Yong T-S, Li E, Clark D P and Stanley S L (1996) Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a novel method for the identification of new anti-amebic drugs. Proc Natl Acad Sci USA 93: 6464-6469
Abstract: The pathogenic protozoan parasite, Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+-and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for the anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasitic culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.
Abstract: 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 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 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 absence of the AdhE protein. This implies that it is the build-up of NADH due to lack of enzyme activity, rather than the absence of the AdhE protein per se, which causes increased induction of the adhE'-lacZ fusion. Moreover, mutations giving elevated levels of active AdhE protein decreased the induction of the adhE'-lacZ fusion. This suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerobic conditions, thus indirectly regulating its own expression.
Abstract: Defects in the acd gene (which may be allelic to ubiH) result in the inactivation of the coenzyme A-linked acetaldehyde dehydrogenase activity of the multifunctional AdhE protein of Escherichia coli. This activity is restored by addition of ubiquinone-0 to cell extracts. However, the alcohol dehydrogenase activity of the AdhE protein is not decreased by an acd mutation. Abolition of ubiquinone biosynthesis by mutation of ubiA or ubiF does not affect either the acetaldehyde dehydrogenase or the alcohol dehydrogenase activity of AdhE. Guaiacol (2-methoxyphenol) which resembles the intermediate that builds up in ubiH mutants, except in lacking the octaprenyl side-chain, was found to inhibit ethanol metabolism in vivo, presumably via inhibition of acetaldehyde dehydrogenase. In vitro assays confirmed that guaiacol inhibited acetaldehyde dehydrogenase. This suggests that the acetaldehyde dehydrogenase activity of AdhE is specifically inhibited by intermediates of ubiquinone synthesis that accumulate in acd mutants and that this inhibition may be relieved by ubiquinone.
We have cloned and sequenced the adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (GenBank accession number U68173). The Salmonella AdhE protein has 619/878 (70 %) amino acid residues identical to AdhE of E. coli. Salmonella AdhE was synthesized only anaerobically. It was present in higher amounts when cells were grown on reduced substrates like sorbitol, instead of glucose. Growth on glucuronate, which generates no net NADH during metabolism, showed the lowest AdhE levels. Analysis of fermentation products by in vivo NMR showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate. The Salmonella enzyme had a lower Km for alcohol substrates than AdhE of E. coli although both enzymes displayed a similar Km for NAD. Although AdhE of E. coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to 8 carbons.
Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and translational levels. To study the AdhE protein, the adhE+ structural gene was cloned into expression vectors under the control of the lacZ and trp promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) were previously isolated that produced high levels of AdhE under both aerobic and anaerobic conditions. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly-functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from adhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.
The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.
We selected a mutant of Salmonella enterica serovar Typhimurium that is capable of growing in air on ethanol as sole carbon and energy source. This adhI mutant expressed high levels of a novel alcohol dehydrogenase (AdhI) that uses ethanol, 1-propanol and 2-propanol as substrates. The fermentative AdhE alcohol dehydrogenase was not expressed aerobically in the adhI mutant. Anaerobically, both the novel AdhI enzyme and the AdhE were expressed simultaneously in the adhI mutant. However, the adhI mutant showed no alteration in the composition of the fermentation products. In addition we found that both the parental Salmonella and its alcohol using adhI mutant expressed substantial levels of a dye-linked aldehyde dehydrogenase that is presumably responsible for conversion of acetaldehyde to acetate. This contrasts with the situation in Escherichia coli where mutants able to grow on ethanol express high aerobic levels of the AdhE enzyme, which performs both the alcohol dehydrogenase and aldehyde dehydrogenase reactions.
Escherichia coli RNase G encoded by the rng gene is involved in degradation of adhE mRNA. Overproduction of the AdhE protein by rng mutants was found to depend on the genetic background of strains derived from DC272 (adhC81) or MC1061. We found that DC272 carried a point mutation in the Cra-binding site of the adhE promoter. The Cra protein encoded by the cra gene is known to act as a repressor of adhE. P1-phage-mediated transduction and lacZ fusion analysis with the mutant adhE promoter confirmed that this mutation is responsible for overproduction. On the other hand, Southern hybridization revealed that MC1061 had a 0.85-kb deletion of the cra gene. Overproduction of AdhE in the MC1061 background was reversed to the wild-type levels by introduction of a plasmid carrying the cra(+) gene. These results indicated that expression of the adhE gene was regulated transcriptionally by Cra and posttranscriptionally by RNase G.
Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA. Consequently, defects in RNase G result in elevation of AdhE levels. Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation. We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng. Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430. DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430. This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2). However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants. We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced. These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.
Bunch P K, Mat-Jan F, Lee N and Clark D P (1997) Molecular cloning of the ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143: 187-195
Abstract: Under anaerobic conditions, especially at low pH, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH). This LDH is present in substantial basal levels under all conditions but increases approximately ten-fold at low pH. The ldhA gene, encoding the fermentative lactate dehydrogenase of E. coli was cloned using l10E6 of Kohara's collection as the source of DNA. The ldhA gene was subcloned on a 2.8 kb MluI-MluI fragment into a multicopy vector and the region encompassing the ldhA gene was sequenced. The ldhA gene of E. coli was highly homologous to genes for other D-lactate specific dehydrogenases but unrelated to those for the L-lactate specific enzymes . We constructed a disrupted derivative of the ldhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the ldhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy, the ldhA gene was greatly overexpressed suggesting escape from negative regulation. Cells expressing high levels of the D-LDH grew very poorly, especially in minimal medium. This poor growth was largely counteracted by supplementation with high alanine or pyruvate concentrations, suggesting that excess LDH converts the pyruvate pool to lactate, thus creating a shortage of 3-carbon metabolic intermediates. Using an ldhA-cat gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the ldhA gene. Some of these appeared to be in the pta gene which encodes phosphotransacetylase, suggesting the possible involvement of acetyl-phosphate in ldhA regulation.
The fermentative lactate dehydrogenase (LDH) of Escherichia coli is induced by low pH under anaerobic conditions. We have now made both translational and transcriptional gene fusions to the ldhA gene, which encodes the fermentative LDH. Both types of ldhA-lacZ fusion were induced by low pH, but only in the absence of air. However, the translational fusions were consistently expressed at a 5- to 10-fold higher level than the transcriptional fusions, perhaps implying some post-transcriptional effect on ldhA expression. Introduction of arcB::Kan decreased expression of both translational and transcriptional ldhA-lacZ fusions by 3- to 5-fold. Disruption of the mlc gene, which encodes a repressor of several genes of the phosphotransferase system, almost abolished expression of ldhA. Disruption of the csrA gene caused a moderate drop in expression of both operon and protein ldhA fusions, whereas insertional inactivation of csrB or glgA had the opposite effect. These effects are probably indirect, resulting from alterations in sugar accumulation versus storage. Mutations in ptsG, cra, fnr, narL, rpoS, osmZ, appY, ack/pta, aceEF, pfl, and ldhA had no effect on expression of the ldhA fusions. The ldhA gene was not induced by the membrane permeant weak acid, benzoate, implying that it does not respond to the internal pH directly. Little pH induction was seen during growth on glycerol plus fumarate, suggesting that products of sugar fermentation are necessary for acid induction. Addition of succinate, acetate or lactate had no effect on ldhA expression. In contrast, pyruvate caused a 2 to 4-fold increase in expression of ldhA-lacZ. This accords with the idea that increased sugar metabolism indirectly induces ldhA.
Clark D P (1994) Chromate reductase activity of Enterobacter aerogenes is induced anaerobically by nitrite. FEMS Microbiol Letters 122: 233-238
Abstract: A chromate resistant mutant of Enterobacter aerogenes manifested its chromate resistance only under aerobic conditions. Both parent and mutant showed substantial levels of anaerobic chromate reductase activity when grown on glycerol plus fumarate. The chromate reductase was further induced by growth in the presence of nitrite but was repressed by nitrate. The chromate reductase activity paralleled that of the formate-linked nitrite reductase. There was no significant difference in chromate reductase levels between the parent and its chromate resistant mutant, indicating that this enzyme activity is not, in fact responsible for chromate resistance as was suggested previously by others.
