Reductase Operon Research Articles

Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli

Fumarate reductase catalyzes the final step of anaerobic electron transport in Escherichia coli when fumarate is used as a terminal electron acceptor. Transcription of the fumarate reductase operon (frdABCD) was repressed when cells were grown in the presence of either of the preferred terminal electron acceptors, oxygen or nitrate, and was stimulated modestly by fumarate. We have previously identified a locus called frdR which pleiotropically affects nitrate repression of fumarate reductase, trimethylamine N-oxide reductase, and alcohol dehydrogenase gene expression and nitrate induction of nitrate reductase expression (L. V. Kalman and R. P. Gunsalus, J. Bacteriol. 170:623-629, 1988). Transformation of various frdR mutants with plasmids identified two complementation groups, indicating that the frdR locus is composed of two genes. One class of mutants was not completely restored to wild-type frdA-lacZ expression or nitrate reductase induction when complemented with multicopy narX+ plasmids, whereas low-copy narX+ plasmid-containing strains were. A second class of frdR mutants was identified and shown to correspond to a previously described gene, narL (frdR2). Complementation of these strains with multicopy narL+ plasmids resulted in superrepression of frdA-lacZ expression and moderate elevation of nitrate reductase expression. Multicopy plasmids containing both narL+ and narX+ or only narL+ were able to complement narL mutants, whereas narX+ plasmids complemented narX mutants only when present in a copy number approximately equal to that of narL. Both narL and narX mutants retained normal oxygen control of frdA-lacZ expression. Both types of mutants are pleiotropic, as evidenced by derepressed levels of the fumarate reductase and trimethylamine N-oxide reductase enzymes and by defective induction of nitrate reductase when cells were grown in the presence of nitrate. These results indicate that both the narL and narX gene products must be present in a defined ratio in the cell. We conclude that these proteins interact to effect normal nitrate control of the anaerobic electron transport-associated operons. From these studies, we propose that narX encodes a nitrate sensor protein while narL encodes a DNA-binding regulatory protein which together function in a manner analogous to other two-component regulatory systems.

The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes.

The nucleotide sequence of a 4.4-kilobase SacII-SspI fragment encoding the narXL operon and a part of the narK gene of Escherichia coli has been determined. The narX and narL genes encode proteins of molecular weight 67,275 and 23,927, respectively, and are transcribed from a common promoter, narXp, locating within 429 bases upstream of narX. Transcription from narXp is not significantly induced by nitrate under anaerobiosis, whereas transcription from narK promoter, which overlaps narXp region and is transcribed divergently, is fully induced by nitrate. The N-terminal two-thirds of the NarL protein has extensive homology with those of a diverse set of prokaryotic regulatory proteins, including OmpR, PhoB, SfrA, UhpA, CheY, CheB, NtrC, DctD, FixJ, VirG, SpoOF, and SpoOA. A segment locating in the C-terminal half of the NarL protein seems to have potential most likely to form the helix-turn-helix structure characteristic of a class of DNA-binding protein. The protein is considered to play a role as a transcriptional activator of the nitrate reductase operon, narCHJI, and the narK gene. The C-terminal region of the NarX protein also has homology with other regulatory proteins known as counterparts of two-component regulatory systems, such as EnvZ, PhoR, PhoM, CpxA, NtrB, DctB, FixL, and VirA. Presence of two copies of hydrophobic segments in the N-terminal half of the NarX protein suggests the role as a transmembrane receptor sensing nitrate.

Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli.

The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87,350, 23,070, and 30,789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single-subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N-terminal amino acids of the dmsA gene product to those of the 82,600 subunit of purified DMSO reductase indicated that post-translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe-4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (Tp R) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (Ap R) on E. coli. The B. subtilis dihydrofolate reductase operon ( dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene ( thyB) and the Tp R-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as dfrA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA ' on pNBLl. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of jS-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RB S, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA ' -lacZ was observed, although the translational efficiency was very low.

Molecular cloning and expression of the Escherichia coli dimethyl sulfoxide reductase operon.

The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molybdoenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from an E. coli gene bank. Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO and trimethylamine N-oxide (TMAO) reductase activities 13- to 15-fold compared with wild-type cells. Amplification was highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reduced levels of enzyme amplification. Growth on nitrate or aerobic growth repressed expression of the enzyme. A 6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vector pBR322, yielding a recombinant DMSO reductase plasmid, pDMS159. Two polypeptides were amplified and identified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E. coli HB101 harboring pDMS159: a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight 23,600. Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 were detected by in vivo transcription/translation studies. The smallest subunit was poorly defined and not detectable by Coomassie blue staining. The DMSO reductase operon was localized to the 20.0-min position on the E. coli linkage map.

RNA polymerase-binding and transcription initiation sites upstream of the methyl reductase operon of Methanococcus vannielii.

