ANALYSIS OF THE CYTOPLASMIC MEMBRANE BINDING REGION OF GUMK, A GLUCURONOSYLTRANSFERASE INVOLVED IN XANTHOMONAS CAMPESTRIS XANTHAN PRODUCTION

BRUKMAN, NG; SALINAS, SR; IELPI, L

Mar del Plata, Pcia de Buenos Aires

Congreso; VIII Congreso Argentino de Microbiología General SAMIGE; 2012

Xanthan gum is an acidic exopolysaccharide produced by the gram negative phytopathogen, Xanthomonas campestris. Xanthan is not essential for the infection but it contributes to Xanthomonas epiphytic survival. This exopolysaccharide is used as thickening and stabilizer in many industrial processes. GumK is a membrane-associated glucuronosyltransferase that participates in the synthesis of the repeating unit of xanthan. GumK transfers a glucuronic acid from UDP-GlcA to a lipid-sugar acceptor embedded in the inner membrane. Functional and structural analyses of GumK are being carried out in our lab. We believe that the association of GumK with the membrane is essential for its activity in order to have a productive interaction with its acceptor substrate. We recently solved the crystallographic structure and a patch of hydrophobic and basic amino acids in the GumK N-domain was found. This region can probably be the one involved in the association to the cytoplasmic membrane. To confirm this inference, deletion mutants were constructed: ΔR55-K60 (Nα2), ΔS97-A112 (Nα4), ΔW85-R96 (Loop), ΔS97-D104 (Nα4N), ΔA105-A112 (Nα4C), ΔW85-H90 (LoopN), ΔP91-R96 (LoopC). Each construction was transformed in gumK- strain (XcK). Functional assays show that xanthan production is drastically reduced when GumK is mutated. Western blots of total extract revealed that GumK mutants are correctly expressed in two-day cultures. However, in five-day cultures expression levels were reduced in different magnitudes for each mutant, suggesting that the stability of the protein is altered. The mutant Nα2 was the most stable and it is associated to the cytoplasmic membrane as it was indicated in the analysis of subcellular localization. Previous studies in our lab showed that mutations of basic amino acids of Nα2 do not affect the activity. Moreover, mutation of the hydrophobic amino acids of the same region abolish activity. Altogether, these results suggest that the region Nα2 analyzed in this study is not essential in GumK-cytoplasmic membrane interaction but it is essential for the enzyme activity and xanthan production. Furthermore, we suppose that Nα2 may be necessary for lipid-sugar binding. We are carrying out similar studies for the other two regions: Nα4 and Loop.