Abstract: The cadA gene, encoding lysine decarboxylase in Escherichia coli, is induced by low pH and anaerobic conditions, as well as by the substrate, lysine. We have used operon fusions of cadA to lacZ to investigate the effects of aeration on cadA regulation. When an insertion mutation in osmZ (= hns) was introduced, a cadA-lacZ fusion was derepressed in the presence of air to approximately the same level as seen under anaerobic conditions. However, the pH-dependent regulation of cadA was not affected by osmZ. Introduction of mutations in rpoS, fur or fnr had no significant effect on cadA expression. However, defects in arcB or arcA largely abolished expression of cadA under anaerobic conditions. Nonetheless, strains defective in both arcB and osmZ showed the same high cadA-lac expression in air as seen in the single osmZ derivatives. Blocking the respiratory chain with mutations or chemical inhibitors also caused derepression of a cadA-lacZ fusion in air, while agents affecting the proton gradient had no effect. Derepression of cadA in air was also mediated by several chelating agents, in particular methoxyindole carboxylic acid. Addition of Fe2+ overcame this effect. Chelating agents also abolished the expression under aerobic conditions of several genes known to be under arcAB control and which are normally repressed under anaerobic conditions but induced in the presence of air. This implies that the effect of chelating agents on cadA expression is mediated via the arcAB regulatory system.
Abstract: Bacillus subtilis can grow anaerobically by respiration with nitrate as terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo NMR scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all of acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.
Abstract: Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx. 2:1:1. Calculations of carbon and electron balances accounted for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E. coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.
Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0. 5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB(glc). Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.
Sato H and Clark D P (1995) Degradation of dibenzothiophene sulphoxide and sulphone by Arthrobacter strain DBTS2. Microbios 83: 145-159
Abstract: A natural isolate, Arthrobacter DBTS2, originally selected for growth on dibenzothiophene (DBT) sulfone was also able to grow on DBT-sulfoxide and, less well on several other sulfur containing aromatic compounds. Although it was unable to grow on naphthalene or biphenyl, the presence of either compound promoted the release of inorganic sulfur from DBT-sulfone. In addition, naphthalene induced oxygen uptake with DBT-sulfoxide as substrate, although it did not induce the oxidation of DBT-sulfone. However, the oxidation of DBT-sulfone was induced both by itself and by DBT-sulfoxide. The oxidation of these heterocyclic compounds by Arthrobacter DBTS2 was subject to catabolite repression by succinate or acetate, as is the case for other bacteria with an oxidatively oriented metabolism. Mutants of DBTS2 defective in growth on DBT-sulfone did not release inorganic sulfur from this compound, whereas mutants of DBTS2 defective in benzoate degradation released sulfur at rates similar to their parent.
Abstract: The thdF gene of Escherichia coli encodes a 48 kDa protein which is involved in the oxidation of derivatives of the sulfur containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by PCR and inserted in front of the lacZ structural gene. Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne, fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2 to 3-fold, but only during stationary phase. Tryptophan, indole, and several indole derivatives had no effect.
Growth of Escherichia coli using the tripeptide glutathione as a sulfur source is well documented, but transport of glutathione into E. coli is uncharacterized. We have found that the ybiK gene, at 18.7 minutes, appears to be involved in the transport of glutathione and have therefore renamed ybiK as spt for sulfur peptide transport. The ybiK/spt gene is the first of what appear to be five cotranscribed genes three of which show high homology to the peptide transport operon, dpp. When the lacZ gene encoding beta-galactosidase was fused to the promoter of ybiK/spt, expression of the ybiK-lacZ fusion was repressed in rich media. This was shown to be due to the presence of exogenous cysteine. The ybiK-lacZ fusion was found to be regulated by cysB, the transcriptional activator for the cysteine regulon. Mutations in the cysB or ybiK genes led to severe growth inhibition when cells were given glutathione as the sole sulfur source. In particular, strains of E. coli containing mutations in both the ybiK and cysA genes were unable to grow when the sole sulfur source provided was glutathione whereas single cysA mutants grew well with glutathione. In contrast, no such defects were seen when L-djenkolic acid or cysteine were used as the sole sulfur source.