Navegando por Palavras-chave "Xanthomonas citri subsp citri"

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    Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri
    (Funpec-editora, 2012-01-01) Soares-Costa, Andrea; Silveira, Roseli Santos da; Novo, Maria Teresa Marques; Alves, Marcio Fernando Madureira [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Belasque Junior, José; Henrique-Silva, Flávio; Universidade Federal de São Carlos (UFSCar); Universidade Federal de São Paulo (UNIFESP); Fundo Def Citricultura
    Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. the aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. the gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. the same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K-m) of 47 mu M-1.s(-1). the purified (HIS)CPXAC displayed maximal catalytic activity at pH 5.5 and 30 degrees C. the recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K-i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.
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    Xanthomonas citri subsp citri surface proteome by 2D-DIGE: Ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction
    (Elsevier Science Bv, 2017) Carnielli, Carolina Moretto; Artier, Juliana; Franco de Oliveira, Julio Cezar [UNIFESP]; Marques Novo-Mansur, Maria Teresa
    Xanthomonas citri subsp. citri (XAC) is the causative agent of citrus canker, a disease of great economic impact around the world. Understanding the role of proteins on XAC cellular surface can provide new insights on pathogen-plant interaction. Surface proteome was performed in XAC grown in vivo (infectious) and in vitro (non-infectious) conditions, by labeling intact cells followed by cellular lysis and direct 2D-DIGE analysis. Seventy-nine differential spots were analyzed by mass spectrometry. Highest relative abundance for in vivo condition was observed for spots containing Dnal(protein, 60 kDa chaperonin, conserved hypothetical proteins, malate dehydrogenase, phosphomannose isomerase, and ferric enterobactin receptors. Elongation factor Tu, OmpA-related proteins, Oar proteins and some Ton-B dependent receptors were found in spots decreased in vivo. Some proteins identified on XAC's surface in infectious condition and predicted to be cytoplasmic, such as DnaK and 60 KDa chaperonin, have also been previously found at cellular surface in other microorganisms. This is the first study on XAC surface proteome and results point to mediation of molecular chaperones in XAC-citrus interaction. The approach utilized here can be applied to other pathogen-host interaction systems and help to achieve new insights in bacterial pathogenicity toward promising targets of biotechnological interest. Biological significance: This research provides new insights for current knowledge of the Xanthomonas sp. pathogenicity. For the first time the 2D-DIGE approach was applied on intact cells to find surface proteins involved in the pathogen-plant interaction. Results point to the involvement of new surface/outer membrane proteins in the interaction between XAC and its citrus host and can provide potential targets of biotechnological interest for citrus canker control. (C) 2016 Elsevier B.V. All rights reserved.