Expression Of Zwf Research Articles

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis.

Riboflavin is an essential vitamin widely used in the food, pharmaceutical, and feed industries. However, the insufficient supply of precursors caused by the imbalance of intracellular metabolic flow limits the riboflavin synthesis by industrial strains. Here, we increase riboflavin production by tuning multiple gene expression to balance intracellular metabolic flow. First, we tuned the expression of mCherry and egfp genes within operons by generating libraries of tunable intergenic regions (TIGRs) and confirmed the relative expression of the two reporter genes. The TIGR library can coordinate the expression ratio of reporter genes more than 180 times in Escherichia coli and more than 70 times in Bacillus subtilis. Next, we used this strategy to tune the expression of zwf, ribBA, and ywlf genes within operons through the TIGR library to increase the intracellular precursor pool for riboflavin biosynthesis. Based on the fluorescence characteristics of riboflavin, 96-well plates were used to screen the optimal combination mutants quickly. The best-engineered strain was selected from the library, which produced 2.7 g/L riboflavin, increasing by 64.35% in the shake flask. Finally, the riboflavin titer increased by 59.27% to 11.77 g/L in fed-batch fermentation. The strategy described here will contribute to the industrial production of riboflavin and related products by B. subtilis.

Induced expression of the zwf gene in the presence of glucose contributes to lowering of glucose 6-phosphate level and consequently reduction of growth rate of Mycobacterium smegmatis.

In Mycobacterium smegmatis (renamed Mycolicibacterium smegmatis), glucose 6-phosphate (G6P) level is exceptionally high as compared to other bacteria, E. coli for example. Earlier investigations have indicated that G6P protects M. smegmatis (Msm) against oxidative stress-inducing agents. G6P is a glycolytic intermediate formed either directly through the phosphorylation of glucose or indirectly via the gluconeogenic pathway. Its consumption is catalysed by several enzymes, one of which being the NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf (msmeg_0314). While investigating the extent to which the carbon sources glucose and glycerol influence Msm growth, we observed that intracellular concentration of G6P was lower in the former's presence than the latter. We could correlate this difference with that in the growth rate, which was higher in glycerol than glucose. We also found that lowering of G6P content in glucose-grown cells was triggered by the induced expression of zwf and the resultant increase in G6PDH activity. When we silenced zwf using CRISPR-Cas9 technology, we observed a significant rise in the growth rate of Msm. Therefore, we have found that depletion of G6P in glucose-grown cells due to increased G6PDH activity is at least one reason why the growth rate of Msm in glucose is less than glycerol. However, we could not establish a similar link-up between slow growth in glucose and lowering of G6P level in the case of Mycobacterium tuberculosis (Mtb). Mycobacteria, therefore, may have evolved diverse mechanisms to ensure that they use glycerol preferentially over glucose for their growth.

Improving l-threonine production in Escherichia coli by elimination of transporters ProP and ProVWX

BackgroundBetaine, an osmoprotective compatible solute, has been used to improve l-threonine production in engineered Escherichia colil-threonine producer. Betaine supplementation upregulates the expression of zwf encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for l-threonine production. In E. coli, betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX. ProP is a H+-osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in E. coli by enzymes encoded by betABIT. However, the influence of ProP, ProVWX and betABIT on l-threonine production in E. coli has not been investigated.ResultsIn this study, the influence of ProP, ProVWX and betABIT on l-threonine production in E. coli has been investigated. Addition of betaine slightly improved the growth of the l-threonine producing E. coli strain TWF001 as well as the l-threonine production. Deletion of betABIT retarded the growth of TWF001 and slightly decreased the l-threonine production. However, deletion of proP or/and proVWX significantly increased the l-threonine production. When proP was deleted, the l-threonine production increased 33.3%; when proVWX was deleted, the l-threonine production increased 40.0%. When both proP and proVWX were deleted, the resulting strain TSW003 produced 23.5 g/l l-threonine after 36 h flask cultivation. The genes betABIT, proC, fadR, crr and ptsG were individually deleted from TSW003, and it was found that further absence of either crr (TWS008) or ptsG (TWS009) improved l-threonine production. TSW008 produced 24.9 g/l l-threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l l-threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001.ConclusionsIn this study, l-threonine-producing E. coli strains TSW008 and TSW009 with high l-threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms.

