Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide

Abstract

In Escherichia coli K-12, transcription of zwf, the gene for glucose 6-phosphate dehydrogenase, is subject to growth rate-dependent regulation and is activated by SoxS in response to superoxide stress. To define genetically the site of SoxS activation, we undertook a detailed deletion analysis of the zwf promoter region. Using specifically targeted 5 and 3 deletions of zwf sequences, we localized the SoxS activation site to a 21-bp region upstream of the zwf promoter. This minimal ''soxbox'' was able to confer paraquat inducibility when placed upstream of a normally unresponsive gnd-lacZ protein fusion. In addition, we used these findings as the basis for resecting unnecessary sequences from the region upstream of the promoters of two other SoxSregulated genes, sodA and nfo. Like the zwf soxbox, the regions required for SoxS activation of sodA and nfo appear to lie just upstream of or overlap the ؊35 hexamers of the corresponding promoters. Importantly, the sequence boundaries established here by deletion analysis agree with the primary SoxS recognition sites of zwf, sodA, and nfo that we previously identified in vitro by gel mobility shift and DNase I protection assays with a purified MalE-SoxS fusion protein.In Escherichia coli, the oxidative branch of the pentose phosphate pathway provides ribose for nucleoside biosynthesis and NADPH for reductive biosynthesis (9, 12). Two of the enzymes of this pathway, glucose 6-phosphate dehydrogenase (encoded by zwf) and 6-phosphogluconate dehydrogenase (encoded by gnd) are subject to growth rate-dependent regulation (40). The specific activities of these enzymes increase in proportion to increased growth rate during steady-state growth on different carbon sources. Studies on the growth rate-dependent regulation of zwf and gnd have shown that at least part of the increase in the specific activities of these enzymes is attributable to an increase in the transcription rates of the respective genes (25,26).Apart from its basic growth rate-dependent regulation, zwf is also a member of at least two other regulons. Thus, unlike gnd, zwf expression is transcriptionally activated by SoxS during episodes of oxidative stress induced by exposure of E. coli to superoxide-generating agents such as paraquat (17,18,39). Indeed, E. coli strains deleted for zwf are hypersensitive to oxidative stress, thus making zwf a pivotal member of the soxRS regulon (18, 21). In addition, treatment of cells with several antibiotics and aromatic weak acids appears to activate zwf transcription through the action of marA, thus making zwf a member of the multiple antibiotic resistance (mar) regulon (2,6,7,14,16).The work presented here has several overlapping long-range objectives. One is to understand the physiological basis for zwf's membership in the soxRS and marRAB regulons. The other is to identify the cis-acting regulatory sites of zwf that are involved in superoxide control for subsequent comparison with the sites required for growth rate control and for induction by antibiotics and aromatic weak...

Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid

“…In prokaryotic cells, G6PD is clustered in the soxR regulon together with other antioxidant enzymes. Upon oxidative stress the expression of these enzymes increases in a coordinated fashion protecting the bacteria from killing by phagocytes [61,62]. A similar response may protect against phagocyte-mediated tissue injury in eukaryotic organisms.…”

Real-time detection of central carbon metabolism in livingEscherichia coliand its response to perturbations

“…Besides being subject to growth-rate dependent regulation, the zwf gene encoding glucose 6-phosphate dehydrogenase is a member of the multiple antibiotic resistance (mar) operon and is transcriptionally induced by various antibiotics and aromatic acids [16,23,24]. 2,4-Dinitrophenol is a particularly strong inducer of the mar promoter at low ($1 mM) amounts and was accordingly used for chemical perturbation of E. coli BL21 cells.…”

“…Formation of d-6-phosphogluconolactone by the glucose 6-phosphate dehydrogenase catalyzed reaction is the first reaction in the oxidative branch of the pentose phosphate pathway. Glucose 6-phosphate dehydrogenase (encoded by the zwf gene) is subject to growth-rate-dependent control [16] and enzyme levels of glucose-6-phosphate dehydrogenase in cellular extracts increase three-to fivefold with increasing growth rate [21] due to transcriptional activation of the zwf gene [16]. Increased in vivo formation of d-6-phosphogluconolactone from hyperpolarized glucose tracer during mid-exponential growth thus agrees well with levels of transcriptional activation of the zwf gene at increasing growth rate and with increased demand for reductive biosynthesis.…”

“…Encouraged by this finding, the current study aimed at establishing fast and background free in vivo visualizations of metabolic network plasticity in the metabolically more flexible model prokaryote Escherichia Abbreviations: 1,3BPG, 1,3 bisphosphoglycerate; 2PG, 2-phosphoglycerate; 2,4 DNP, 2,4 dinitrophenol; 3PG, 3-phosphoglycerate; 6-PG, 6-phosphogluconate; 6-PGL, 6-phospho-d-gluconolactone; AAT, alanine aminotransferase; Ac, acetate; Ac-CoA, acetyl-CoA; ACK, acetyl kinase; ADH, alcohol dehydrogenase; ALD, aldolase; DHAP, dihydroxyacetone phosphate; ENO, enolase; Frc-1,6P 2 , fructose-1, 6-bisphosphate; Frc-6P, fructose-6phosphate; G6PDH, glucose-6phosphate dehydrogenase; GA3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; Glc, glucose; Glc-6P, glucose-6phosphate; LB, lysogeny broth; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; PDH E1/E2, pyruvate dehydrogenase components E1 and E2; PEP, phosphoenolpyruvate; PFL, pyruvate formate lyase; PFK, phosphofructokinase; PGD, 6-phosphogluconate dehydrogenase; PGL, 6-phosphogluconate lactonase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PTA, phosphotransacetylase; PTS, glucose-specific enzyme II of the phosphotransferase system; Pyr, pyruvate; PYK, pyruvate kinase; TPI, triosephosphate isomerase; YPD, yeast peptone dextrose coli. E. coli is known to modulate its metabolism at various catabolic branch points in response, e.g., to aeration, growth rate or oxidative and chemical stress [16,17]. Such adjustments of E. coli to varying growth phases and chemical perturbations are tracked by fast and easy-to interpret in vivo assays of the reaction network shown in Fig.…”

Underinvestigated Roles of Glucose-6-Phosphate Dehydrogenase

“…However, it is known that the gene coding for GR in Escherichia coli, gorA, is regulated by the OxyR transcriptional factor responding to hydrogen peroxide [40]. In contrast, G6PDH has so called "soxbox" and is regulated by the SoxS regulatory protein responding to superoxide anionradical [11,79]. Hydrogen peroxide may activate also SoxS target genes but there was no zwf among them [43,81].…”