RNA polymerase, purified from Methanococcus vannielii, was shown by exonuclease III footprinting to bind to a 49-base-pair (bp) region of DNA in the intergenic region upstream of mcrB. S1 nuclease protection experiments demonstrated that transcription initiation in vivo occurs within this region at 32 or 33 bp 5' to the ATG translation initiation codon of mcrB and 19 or 20 bp 3' to a TATA box.

The frdR gene of Escherichia coli globally regulates several operons involved in anaerobic growth in response to nitrate.

Fumarate reductase catalyzes the terminal step of anaerobic electron transport with fumarate as a terminal electron acceptor. Transcription of the fumarate reductase (frdABCD) operon in Escherichia coli is repressed in the presence of the preferred terminal electron acceptors, oxygen and nitrate. To identify trans-acting genes involved in regulation by nitrate, a number of E. coli mutants were generated in which expression of a frdA'-'lacZ protein fusion was no longer fully repressed by nitrate. One of these mutants, strain LK23R35, exhibited 17-fold higher beta-galactosidase activity than the wild-type strain when grown anaerobically in the presence of nitrate. When grown aerobically in the presence of nitrate, it contained three- to fourfold more beta-galactosidase activity than the wild-type strain did. Oxygen regulation of frd expression, however, was unaffected by the mutation, since the level of beta-galactosidase activity in both strains was nearly identical when they were grown in the absence of nitrate either aerobically or anaerobically. To confirm that the mutation acts in trans to frdABCD, we measured fumarate reductase levels and found them to parallel FrdA'-beta-galactosidase activity under all growth conditions tested. The effect of the mutation is pleiotropic, since the levels of nitrate reductase in LK23R35 were not induced by the addition of nitrate. The frdR mutant was also derepressed for nitrate control of the trimethylamine-N-oxide reductase and alcohol dehydrogenase enzymes. The mutation maps in a region between trp and hemA at 27 min on the E. coli chromosome. This gene, where we call frdR, is involved in both positive and negative regulation of electron transport and fermentation associated genes. A cloned 4.9-kilobase fragment of chromosomal DNA was found to complement the frdR mutation; both repression of fumarate reductase gene expression and activation of nitrate reductase gene expression were restored.

Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product.

The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E. coli chromosome at the lambda attachment site. Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic cell growth, increased an additional 1.5-fold by the presence of fumarate, the substrate, and decreased 23-fold by nitrate, a preferred electron acceptor. The addition of trimethylamine-N-oxide as an electron acceptor did not significantly alter frdA'-'lacZ expression. Control of frd operon expression is therefore exerted at the transcriptional level in response to the availability of the electron acceptors oxygen, fumarate, and nitrate. Anaerobic induction of frdA'-'lacZ expression was impaired in an fnr mutant and was restored when the fnr+ gene was provided in trans, thus establishing that the fnr gene product, Fnr, is responsible for the anaerobic activation of frd operon expression. Nitrate repression of frdA'-'lacZ expression was observed under either aerobic or anaerobic cell growth conditions in both wild-type and fnr mutant strains, demonstrating that the mechanism for nitrate repression is independent of nitrate respiration and oxygen control imparted by Fnr. Studies performed with a fnr'-'lacZ protein fusion confirmed that the fnr gene is expressed both aerobically and anaerobically. A model is proposed for the regulation of frdABCD operon expression in response to the availability of the alternate terminal electron acceptors oxygen, nitrate, and fumarate.

Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells.

Escherichia coli cells grown anaerobically in the presence of nitrate reduce the nitrate as a terminal electron acceptor in place of molecular oxygen by an induced respiratory-type electron transferring system residing in the inner membrane structure. When oxygen is introduced to a suspension of nitrate-respiring cells, the oxygen is immediately reduced preferentially and the cellular uptake of nitrate ceases abruptly. In contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduction of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the "inducer exclusion mechanism."

The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli.

Escherichia coli, which can utilize O2, nitrate, fumarate, or trimethylamine N-oxide (Me3NO) as terminal electron acceptor, preferentially utilizes the one with the highest redox potential. Thus O2 prevents induction of nitrate, fumarate, and Me3NO reductases, and nitrate curtails the induction of fumarate and Me3NO reductases. Under anaerobic conditions the narL gene product, in the presence of nitrate, is known to activate transcription of the narC operon, which encodes nitrate reductase. This study shows that the same product plays a role in the repression by nitrate of the operons (frd and tor) that encode fumarate and Me3NO reductases. In contrast, the anaerobic repression of ethanol dehydrogenase by nitrate does not require the narL product. Expression of narL does not require the fnr gene product, a pleiotropic activator that is required for full expression of narC, frd, and tor.