Betaine supplementation improved l-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

Abstract Background The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an l -threonine producer, was used to examine the production of l -threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. Results Betaine supplementation in the medium of E. coli THRD significantly improved l -threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for l -threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while l -threonine fermentation of the new strain was promoted by betaine addition. Conclusions We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of l -threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains. How to cite: Li Y, Zhang D, Cai N, et al. Betaine supplementation improved l -threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.03.004 .

Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Hyaluronic acid (HA) is a high-value biopolymer that is produced in large scales using attenuated strains ofgroup C streptococci. However, due to the pathogenicity and fastidious nature of these bacteria, the development of bioprocesses for HA production centered on robust ‘Generally Recognized as Safe (GRAS)’ organisms, such as Bacillus subtilis, is of increased interest. Here, we report metabolic engineering of novel B. subtilis strains in which the carbon flux has been partially diverted from central metabolism, i.e. the pentose phosphate pathway (PPP) and glycolysis, into HA biosynthesis. First, an improved base strain of B. subtilis was engineered for more effective HA production with less susceptibility to catabolite repression when expressing genes from a xylose-inducible promoter. Subsequently, Clustered Regularly Interspaced Palindromic Repeats interference (CRISPRi) was applied to reduce the expression of individual pfkA or zwf in the base strain, leading to substantial improvements to the HA titer with a concomitant decrease in the molecular weight (MW). On the other hand, multiplexed repression of both pfkA and zwf expression resulted in increases to the HA titer of up to 108% and enhancements to the MW, compared to the base strain. Moreover, the addition of exogenous HA monomers, i.e. glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc), to B. subtilis cultures markedly improved the HA MW but decreased the HA titer, providing insights into the mechanism of HA biosynthesis by streptococcal hyaluronan synthase (SeHAS) in B. subtilis. Our study demonstrates the successful application of metabolic engineering strategies to establish B. subtilis as an effective platform for high-level HA production.

The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae

The NTR system is the major regulator of nitrogen metabolism in Bacteria. Despite its broad and well-known role in the assimilation, biosynthesis and recycling of nitrogenous molecules, little is known about its role in carbon metabolism. In this work, we present a new facet of the NTR system in the control of NADPH concentration and the biosynthesis of molecules dependent on reduced coenzyme in Herbaspirillum seropedicae SmR1. We demonstrated that a ntrC mutant strain accumulated high levels of polyhydroxybutyrate (PHB), reaching levels up to 2-fold higher than the parental strain. In the absence of NtrC, the activity of glucose-6-phosphate dehydrogenase (encoded by zwf) increased by 2.8-fold, consequently leading to a 2.1-fold increase in the NADPH/NADP+ ratio. A GFP fusion showed that expression of zwf is likewise controlled by NtrC. The increase in NADPH availability stimulated the production of polyhydroxybutyrate regardless the C/N ratio in the medium. The mutant ntrC was more resistant to H2O2 exposure and controlled the propagation of ROS when facing the oxidative condition, a phenotype associated with the increase in PHB content.

Effect of Tween 40 and DtsR1 on L-arginine overproduction in Corynebacterium crenatum.

Backgroundl-Glutamate is an important precursor in the l-arginine (l-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and dtsR1 disruption, have been widely used to induce l-glutamate overproduction in Corynebacterium glutamicum. In this study, a novel strategy for l-Arg overproduction through Tween 40 trigger and ΔdtsR1 mutant were proposed in Corynebacterium crenatum.ResultsCorynebacterium crenatum mutant (CCM01) was selected as a host strain, whose argR was lethal via mutagenesis screening, the proB gene was knocked out, and argB was replaced by argB M4 (E19R, H26E, D311R, and D312R) to release l-Arg feedback resistance. After Tween 40 trigger in the logarithmic period, l-Arg production increased from 15.22 to 17.73 g/L in CCM01 strain. When NCgl1221 and dtsR1 disruption (CCM03), l-Arg production drastically increased to 27.45 g/L and then further to 29.97 g/L after Tween 40 trigger. Moreover, the specific activity of α-oxoglutarate dehydrogenase complex (ODHC) decreased, whereas the regeneration of NADP+/NADPH significantly increased after dtsR1 disruption and Tween 40 trigger. Results of real-time PCR showed that the transcriptional levels of odhA, sucB, and lpdA (encoding three subunits of the ODHC complex) were downregulated after Tween 40 trigger or dtsR1 disruption. By contrast, zwf transcription (encoding glucose-6-phosphate dehydrogenase) showed no significant difference among CCM01, CCM02 (ΔNCgl1221), and CCM03 (ΔNCgl1221ΔdtsR1) strains without Tween 40 trigger but evidently increased by 5.50 folds after Tween 40 trigger.ConclusionA novel strategy for l-Arg overproduction by dtsR1 disruption and Tween 40 trigger in C. crenatum was reported. Tween 40 addition exhibited a bifunctional mechanism for l-Arg overproduction, including reduced ODHC activity and enhanced NADPH pools accumulation by downregulated dtsR1 expression and upregulated zwf expression, respectively.

