title: In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa.
abstract: An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Deltaalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. (1)H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.
authors.affiliation: Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
authors.forename: Uwe
authors.initials: U
authors.lastname: Remminghorst
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000464:Alginates / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D017353:Gene Deletion; D020723:Glucuronic Acid / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006603:Hexuronic Acids / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011108:Polymers / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D011108:Polymers; D020723:Glucuronic Acid; D016695:Glycosyltransferases