| Chronic pulmonary infection by Pseudomonas aeruginosa is a serious complication for Cystic Fibrosis patients. Alginate, a capsule-like exopolysaccharide, is an important virulence factor of CF clinical isolates. Alginate is composed of D-mannuronate and L-guluronate, with beta, 1-4 linkages. The genes for alginate biosynthesis, polymerization and secretion are located in an 18 kb operon. The alg8 and alg44 products appear to be important in polymerization and transport. Alg8, encoded by the second gene in the operon, shows structural similarity to beta-glycosyltransferases and is the putative enzyme involved in the polymerization of D-mannuronate from GDP-mannose. Beta-glycosyltransferases are often inner membrane proteins with a large cytoplasmic loop that contains the enzymatic domains. To determine if Alg8 fits this pattern, topology modeling using transmembrane prediction program TMpred and hydrophobicity plot analysis was performed. These results indicated that Alg8 is a cytoplasmic membrane protein that spans the membrane five times and has a large cytoplasmic loop containing the enzymatic domains A and B. Site directed mutagenesis of the enzymatic domains identified amino acids that were required for the activity of Alg8. In addition, a mutant analysis of Alg8, in which the C-terminus was deleted, suggests an important role for this domain. Alg44 is the third gene in the alginate biosynthesis operon and shares limited homology to membrane fusion proteins (MFP). Topology modeling suggested that unlike MFPs, Alg44 contained two transmembrane domains, and this was tested by translational gene fusions. The results indicated that Alg44 contained two transmembrane domains with the N and C termini located in the periplasm where over 60% of the protein is localized. Western blot analysis revealed that the alg44 mutant did not contain detectable levels of AlgK and AlgE. However, complementation of the mutant with an alg44-phoA expression construct restored alginate expression. Western blot analysis revealed that the restored alginate phenotype included the appearance of AlgE but not AlgK. Quantitative analysis showed that the complemented mutant secreted approximately 60% less alginate than the parental strain FRD1. |
