Expression, purification and characterization of the enzymes GumD and GumC involved in the biosynthesis of the exopolysaccharide produced by the bacteria Xyllela fastidiosa

Author(s)

Celina de Pieri

• Document type: Master's Dissertation
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2002-06-24

Examining board members

Glaucius Oliva; Heloisa Sobreiro Selistre de Araújo; Celso Eduardo Benedetti

• Advisor: Glaucius Oliva

Abstract

The Citrus Variegated Chlorosis (CVC) is a serious disease of orange trees in countries like Brazil, USA, France and Spain. Typical disease symptoms include conspicuous variegations with chlorotic areas on the upper side and small necrotic lesions on the lower side of the leaves. The affected fruits are smaller, hardened and without commercial value. Citrus variegated chlorosis is caused by Xylella fastidiosa, which is a xylem-limited bacterium. X fastidiosa is transmitted by specific sharpshooter leafhoppers when the insect feeds on the xylem sap. X fastidiosa has a nine-gene operon (B, C, D, E, F, H, J, K, and M genes) responsible for the biosynthesis of exopolysaccharides, denoted fastidian gum, which can be involved in its pathogenicity. GumD glycosyltransferase enzyme adds the first sugar, glycose-l-phosphate, to the prenol lipid. GumC enzyme probably is involved in the polymerization and/or exportation of the fastidian gum through the membrane of the bacterium. Studies were done on the GumD and GumC enzymes with the aim of getting a better understanding of the exopolysaccharide biosynthetic pathway. The gumD gene was cloned into the pMAL-c2x and pKK223-3 expression vectors, and GumD protein was purified through ion exchange and size exclusion chromatography. Then GumD molecular mass and pl were determined using, respectively, size exclusion chromatography and electrophoresis. In addition, according to circular dichroism spectroscopy, GumD prevalent secondary structure is u-helix. On the other hand, the gumC gene was cloned in two expression vectors: pMAL-c2x (the protein is expressed in fusion with Maltose Binding Protein ¬MBP) and pET29a (the protein is expressed without fusion in our strategy). GumC¬MBP, the protein in fusion, was partially purified using an amylose column and the fusion between GumC and MBP was confirmed with imunoblotting technique. Purification trials of GumC enzyme expressed in pET29a are in course. Anti-GumC¬MBP antibodies were already produced in mice and they will be used to characterize the GumC protein expression without fusion. Structural studies of GumD and GumC enzymes will provide information about the fastidian gum biosynthetic pathway, as well as to the development of specific inhibitors (AU)