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.

Pulchellin is a type 2 ribosome‐inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A‐chain was cloned and inserted into pGEX‐5X to express the recombinant pulchellin A‐chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A‐chain of abrin‐c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of ≈ 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 µg·kg−1. Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.

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Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome‐inactivating pulchellins, each consisting of a toxic A‐chain linked to a sugar binding B‐chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B‐chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B‐chain‐mediated cell surface interactions that precede and determine toxin uptake pathways.