Dual regulation of zwf-1 by both 2-keto-3-deoxy-6-phosphogluconate and oxidative stress in Pseudomonas putida

Abstract

Northern blot analysis and a GFP-based reporter assay showed that zwf-1, which encodes glucose-6-phosphate dehydrogenase, was highly induced when Pseudomonas putida KT2440 was cultured in minimal medium containing glucose or gluconate. However, zwf-1 expression was not detected in the presence of pyruvate or succinate. The use of a knockout mutant of HexR, a putative transcription regulator, resulted in constitutively high expression of zwf-1, regardless of the carbon source. An electrophoretic mobility shift assay showed that HexR protein binds to the zwf-1 promoter region and that HexR binding is inhibited by 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite the presence of gluconate, the edd mutant (non-KDPG producer) was not able to induce the zwf-1 gene. The eda mutant (KDPG overproducer) featured a constitutively high level of zwf-1 expression. Interestingly, zwf-1 was also highly expressed in the presence of oxidative stress-inducing reagents. The level of zwf-1 induction in wild-type cells undergoing oxidative stress did not differ significantly from that observed with the hexR mutant under normal conditions. Interestingly, the hexR mutant was more tolerant of oxidative stress than the wild-type. Expression of zwf-1 was induced by oxidative stress in the edd mutant. Thus, KDPG, a real inducer of zwf-1 gene expression, was not necessary for oxidative-stress induction. In vitro binding of HexR to its cognate promoter region was diminished by menadione and cumene hydroperoxide. The data suggested that HexR might be a dual-sensing regulator of zwf-1 induction that is able to respond to both KDPG and oxidative stress.