Genetic and physical analyses of the growth rate-dependent regulation of Escherichia coli zwf expression

Abstract

Growth rate-dependent regulation of the level of Escherichia coli glucose 6-phosphate dehydrogenase, encoded by zwf, and 6-phosphogluconate dehydrogenase, encoded by gnd, is similar during steady-state growth and after nutritional upshifts. To determine whether the mechanism regulating zwf expression is like that of gnd, which involves a site of posttranscriptional control located within the structural gene, we prepared and analyzed a set of zwf-lacZ protein fusions in which the fusion joints are distributed across the glucose 6-phosphate dehydrogenase coding sequence. Expression of j8-galactosidase from the protein fusions was as growth rate dependent as that of glucose 6-phosphate dehydrogenase itself, indicating that regulation does not involve an internal regulatory region. The level of ,-galactosidase in zwf-lac operon fusion strains and the level of zwf mRNA from a wild-type strain increased with increasing growth rate, which suggests that growth rate control is exerted on the mRNA level. The half-life of the zwfmRNA mass was 3.0 min during growth on glucose and 3.4 min during growth on acetate. Thus, zwf transcription appears to be the target for growth rate control of the glucose 6-phosphate dehydrogenase level.A model system for studying growth rate-dependent regulation of nonribosomal genes is the Escherichia coli gnd gene, which encodes 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), an enzyme of the pentose phosphate pathway. An interesting feature of gnd regulation is that it occurs at the posttranscriptional level and requires a negative control site that lies deep within the structural gene (3, 4). The site, called the internal complementary sequence (ICS), appears to function by forming a long-range mRNA secondary structure that sequesters the ribosome-binding region of gnd mRNA (9). The proposed regulatory role for the secondary structure is to reduce the translation initiation frequency and/or the stability of the mRNA. The effector of the regulation is unknown.Although the formation of long-range mRNA secondary structures that sequester a translation initiation region on mRNA has been shown in other systems to be involved in mediating translational coupling (6,21,25), no other example of a role for such a structure in growth rate-dependent regulation has yet been reported. Since we were interested in determining whether the mechanism is used for growth rate control of other genes, particularly central metabolism genes, we decided to study the zwf gene, which codes for glucose 6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), the enzyme catalyzing the first step in the pentose phosphate pathway. The physiological response of zwf to changes in growth rate is similar to that of gnd. The specific activity of G6PD increases with increasing growth rate during steadystate growth in minimal media, although the extent of the increase is less than that of 6PGD (33). Also, the accumulation of G6PD after a nutritional upshift follows the same kinetics as that of 6PGD (11).In a previous publication, we ...

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

“…Similarly, it was determined that HB301( DR101) (26), which carries zwf sequences from Ϫ646 to ϩ119, was induced 6.4-fold. The data obtained with these two zwf-lacZ protein fusions indicate that zwf sequences between Ϫ140 and ϩ119 are sufficient for paraquat inducibility.…”

“…Our initial approach to delineating the cis-acting site in zwf that is involved in the soxRS-mediated response to oxidative stress was based on the observation that a zwf-lacZ operon fusion, DR52 (26), was regulated by soxRS at the transcriptional level (18). This operon fusion carries a 686-bp BclI promoter-containing zwf fragment (positions Ϫ140 to ϩ546) fused to ЈtrpA-ЈlacOZYAЈ (26). Fortuitously, the BclI site within the zwf coding sequence is in frame, and taking advantage of this fact, we isolated the 686-bp BclI fragment from NF3079(pDR17) and subcloned it into the protein fusion expression vector pMLB1034 (34).…”

“…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).…”

“…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).…”

Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon

“…The complete nucleotide sequence of the E. coli zwfgene has been published, and the promoter has been located (30). Genetic and physical analyses of zwf expression have established that the zwf gene is monocistronic and subject to growth rate-dependent regulation (29). Expression of edd, as well as gluconate transport and gluconokinase, is controlled in a negative fashion by the product of gntR, which presumably encodes a repressor CHARACTERIZATION OF E. COLI edd-eda OPERON 4639 accomplished by complementation of a defect in glucuronic acid metabolism in E. coli DF214 as described previously (5).…”