Studies of two enzymes from Xyllela fastidiosa, GumC and GumK, involved in the biosynthesis of fastidian gum

Author(s)

Celina de Pieri

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2006-04-17

Examining board members

Glaucius Oliva; Leila Maria Beltramini; Raquel Kely Bortoleto Bugs; Lucile Maria Floeter Winter; Nelson Arno Wulff

• Advisor: Glaucius Oliva

Abstract

The bacterium Xylella fastidiosa has been associated with diseases of economically importante crop as the citrus variegated chiorosis (CVC) in citrus in Brazil and the Pierce\'s disease in grapevines in the United States (Hopkins. 1989 and Purcell et al., 1997). The economic importance of the citrus industn - in Brazil and the high level of damage caused bv CVC in brazilian orchards hm -e resulted in an extensive research program starting with the sequencing of the entire genome of X. fastidiosa (Simpson et al.. 2000). The bacterium is transmitted bv specific sharpshooter lealloppers when the insect feeds on the talem sap. Tvpical disease svmptoms include conspicuous variegations. with chlorotic amas on the upper side and small necrotic lessions on the lower side of the leaves. The affected fruits are smaller. often no more than one-third of the diameter of healthy fruits, hardened and without commercial value. X fastidiosa has a nine-gene operou (B, C. D. E. F, H. J. K. and M genes) responsible for the biosvnthesis of exopolysaccharides, denoted fastidian gum, that mas be linked directly to the pathogenecin - of the microorganism. In this work our main aim is to study the GLIM(\' enzyme, that may be responsible for gum polymerization or secretion through the membrane of X fástidiosa and GUMK enzyme, a glucuronosvltransferase or glycosyltransferase IV. that adds a glucuronic acid to tetrasaccharide manose-α-1,3- glucose-β-1,4-glucose-P-P-po1visoprenyl. The gume gene was cloned into the pMAL-c2x expression vector, and GIIMC-MBP fusion protein was expressed with estimate molecular mass 98kDa. was purified through affinin - column and was characterized through the reaction of antiMBP antibodv by imunoblotting. Molecular aggregation studies of GII.MC-MBP fusion protein were measured by Dynamic Light-Scattering (DLS). Crystallization screens were made and microcrvstals were obtained. The GUMC-MBP fusion protein were cleavaged with Factor Xa and the GIM\' enzyme purified through ion exchange chromatography. The GUMC molecular mass were determined using size exclusion chromatography, superosel2. Anti-GUMC-MBP antibodies were already produced in mice and were used to characterize the GUMC protein. by imunoblotting technique. In addition, according to circular dichroism spectroscopy, GUMC protein prevalent secondary structure is α-hefix. Nine constructions for the gtmiC gene. without 5 - and 3° transmembrane regions (one for cloning in pET29b and 8 for cloning in pMALc-2x) were carried out. One of the constructions. the modified fusion protein GIIMC-MBP C5. was expressed into the pMALc-2x expression. vector. The modified fusion protein GLIMC-MBP 5C were purified through affmin - resin and cleavaged with the Factor Xa enzvme. In the future. aggregation states of GIIMC-MBP and modifiecl GUMC -MBP C5 fusion proteins. and GII.MC and modificai GUMC C5 enzymes could be measured by DLS and compared. The gumK gene. cloned into the pMALc-2x expression. vector. expressed a GIIMK-MBP fusion protein soluble with molecular mass 86kDa. This fusion protein was purified through affmin - resin. The GIIMK-MBP fusion protein was characterized for imunoblotting and CD measurements. The GUMK-MBP fusion protein was cleavaged with Factor Xa enzvme and the GUMK enzyme was purified through cationic Exchange chromatography. Crvstallization screens were made and micmcrystals and crystals in needle form were obtained. Future enzvmes characterization three-dimensional structure will allow - further structural and functional studies on the pathogenicity of X. fastidiosa. as well as to development of specific inhibitors (AU)