Metabolic engineering of Escherichia coli for the production of riboflavin.

BackgroundRiboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions.ResultsThe basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose.ConclusionsThe engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

Metabolic engineering of

Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

Expression of NAD(H) Kinase and Glucose-6-Phosphate Dehydrogenase Improve NADPH Supply and l-isoleucine Biosynthesis in Corynebacterium glutamicum ssp. lactofermentum

Corynebacterium glutamicum is the workhorse for the production of amino acids, including L-isoleucine (Ile). During Ile biosynthesis, NADPH is required as a crucial cofactor. In this study, four NADPH-supplying strategies based on NAD kinase, NADH kinase, glucose-6-phosphate dehydrogenase, and NAD kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on Ile biosynthesis were examined. PpnK is a NAD kinase of C. glutamicum ssp. lactofermentum JHI3-156 that predominantly phosphorylates NAD(+) to produce NADP(+). Pos5 is a NADH kinase of Saccharomyces cerevisiae that predominantly phosphorylates NADH to produce NADPH. Zwf is a glucose-6-phosphate dehydrogenase of JHI3-156. The ppnK, POS5, zwf, and zwf-ppnK genes were overexpressed in the Ile-producing strain JHI3-156. The expression of all four genes increased intracellular NADPH concentration and Ile production. The increase of NADPH concentration and Ile production in a POS5-expressing strain (229 and 75.6%, respectively) was higher than that in a ppnK-expression strain. The expression of zwf also increased NADPH supply and Ile biosynthesis, but the constitutive expression of zwf was not as effective as the inducible expression of zwf. Coexpression of zwf and ppnK genes greatly enhanced NADPH supply and thus improved Ile production by up to 85.9%, indicating that this strategy was the most effective one. These results are helpful for improving Ile biosynthesis and other biosynthetic processes.

Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be efficient osmoprotectants in Sinorhizobium meliloti

Inactivation of the zwf gene in Sinorhizobium meliloti induces an osmosensitive phenotype and the loss of osmoprotection by trehalose and sucrose, but not by ectoine and glycine betaine. This phenotype is not linked to a defect in the biosynthesis of endogenous solutes. zwf expression is induced by high osmolarity, sucrose and trehalose, but is repressed by betaine. A zwf mutant is more sensitive than its parental strain to superoxide ions, suggesting that glucose 6-phosphate dehydrogenase involvement in the osmotic response most likely results from the production of reactive oxygen species during osmotic stress.

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli.

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity in Escherichia coli. We constructed a new E. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression of soxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress in E. coli. The downregulation of soxS expression by polyamines was not shown in the soxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded by zwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded by sodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of the zwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a higher soxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 micromol/L. While the growth rate of the mutant was decreased, soxS expression was increased in a concentration-dependent manner above 0.01 micromol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, and soxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulate soxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results in soxS downregulation.

Purification of a MalE-SoxS fusion protein and identification of the control sites of Escherichia coli superoxide-inducible genes.

In Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA-binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to malE, which encodes maltose-binding protein, and demonstrated that the in vivo expression of the MalE-SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated in vitro expression of zwf in a coupled transcription-translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE-SoxS protected from DNase I attack 22-27 bp segments immediately adjacent to or overlapping the -35 hexamers of the zwf, sodA, nfo, micF, and fumC promoters. The protected regions revealed a consensus 'soxbox' sequence.

Molecular analysis of the Erwinia chrysanthemi region containing the kdgA and zwf genes.

