Purification, crystallization and preliminary X-ray diffraction analysis of GumB, an outer membrane lipoprotein essential for the secretion of xanthan

MELISA JACOBS AND LUIS IELPI

Salta

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Biofísica y 3er Reunión de la Sociedad Latino Americana de Proteínas.; 2010

Sociedad Argentina de Biofísica, Latin American Protein Society

Most bacteria produce different kinds of extracellular polysaccharides whichare essential for the cellular viability and for interactions between bacteria and eukaryoticcells. The phytopathogenic bacterium Xanthomonas campestris secretes xanthan, anexopolysaccharide essential for pathogenesis. Besides, xanthan has diverse applicationsin the industry as thickener and viscosifier. GumB from X. campestris is a 23 kDa outermembrane lipoprotein involved in the secretion of xanthan1. GumB belongs to the outermembrane polysaccharide export protein family, which only shares PES motif (Pfam02563) as primary sequence similarity2. Overexpression of GumB results toxic to the cellprobably because of its localization in the outer membrane. An efficient overexpressionof GumB was obtained from gumB ORF lacking the signal peptide for transport to themembrane. Recombinant cytoplasmic GumB (GumBcyt) was purified to homogeneity byaffinity chromatography followed by size exclusion chromatography. Crystals of recombinantGumBcyt were obtained, diffracting to 3.0 Å. Data analysis suggest that GumBcytcrystals belong to the orthorhombic space group P212121 (a=84.27, b=89.31, c=119.21,α=β=γ=90º). Knowledge of the crystal structure of GumB will help to understand thestructure and function of the xanthan assembly system.