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 14 C-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 ⌬alg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. 1H-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.

Production of polyhydroxybutyrate and alginate from glycerol byAzotobacter vinelandiiunder nitrogen-free conditions

“…6,9,[12][13][14] The biosynthetic pathways for alginate and PHB production in A. vinelandii have previously been reviewed by Galindo et al 6 As shown in Fig. 1, alginate is synthesized from fructose 6-phosphate by many enzymes encoded by the alg cluster, 8,15 whereas PHB is synthesized in 3 steps from acetyl-CoA and 3 phb genes are essential for synthesis. 16 The regulatory mechanisms for the production of these biopolymers have been analyzed.…”

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

“…Alg8 shares structural similarity with glycosyltransferases (GTs) of family 2, making Alg8 the probable alginate polymerase (Saxena et al, 1995). This was confirmed with an in vitro assay showing that Alg8-containing envelope fractions of mucoid P. aeruginosa could utilize GDP-mannuronate as a substrate for alginate polymerase activity (Remminghorst & Rehm, 2006a). Another protein required for alginate biosynthesis is Alg44 (Maharaj et al, 1993;Remminghorst & Rehm, 2006b).…”

“…Unfortunately, the substrate of Alg8, GDP-mannuronate, is not commercially available. However, radio-labelled GDP-mannuronate was recently synthesized to show in vitro that Alg8-containing membranes of P. aeruginosa can use GDPmannuronate for polymer production, thus confirming the role of Alg8 in alginate polymer formation (Remminghorst & Rehm, 2006a).…”

“…1B. Others have predicted a similar membrane topology for Alg8 from P. aeruginosa Rehm, 2006a) andA. vinelandii (Mejia-Ruiz et al, 1997).…”

“…However, unlike an DalgG mutant, neither released degraded alginate, which would be detected as small (dialysable) uronic acids ( Table 2). As Alg8 is the GT for alginate, utilizing GDP-mannuronate as substrate to synthesize the polymer (Remminghorst & Rehm, 2006a), an alg8 mutant would not be expected to produce alginate or alginate fragments. Interestingly, the same phenotype was observed alg44 2 0 *Secretion of alginate fragments was determined as previously described (Jain & Ohman, 2005) by the demonstration of uronic acids in culture supernatants of non-mucoid strains that are lost by exhaustive dialysis.…”