The pathways of pectin and galacturonate catabolism in Erwinia chrysanthemi converge to form a common intermediate, 2-keto-3-deoxygluconate, which is phosphorylated to form 2-keto-3-deoxy-6-phosphogluconate (KDGP) and then cleaved by the aldolase encoded by the kdgA gene. We cloned the kdgA gene of the E. chrysanthemi strain 3937 by complementing an Escherichia coli kdgA mutation, using an RP4-derivative plasmid. Restriction mapping of the kdgA region and isolation of kdgA-lac fusions allowed the more precise localization of the kdgA gene and determination of its transcriptional direction. The nucleotide sequence of the kdgA region indicated that the kdgA reading frame is 639 bases long, corresponding to a protein of 213 amino acids with a molecular mass of 22,187 Da. Comparison of the deduced primary amino acid sequences of the E. chrysanthemi KDGP-aldolase to the E. coli, Zymomonas mobilis and Pseudomonas putida enzymes showed that they are highly conserved. The E. chrysanthemi kdgA structural gene begins 153 bases downstream of an open reading frame that has a high homology with the zwf E. coli gene encoding glucose-6-phosphate dehydrogenase. The zwf gene is also linked to eda (kdgA) in E. coli and P. putida but genetic organization is different. Regulation of zwf and kdgA expression in E. chrysanthemi was analysed using lacZ fusions. The expression of zwf is independent of the growth rate, but is repressed in the presence of glucose. Induction of kdgA by pectin-degradation products is mediated in vivo by the negative regulatory gene kdgR, which also controls all the steps of pectin degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

The polycistronic mRNA of the Zymomonas mobilis glf-zwf-edd-glk operon is subject to complex transcript processing.

The full-length 6.14-kb polycistronic glf-zwf-edd-glk mRNA from Zymomonas mobilis appears to be processed by endonucleolytic cleavage, resulting in the formation of several discrete transcripts. Northern analysis and transcript mapping revealed that the processed transcripts correspond to functional mono-, di-, or tricistronic messages. The relative abundance of the gene-specific, functional messages was measured. Expression of zwf and edd correlated well with functional message levels. Disproportionally high levels of the glk-specific mRNAs might compensate for the instability of glucokinase by allowing increased translation. The relative abundance of the discrete transcripts was shown to be a function of their respective decay rates. Northern analysis of the fate of the 6.14-kb transcript after inhibition of transcription by rifampin showed that the abundance of shorter, more stable transcripts increased at the expense of longer, less stable transcripts. This is suggestive of endonucleolytic mRNA processing. The most abundant 5' and 3' transcript ends were found to lie within secondary structures that probably impart stability to the most abundant mRNAs.

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

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 beta-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 beta-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 zwf mRNA 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.

Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase

In Escherichia coli K-12, expression of zwf, the gene for glucose 6-phosphate dehydrogenase, is coordinated with the cellular growth rate and induced by superoxide-generating agents. To initiate the study of the molecular mechanisms regulating its expression, the gene was cloned and its DNA sequence was determined. The 5' ends of zwf mRNA isolated from cells growing in glucose and acetate minimal media were mapped. The map was complex in that transcripts mapped to -45, -52, and -62, with respect to the beginning of the coding sequence. Three analytical methods were used to search the DNA sequence for putative promoters. Only one sequence for a promoter recognized by the sigma 70 form of RNA polymerase was found by all three search routines that could be aligned with a mapped transcript, indicating that the other transcripts arise by processing of the mRNA. A computer-assisted search did not reveal a thermodynamically stable long-range mRNA secondary structure that is capable of sequestering the translation initiation region, which suggests that growth-rate-dependent regulation of glucose 6-phosphate dehydrogenase level may not be carried out by a mechanism similar to the one for the gene (gnd) for 6-phosphogluconate dehydrogenase. The DNA segment between the -10 hexamer and the start point of transcription resembles the discriminator sequence of stable RNA genes, which has been implicated in stringent control and growth-rate-dependent regulation.

“Up-promoter” mutations of glucose 6-phosphate dehydrogenase in Escherichia coli

The “up-promoter” mutation, zwfL1, of the constitutive gene ( zwf) for glucose 6-phosphate dehydrogenase of Escherichia coli (Fraenkel & Banerjee, 1971), has been mapped with respect to point mutations in the structural gene. Both zwfL1 and two other high-level mutations are at one end of the gene. The direction of transcription of zwf is probably counter-clockwise, as the map is usually drawn. The level of expression of zwf in the wild-type is about 0.1 that of fully induced lac, while one of the promoter mutants, zwfL2, increases its rate of expression by 18-fold, giving perhaps the highest rate for any known bacterial gene.