Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named "pathogenicity, virulence and adaptation". One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a severe disease of citrus. Xcc presents broad spectrum of citrus hosts including economically important species whereas X. fuscans subsp. aurantifolii–type C (XauC) causes a milder disease and only infects Citrus aurantifolia. Trehalase catalyzes hydrolysis of the disaccharide trehalose, a sugar that has been reported to be related to Xcc pathogenicity. We expressed the recombinant gene product and assessed Xcc trehalase structural and kinetics data. The recombinant protein presented 42.7% of secondary structures in α-helix and 13% in β-sheets, no quaternary structure in solution, and Michaelis-Menten constant (KM) of 0.077 mM and Vmax 55.308 μMol glucose.min-1.mg protein-1 for trehalose. A Xcc mutant strain (XccΔtreA) was produced by gene deletion from Xcc genome. Enzymatic activity of trehalase was determined in Xcc, XauC and XccΔtreA cellular lysates, showing the highest values for XauC in in vitro infective condition and no activity for XccΔtreA. Finally, leaves of Citrus aurantifolia infected with XccΔtreA showed much more drenching and necrosis than those infected by wild type Xcc. We concluded that trehalase contributes to alleviate bacterial virulence and that inability for trehalose hydrolysis may promote higher Xcc infectivity.

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase β subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.

Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.

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