Lack of redox control of the anaerobically-induced nirB+ gene of Escherichia coli K-12.

Operon fusion strains of Escherichia coli K-12 have been used to demonstrate that transcription of the structural gene for NADH-dependent nitrite reductase is regulated by oxygen repression and induction by its substrate, nitrite. This two-stage regulation of nirB is totally dependent upon a functional Fnr protein. Unlike the Fnr-dependent fumarate reductase operon, nirB transcription is not repressed by nitrate. These results suggest that the Fnr protein is simply a positive control protein essential for the derepression of some, but not all, anaerobically-induced operons rather than a more general redox-sensitive regulator, as suggested by the redox control hypothesis for the regulation of gene expression in facultatively anaerobic bacteria.

Regulatory components in Citrobacter freundii ampC beta-lactamase induction.

Citrobacter freundii encodes an inducible chromosomal beta-lactamase similar to the constitutively expressed ampC beta-lactamase of Escherichia coli. In the latter species the ampC gene is located next to the fumarate reductase (frd) operon, whereas in C. freundii the ampC gene is known to be separated from frd by 1100 base pairs. This intervening DNA segment carries a gene, ampR, coding for a 31-kilodalton polypeptide. The cloned C. freundii OS60 ampC gene is inducible by beta-lactam antibiotics in E. coli, but only in the presence of an intact ampR gene. In the absence of inducer the AmpR protein represses C. freundii ampC synthesis 2.5-fold. Addition of beta-lactams induced expression from the cloned ampC beta-lactamase gene 11-fold. Thus, the AmpR protein has a positive effect on ampC expression in the presence of inducing beta-lactams. Two spontaneous mutants of C. freundii were isolated that constitutively overproduce the ampC beta-lactamase. The mutations in both these strains occurred outside the frd-amp region, suggesting that there is at least one additional component in the regulatory system. With the cloned C. freundii ampC gene in E. coli, mutants with the same phenotype could be obtained. These mutations were located on the E. coli chromosome. The constitutive beta-lactamase overproduction in these mutants requires the presence of an intact ampR gene.

Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle.

The expression of fumarate reductase in Escherichia coli has been amplified over 30-fold by utilizing a recombinant plasmid, pFR63 , carrying the fumarate reductase operon. More than 50% of the inner-membrane protein could be accounted for by the enzyme, whereas the total amount of protein associated with the membrane fraction doubled. The membrane accommodated this excess fumarate reductase without reducing the levels of other membrane-associated enzymes. At the same time, the amount of membrane lipid increased such that the lipid/protein ratio remained constant, indicating that the total amount of membrane had doubled. Small alterations in fatty acid composition as well as a large increase in cardiolipin were detected in the fumarate reductase-enriched membranes. The excess membrane was localized in novel tubular structures which were observed in thin-section and negatively stained electron-microscopic preparations. The tubules only appeared after the cytoplasmic membrane became highly enriched in fumarate reductase. They branched from the cytoplasmic membrane and were fumarate reductase. They branched from the cytoplasmic membrane and were composed of an aggregate of fumarate reductase and lipid.

Regulation of the nitrate reductase operon: effect of mutations in chlA, B, D and E genes.

Introduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less efficient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added.

Involvement of a gene of the chl E locus in the regulation of the nitrate reductase operon.

A strain of E. coli carrying a Mudl insertion leading to chlorate resistance was found to lack nitrate reductase and formate dehydrogenase activities, but to synthesize b-type cytochrome constitutively. Introduction of this insertion mutation into a strain bearing a fusion between the nitrate reductase operon (chl C, chl I) and the lac structural genes resulted in the constitutive expression of the lac genes of this last fusion. Identical results were found when the Mudl was eliminated promoting a deletion in the original insertion site. This mutation was located midway between gal and aro A, at the Chl E locus. Study of a chl E strain already described revealed similar behaviour. Absence of nitrate reductase activity in these strains which constitutively express the structural genes of the nitrate reductase operon was tentatively attributed to the simultaneous lack of a cofactor of the nitrate reductase terminal enzyme, possibly cofactor Mo-X, and of a repressor of the operon.

Overlap between ampC and frd operons on the Escherichia coli chromosome.

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli.

Strains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize beta-galactosidase constitutively in anaerobiosis; (ii) mutants in which beta-galactosidase synthesis is no longer repressed by oxygen. Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of beta-galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.

Nitrate reductase and cytochrome bnitrate reductase structural genes as parts of the nitrate reductase operon

The existence of a nitrate-reductase operon in the tryptophane region was deduced from the effects of prophage insertion in each of chlI and chlC genes and from transposition of the Mu-mediated host DNA fragments of F-prime. This operon appears to be polarized from chlC to chlI and the gene order in the region is trp -- chlI -- chlC -- purB.