Crystallization and crystallographic analysis of Bacillus subtilis xylanase C

XynC, a glycosyl hydrolase family 5 xylanase from B. subtilis, has been purified and crystallized with the primary goal of structural determination and characterization of a second xylanase from this large catalytically diverse glycosyl hydrolase family

Identification of Genes in Xanthomonas euvesicatoria pv. rosa That Are Host Limiting in Tomato

Xanthomonas euvesicatoria pv. rosa strain Xer07 causes a leaf spot on a Rosa sp. and is closely related to X. euvesicatoria pv. euvesicatoria (Xee) and X. perforans (Xp), causal agents of bacterial spot of tomato. However, Xer07 is not pathogenic on tomato and elicits a hypersensitive reaction (HR). We compared the genomes of the three bacterial species to identify the factors that limit Xer07 on tomato. Comparison of pathogenicity associated factors including the type III secretion systems identified two genes, xopA and xer3856, in Xer07 that have lower sequence homology in tomato pathogens. xer3856 is a homolog of genes in X. citri (xac3856) and X. fuscans pv. aurantifolii, both of which have been reported to elicit HRs in tomato. When xer3856 was expressed in X. perforans and infiltrated in tomato leaflets, the transconjugant elicited an HR and significantly reduced bacterial populations compared to the wildtype X. perforans strain. When xer3856 was mutated in Xer07, the mutant strain still triggered an HR in tomato leaflets. The second gene identified codes for type III secreted effector XopA, which contains a harpin domain that is distinct from the xopA homologs in Xee and Xp. The Xer07-xopA, when expressed in X. perforans, did not elicit an HR in tomato leaflets, but significantly reduced bacterial populations. This indicates that xopA and xer3856 genes in combination with an additional factor(s) limit Xer07 in tomato

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modeling and in silico docking studies for the cyclophilin–AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.EEA Bella VistaFil: Gochez, Alberto Martín. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina. University of Florida. Department of Plant Pathology; Estados UnidosFil: Shantharaj, Deepak. University of Florida. Department of Plant Pathology; Estados UnidosFil: Potnis, Neha. University of Florida. Department of Plant Pathology; Estados UnidosFil: Zhou, Xiaofeng. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Minsavage, Gerald V. University of Florida. Department of Plant Pathology; Estados UnidosFil: White, Frank F. University of Florida. Department of Plant Pathology; Estados UnidosFil: Wang, Nian. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Hurlbert, Jason C. Winthrop University. Department of Chemistry, Physics and Geology, Citrus Research and Education Center; Estados UnidosFil: Jones, Jeffrey B. University of Florida. Department of Plant Pathology; Estados Unido

Functional Characterization of a Novel Xylanase from a Corn Strain of Erwinia chrysanthemi

A β-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35°C, with a secondary structure under those conditions that consists of approximately 10 to 15% α-helices. The enzyme was still active at temperatures higher than 40°C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45°C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-d-glucuronic acid and appears to require two 4-O-methyl-d-glucuronic acid residues for substrate recognition and/or cleavage of a β-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process

Characterization of three novel genetic loci encoding bacteriocins associated with Xanthomonas perforans.

Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three (ORFA, ORF2 and ORF4) were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases