Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

Abstract

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

FIG 1

Uptake and utilization of glucose, fructose, and glycerol in P. fluorescens. Abbreviations: G6P, glucose-6-phosphate; 6PGA, 6-phosphogluconate; 2KGP, 2-keto-6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; F1P, fructose-1-phosphate; GAP, glyceraldehyde-3-phosphate; Pyr, pyruvate; DPGA, 1,3-diphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; G3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; Gad, gluconate dehydrogenase; Gcd, glucose dehydrogenase; Kgk, 2-ketogluconate kinase; Gnk, gluconokinase; Glk, glucokinase; Kgr, 2KGP reductase; Gnd, 6PGA dehydrogenase; GlpK, glycerol kinase; GlpD, G3P dehydrogenase; Zwf1/2, G6P dehydrogenases; Pgi, phosphoglucose isomerase; Pgl, 6-phosphogluconolactonase; Edd, 6PGA dehydratase; Eda, KDPG aldolase; Fpk, 1-phosphofructokinase; Fdp, fructose-1,6-bisphosphatase; Fda, fructose-1,6-bisphosphate aldolase.

FIG 2

Coenzyme preference and ATP-induced inhibition of the glucose-6-phosphate dehydrogenases Zwf-1 (shaded bars) and Zwf-2 (open bars). Enzyme activities are given as average values measured in cell extracts obtained from three independently grown cultures of strains expressing only one of the two G6PDH isoenzymes. Extracts were assayed using NAD⁺ or NADP⁺ as the coenzyme, and with or without the addition of ATP. (See Materials and Methods for details.) One unit of enzyme is defined as the amount of total soluble protein that catalyzes the reduction of 1 μmol NAD(P)⁺ per min.

FIG 3

Growth of P. fluorescens NCIMB 10525 and its mutant derivatives on various carbon sources. The strains were cultivated in minimal medium using glucose (a), fructose (b), or glycerol (c) as the carbon source, and growth was measured as the absorbance at 660 nm. The following strains were investigated: P. fluorescens NCIMB 10525 (◆) and its Δzwf-1 (■), Δzwf-2 (▲), Δgcd (●), Δzwf-1 Δzwf-2 (□), Δzwf-1 Δgcd (△), and Δzwf-2 Δgcd (○) mutants. Each cultivation was repeated at least three times, each time resulting in growth profiles essentially the same as those displayed here.

FIG 4

Effects of zwf-2 and gcd inactivation on growth and alginate production in cells cultivated with glucose as the carbon source. (a) Growth, expressed as OD₆₆₀. Data are averages for three biological replicates, and error bars indicate standard deviations. (b) Alginate production, expressed as grams per liter of culture. Data are averages for three biological replicates. (c) Growth, expressed as CFU per milliliter of culture. Data are averages for at least two biological replicates. Strains: Pf201 (◆, open bars), Pf201 Δzwf-2 (▲, dark shaded bars), Pf201 Δgcd (●, striped bars), Pf201 Δgcd::TnHE418 (△, light shaded bars), and the non-alginate-producing wild-type (wt) strain NCIMB 10525 (stippled bars).

FIG 5

Effects of zwf-1 disruption on growth and alginate production. P. fluorescens strains Pf201 (◆), Pf201 ΔalgC Δzwf-1::TnKB60 (■), and Pf201 ΔalgC::TnKB60 MM27 (▲) were grown in minimal medium containing glucose (a and b), fructose (c and d), or glycerol (e and f). Growth and alginate data are shown as averages for at least two biological replicates. Error bars indicate standard deviations.