[show abstract][hide abstract] ABSTRACT: The Pel polysaccharide serves as an intercellular adhesin for the formation and maintenance of biofilms in the opportunistic pathogen Pseudomonas aeruginosa. Pel biosynthesis requires the products of a seven-gene operon, pelA-G, all of which are necessary for Pel-dependent biofilm formation and Pel-related phenotypes. One of the genes, pelA, encodes a protein with a predicted polysaccharide deacetylase domain. In this work, the role of the putative deacetylase domain of PelA in Pel production was examined. First, we established that purified recombinant PelA hydrolyzed the pseudo-substrate p-nitrophenyl acetate in vitro and site-specific mutations of predicted deacetylase active site residues reduced the hydrolysis of p-nitrophenyl acetate greater than 10-fold. Additionally, the PelA deacetylase mutants were deficient for Pel-dependent biofilm formation and wrinkly colony morphology in vivo. Subcellular fractionation experiments demonstrate that PelA localizes to both the membrane and periplasm fractions. Finally, antiserum against the Pel polysaccharide was generated and PelA deacetylase mutants do not produce Pel-reactive material. Taken together, these results suggest that the deacetylase activity of PelA is important for the production of the Pel polysaccharide.
Journal of bacteriology 03/2013; · 3.94 Impact Factor