postgenomic and genetic approaches, a reliable list of 306 new CDSs was identified and a more thorough understanding of some cellular processes was gained.

Abstract Background In bacteria, small non-coding RNAs (sRNAs) have been recognized as important regulators of various cellular processes. Approximately 200 bacterial sRNAs in total have been reported. However, very few sRNAs have been identified from phytopathogenic bacteria. Results Xanthomons campestris pathovar campestris (Xcc) is the causal agent of black rot disease of cruciferous crops. In this study, a cDNA library was constructed from the low-molecular weight RNA isolated from the Xcc strain 8004 grown to exponential phase in the minimal medium XVM2. Seven sRNA candidates were obtained by sequencing screen of 2,500 clones from the library and four of them were confirmed to be sRNAs by Northern hybridization, which were named sRNA-Xcc1, sRNA-Xcc2, sRNA-Xcc3, and sRNA-Xcc4. The transcription start and stop sites of these sRNAs were further determined. BLAST analysis revealed that the four sRNAs are novel. Bioinformatics prediction showed that a large number of genes with various known or unknown functions in Xcc 8004 are potential targets of sRNA-Xcc1, sRNA-Xcc3 and sRNA-Xcc4. In contrast, only a few genes were predicted to be potential targets of sRNA-Xcc2. Conclusion We have identified four novel sRNAs from Xcc by a large-scale screen. Bioinformatics analysis suggests that they may perform various functions. This work provides the first step toward understanding the role of sRNAs in the molecular mechanisms of Xanthomonas campestris pathogenesis.

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health. [ABSTRACT FROM AUTHOR]