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.

FIG. 1.

Schematic view of alg8 knockout construct of plasmid pEX100TΔalg8Gm used for homologous recombination and the alginate biosynthesis operon after replacement of native alg8 gene with Δalg8.

FIG. 2.

Predicted membrane topology of Alg8 based on different HMM-based algorithms (Phobius, SMART, and TMHMM2) of the processed Alg8. Numbers represent the location of the amino acids in the processed form starting with first N-terminal amino acid after the predicted signal peptide cleavage site with number 1. The threading model was developed based on the SAM-T02 alignment of Alg8 with SpsA (1qg8). Cylinders represent α-helical structures. Big arrows represent β-strands. The putative catalytic residues are given as stick side chains and indicated by arrows. N, N terminus of the structural Alg8 model; C, C terminus of the structural Alg8 model.