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Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases. The crystal structure of a bacterial NAMPT with high enzymatic activity reveals a novel NAM-binding tunnel, which is essential for both catalysis and inhibitor binding. The binding of NAM in the tunnel positively cooperates with the catalytic site. [ABSTRACT FROM AUTHOR]

The bacterial pathogens Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) cause leaf blight and leaf streak diseases on rice, respectively. Pathogenesis is largely defined by the virulence genes harboured in the pathogen genome. Recently, we demonstrated that the protein HpaP of the crucifer pathogen Xanthomonas campestris pv. campestris is an enzyme with both ATPase and phosphatase activities, and is involved in regulating the synthesis of virulence factors and the induction of the hypersensitive response (HR). In this study, we investigated the role of HpaP homologues in Xoo and Xoc. We showed that HpaP is required for full virulence of Xoo and Xoc. Deletion of hpaP in Xoo and Xoc led to a reduction in virulence and alteration in the production of virulence factors, including extracellular polysaccharide and cell motility. Comparative transcriptomics and reverse transcription‐quantitative PCR assays revealed that in XVM2 medium, a mimic medium of the plant environment, the expression levels of hrp genes (for HR and pathogenicity) were enhanced in the Xoo hpaP deletion mutant compared to the wild type. By contrast, in the same growth conditions, hrp gene expression was decreased in the Xoc hpaP deletion mutant compared to the wild type. However, an opposite expression pattern was observed when the pathogens grew in planta, where the expression of hrp genes was reduced in the Xoo hpaP mutant but increased in the Xoc hpaP mutant. These findings indicate that HpaP plays a divergent role in Xoo and Xoc, which may lead to the different infection strategies employed by these two pathogens. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, one of the most serious rice diseases. The xrvA gene from Xoo strain 13751 encodes a protein containing a histone-like nucleoid-structuring protein (H-NS) domain. The expression of xrvA in strain 13751 was enhanced in XOM2 minimal medium. Mutation of the xrvA gene of strain 13751 led to a significant reduction in virulence in the host plant rice, a delayed hypersensitive response in the nonhost castor-oil plant, a decrease in extracellular polysaccharide and diffusible signal factor production, and an increase in intracellular glycogen accumulation. Northern hybridization analyses revealed that the virulence-associated genes hrpG, hrpX, rpfC, rpfF, rpfG and gumB were downregulated in the xrvA mutant compared to the wild-type and complemented strains. Interestingly, increase of copy number of xrvA in the wild-type strain 13751 resulted in a strain showing similar phenotypes as the xrvA mutant and a reduction of the expression of gumB, hrpX, rpfC, rpfF and rpfG. These findings indicate that the xrvA gene, which is highly conserved in the sequenced strains of Xanthomonas, encodes an important regulatory factor for the virulence of Xoo.

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.