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. K nowing how carbon sources are channeled through different metabolic pathways to provide the energy and precursors needed to sustain growth is crucial for optimizing an organism for industrial bioproduction purposes. It is known that in pseudomonads, glucose is not channeled to the tricarboxylic acid (TCA) cycle via glycolysis, since these bacteria lack the phosphofructokinase gene. Instead, glucose is metabolized via the Entner-Doudoroff (ED) pathway (see Fig. 1) (1). Previous studies have also indicated strongly that fructose and glycerol are mainly converted to glucose-6-phosphate (G6P) via fructose-1,6-bisphosphate (FBP). G6P is then metabolized through the ED pathway (2).Pseudomonas fluorescens is a diverse species that occupies many niches in nature (3). Like several other species of this genus, it has the ability to produce the linear polymer alginate. This industrially important, nonrepetitive polysaccharide, containing 1,4-linked ␣-L-guluronic acid and ␤-D-mannuronic acid, is currently commercially manufactured from brown algae. Bacterial alginate bioproduction is interesting, however, especially because bacteria may be engineered to produce alginates with properties that are well suited for medical applications. By using a nonpathogenic wild-type strain of P. fluorescens, we have been able to obtain mutant strains...

Identification of a New Phosphatase Enzyme Potentially Involved in the Sugar Phosphate Stress Response in Pseudomonas fluorescens

“…Moreover, alginate production may lower the pH of the medium, leading to a stress response, and this makes the cell more vulnerable to additional stress (10). Similarly, none of the E. coli HADs, including YigL, were essential for growth when the cells experienced no sugar phosphate stress (11,20).…”

“…In our recent study focusing on the role of genes involved in central carbon metabolism of Pf201 on alginate production (10), it was hypothesized that the observed growth deficiencies of the glucose-6-phosphate dehydrogenase (G6PD) (encoded by zwf-1 and zwf-2) mutants of the NCIMB10525 strain grown on fructose resulted from accumulation of hexose phosphates (G6P and F6P) causing sugar phosphate stress. It was shown that the growth defect was less severe in a zwf-1 mutant of an alginate-producing strain, possibly because alginate production may drain away a portion of the excessive sugar phosphates (10). This hypothesis motivated our current study to investigate any possible sugar phosphate stress response in Pf201 further.…”

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

“…This finding is in agreement with our previous study focusing on the central carbon metabolism of P. fluorescens. There, we showed that inactivation of zwf-1 (encoding glucose-6-phosphate dehydrogenase in the Entner-Doudoroff [ED] pathway) was beneficial for alginate production when glycerol was used as carbon source due to decreased carbon flow through the ED pathway and accumulation of F6P, which could be converted to alginate precursor (28).…”

“…For the detection of LacZ activity, 60 l of X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) solution (20 mg/ml in dimethyl sulfoxide) was added to each agar plate 30 min before the plating of bacteria. Alginate was harvested and quantified using M-and G-specific lyases as described earlier (28)(29)(30). All alginate quantifications were performed using three technical replicates.…”

“…Routine growth of Escherichia coli and Pseudomonas fluorescens strains was carried out in L broth (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) in 250-ml shake flasks at 225 rpm at 37 and 30°C, respectively. For growth experiments, alginate production, and TEM, 1% precultures of respective bacterial strains were inoculated in DEF3 minimal medium (28), containing fructose as carbon source. When appropriate, antibiotics were added in the following concentrations: triclosan (Tric), 25 g/ml; apramycin (Am), 50 g/ml; ampicillin (Ap), 200 g/ml; kanamycin (Km), 50 g/ml; and tetracycline (Tc), 15 g/ml.…”