Cloning and Characterization of the Pseudomonas aeruginosa zwf Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat)

In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD⁺- or NADP⁺-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of ∼220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be ∼220 kDa by size exclusion chromatography. Estimated K_m values of purified G6PDH acting on glucose-6-phosphate, NADP⁺, and NAD⁺ were 530, 57, and 333 μM, respectively. The specific activities with NAD⁺ and NADP⁺ were calculated to be 176 and 69 μmol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.Pseudomonas aeruginosa is a gram-negative bacillus that is virtually ubiquitous throughout nature. It is also an opportunistic pathogen of humans, most notably those who have cystic fibrosis or whose immune systems have been compromised (e.g., as a result of burns or cancer chemotherapy) (11, 16, 55). The organism has a remarkable capacity to utilize a wide range of carbon sources for growth under a variety of environmental conditions. Although tricarboxylic acid (TCA) cycle intermediates such as succinate are preferentially utilized by P. aeruginosa, the organism readily catabolizes glucose. In contrast to the facultative organism Escherichia coli, P. aeruginosa does not metabolize glucose via the Embden-Meyerhof pathway because it does not possess 6-phosphofructokinase (36). Thus, the catabolism of glucose by P. aeruginosa requires its conversion to glyceraldehyde-3-phosphate and pyruvate via the Enter-Doudoroff enzymes 6-phosphogluconate dehydratase (Edd) and 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) (36). Depending on the physiological conditions, glucose is converted to 6-phosphogluconate by one of two routes, one of which is oxidative and the other of which is phosphorylative. The direct oxidative route involves oxidation of glucose to gluconate and 2-ketogluconate in the periplasm via membrane-bound glucose and gluconate dehydrogenases (29). Recently both oxidative routes have been shown to be physiologically significant with the isolation of mutants blocked in either gluconate or 2-ketogluconate utilization (67a). Alternatively, the phosphorylative route involves uptake of glucose by an inducible transport system where, once inside the organism, it is phosphorylated by glucokinase and next converted to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), the product of the zwf gene (29).Interestingly, a zwf mutant of E. coli was reported to be more sensitive to the redox-active, superoxide (O₂⁻)-generating agent methyl viologen (paraquat) (19). It was then postulated by Liochev and Fridovich (38) that this sensitivity was attributed to a reduced level of NADPH, a cofactor necessary for the activity of glutathione reductase (3) and alkylhydroperoxide reductase (32), enzymes which combat paraquat-mediated oxidative stress.In this study, we describe the cloning and characterization of the zwf gene of P. aeruginosa PAO1. We demonstrate that inactivation of the zwf gene does not allow mutant organisms to grow on mannitol as the sole carbon source. In addition, we demonstrate that the zwf gene is under tight control, being highly transcribed in the presence of inducing agents such as glycerol and glucose and weakly transcribed in the presence of the TCA cycle intermediate succinate. In addition, we demonstrate that G6PDH activity is important in resistance to the O₂⁻-generating agent paraquat.