GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa: evidence from metabolite studies

Abstract

PO Box 903, Cardiff CF1 lST, UKThe Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. 1. Russell & P. Gacesa (1993), I Gen Microbiol 139,119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of a/gD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDPmannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

The alternative sigma factor AlgT, but not alginate synthesis, promotes in planta multiplication of Pseudomonas syringae pv. glycinea

“…The characterization of the two algD mutants showed that disruption of algD led to an alginate-negative phenotype as expected, since algD had been shown to be indispensable for alginate biosynthesis in P. aeruginosa (Tatnell et al, 1994). We did not observe a significant difference in alginate production between the AlgT-deficient strain PG4180 and its algD mutant, PG4180.alg.…”

“…Genes encoding enzymes of alginate biosynthesis in P. aeruginosa and P. syringae are organized in an operon (algD-algA), with the exception of algC (Chitnis & Ohman, 1993;Peñaloza-Vázquez et al, 2004Zielinski et al, 1991). The first gene of the operon, algD, encodes a GDP-mannose dehydrogenase, whose activity is the kinetic control point and is indispensable for the alginate pathway (Tatnell et al, 1994). In P. syringae, algD is expressed during colonization of susceptible and resistant plant hosts (Keith et al, 2003).…”

Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281

“…In G. xylinus I-2281, the overexpression of genes coding for mannose-6-phosphate isomerase should also lead to a higher level of EPS. Given the fact that only very low mannose-P isomerase activities were measured in G. xylinus I-2281 (Table 2), the overexpression of this gene could also lead to higher levels of EPS synthesis, as is true for microbial alginate production (34).…”

Identification and Characterization of the Capsular Polysaccharide (K-Antigen) Locus of Porphyromonas gingivalis

“…The monosaccharide composition of the capsular polysaccharide of P. gingivalis ATCC 53978 (W50) was determined to be mannuronic acid, glucuronic acid, galacturonic acid, galactose, and 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) in a molar ratio of 1:2:1:1:2, respectively (13). Synthesis of mannuronic and glucuronic acid could occur directly from GDP-Man and UDP-Glc by a GDP-Man-dehydrogenase (a key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa [49]) and UDP-Glc-dehydrogenase (an enzyme required for virulence in the yeast Cryptococcus neoformans [17]), respectively. PG0108 (see legend to Fig.…”