Isolation and expression of the Escherichia coli gene encoding malate dehydrogenase.

An oligodeoxynucleotide specific for a pentapeptide sequence corresponding to amino acid residues 32 through 36 of Escherichia coli malate dehydrogenase was chemically synthesized and used to identify the mdh gene on plasmid pLC32-38 from the Clarke-Carbon recombinant library. Cells transformed with this plasmid exhibited a 10-fold increase in malate dehydrogenase activity. A 1.2-kilobase PvuII fragment which hybridized with the oligodeoxynucleotide probe was subcloned, and the identity of the mdh structural gene was confirmed by partial nucleotide sequence analysis. The expression of the mdh gene, as measured by both Northern blotting and enzyme assays, was found to vary over a 20-fold range with different culture conditions.     

Role of NAD in regulating the adhE gene of Escherichia coli.

The fermentative alcohol dehydrogenase of Escherichia coli is encoded by the adhE gene, which is induced under anaerobic conditions but repressed in air. Previous work suggested that induction of adhE might depend on NADH levels. We therefore directly measured the NAD+ and NADH levels for cultures growing aerobically and anaerobically on a series of carbon sources whose metabolism generates different relative amounts of NADH. Expression of adhE was monitored both by assay of alcohol dehydrogenase activity and by expression of phi(adhE'-lacZ) gene fusions. The expression of the adhE gene correlated with the ratio of NADH to NAD+. The role of NADH in eliciting adhE induction was supported by a variety of treatments known to change the ratio of NADH to NAD+ or alter the total NAD+-plus-NADH pool. Blocking the electron transport chain, either by mutation or by chemical inhibitors, resulted in the artificial induction of the adhE gene under aerobic conditions. Conversely, limiting NAD synthesis, by introducing mutational blocks into the biosynthetic pathway for nicotinic acid, decreased the expression of adhE under anaerobic conditions. This, in turn, was reversed by supplementation with exogenous NAD or nicotinic acid. In merodiploid strains carrying deletion or insertion mutations abolishing the synthesis of AdhE protein, an adhE-lacZ fusion was expressed at nearly 10-fold the level observed in an adhE+ background. Introduction of mutant adhE alleles producing high levels of inactive AdhE protein gave results equivalent to those seen in absence of the AdhE protein. This finding implies that it is the buildup of NADH due to lack of enzyme activity, rather than the absence of the AdhE protein per se, which causes increased induction of the phi(adhE'-lacZ) fusion. Moreover, mutations giving elevated levels of active AdhE protein decreased the induction of the phi(adhE'-lacZ) fusion. This finding suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerobic conditions, thus indirectly regulating its own expression.     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Cloning, mapping, and sequencing of the gene encoding Escherichia coli quinoprotein glucose dehydrogenase.

Escherichia coli contains pyrroloquinoline quinone-dependent glucose dehydrogenase. We cloned and sequenced the gene (gcd) encoding this enzyme and showed that the derived amino acid sequence is highly homologous to that of the gdhA gene product of Acinetobacter calcoaceticus. Stretches of homology also exist between the amino acid sequence of E. coli glucose dehydrogenase and other pyrroloquinoline quinone-dependent dehydrogenases from several bacterial species. The position of gcd on the chromosomal map of E. coli was determined to be at 3.1 min.     

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.     

Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species.

The gene adh encoding a NAD-dependent alcohol dehydrogenase from the novel strain RC3 of Sulfolobus sp. was cloned and sequenced. Both the adh gene from Sulfolobus sp. strain RC3 and the alcohol dehydrogenase gene from Sulfolobus solfataricus (DSM 1617) were expressed at a high level in Escherichia coli, and the recombinant enzymes were purified, characterized, and compared. Only a few amino acid replacements were responsible for the different kinetic and physicochemical features investigated.     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

Comparison of the fermentative alcohol dehydrogenases of Salmonella typhimurium and Escherichia coli

The adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (Genbank accession number U68173) was cloned and sequenced. The Salmonella AdhE protein has 619/878 (70%) amino acid residues identical to the AdhE protein of Escherichia coli. Salmonella AdhE was synthesized only anaerobically. It was present in higher amounts when cells were grown on reduced substrates such as sorbitol, instead of glucose. Growth on glucuronate, which generated no net nicotinamide-adenine dinucleotide reduced (NADH) during metabolism, showed the lowest AdhE levels. Analysis of fermentation products by in vivo nuclear magenetic resonance showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate. The Salmonella enzyme had a lower Michaelis-Menten constant (Km) for alcohol substrates than AdhE of E. coli although both enzymes displayed a similar Km for nicotinamide-adenine dinucleotide (NAD+). Although AdhE of E. coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to eight carbons.     

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.