Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.     

Electron paramagnetic resonance spectroscopic characterization of dimethyl sulfoxide reductase of Escherichia coli

The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme. Iron-sulfur clusters of the [4Fe-4S] type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C. The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule. The dithionite-reduced clusters were reoxidized by DMSO or TMAO. The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E. coli nitrate reductase. The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor. In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased. Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur. These results are in agreement with predictions from amino acid sequence comparisons, that the molybdenum center is located in DmsA, while four iron-sulfur clusters are in DmsB. The midpoint potentials of the molybdenum and iron-sulfur clusters in the various preparations were determined by mediator titrations. The iron-sulfur signals could be best fitted by four clusters, with midpoint potentials spread between -50 and -330 mV. The midpoint potentials of the iron-sulfur clusters and Mo(V) species were pH dependent. In addition, all potentials became less negative in the presence of the detergent Triton X-100. Observation of relaxation enhancement of the Mo(V) species by the reduced [4Fe-4S] clusters indicated that the centers are in proximity within the protein.     

Location of anaerobic respiratory enzyme trimethylamine N-oxide reductase in the cytoplasmic membrane ofEscherichia coli strain K10

Trimethylamine N-oxide (TMAO) reductase, which is anaerobically induced by TMAO, is a terminal enzyme in anaerobic electron transport inEscherichia coli. When the organism was anaerobically grown with TMAO, a marked increase in the specific activity of TMAO reductase was observed mainly in a cell membrane fraction and stopped after exhausting TMAO. On the other hand, activity was moderately increased in a soluble fraction of the cell even after exhaustion of TMAO. Immunoblot analysis with an antiserum against the TMAO reductase purified from the soluble fractions showed that the cells growing with TMAO contained only a membrane-bound enzyme, which has a molecular mass of 94 kDa, while a soluble enzyme with 92 kDa appeared in the stationary growth phase lacking TMAO. Experiments with right-side-out and inside-out vesicles of cytoplasmic membrane indicated that the membrane-bound enzyme faces the cytoplasm. The soluble enzyme was mainly found in the cytoplasm of the cell, but also at a negligible amount in the periplasm. The membrane-bound form of TMAO reductase functioning in anaerobic electron transport seems to be cleaved and released into the cytoplasm as soluble enzyme after exhaustion of TMAO.     

Nucleotide sequence and comparative analysis of the frd operon encoding the fumarate reductase of Proteus vulgaris. Extensive sequence divergence of the membrane anchors and absence of an frd-linked ampC cephalosporinase gene

The fumarate reductase of Escherichia coli is a bioenergetically important membrane-bound flavoenzyme consisting of four subunits. A and B comprise a membrane-extrinsic catalytic domain whereas C and D are hydrophobic polypeptides which link the catalytic centres to the electron-transport chain. The nucleotide sequence of the frd operon encoding the fumarate reductase of the distantly related bacterium, Proteus vulgaris has been determined and used to predict the primary structures of the respective subunits. Extensive amino acid sequence identity (greater than 80%) was found between the fumarate reductase A and B subunits of P. vulgaris and E. coli. In contrast, the primary structures of the P. vulgaris and E. coli C and D proteins are much less closely related (about 60% homology) although the overall hydrophobicity of their three membrane-spanning segments has been conserved. In most enteric bacteria, the frd operon is followed by genes, ampR and/or ampC, required for the genetic regulation and biosynthesis of a cephalosporinase. The corresponding region of the P. vulgaris genome is occupied by an operon (orf A'BCD) containing at least four genes which are clearly unrelated to the ampC system. Intriguingly the primary structures of the OrfA and OrfD proteins suggest that, like fumarate reductase, they may be components of a membrane-bound enzyme complex involved in energy metabolism.     

A Single enzyme is Responsible for Both Dimethylsulfoxide and Trimethylamine-N-oxide Respirations as the Terminal Reductase in a Photodenitrifier: Rhodobacter sphaeroides f.s. denitrificans

Polyacrylamide gel electrophoresis of the periplasmic fractionfrom Rhodobacter sphaeroides f.s. denitrificans grown with dimethylsulfoxide(DMSO) and trimethylamine-Af-oxide (TMAO) showed that only onepolypeptide was stained for both DMSO and TMAO reductase activities,and it was the same as the purified DMSO redutase. Determinationof DMSO and TMAO reductase activities of intact cells grownwith DMSO or TMAO showed that each reagent induced both DMSOand TMAO activities and that DMSO showed much higher inductionactivity than TMAO. These results indicate that a single enzymeis responsible for both DMSO and TMAO respirations as the terminalreductase. (Received July 15, 1987; Accepted November 24, 1987)     

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.     

Nucleic acid spot hybridization for detection of Chlamydia trachomatis

Abstract The TMAO reductase activity of Escherichia coli grown anaerobically in the presence or absence of TMAO was analysed on linear sucrose gradients and on non-denaturing polyacrylamide gels. The results, together with those obtained by analysis of some other properties of TMAO reductase, showed that there are significant differences between the enzyme synthesized in the absence of TMAO ("constitutive" enzyme) and that synthesized in its presence ("inducible" enzyme).
A similar study of a tor mutant specifically altered in the structural gene for TMAO reductase, showed that the enzymes synthesized under the 2 growth conditions are probably 2 distinct enzymes encoded by different genes.     

Differentiation of the multiple S- and N-oxide-reducing activities ofEscherichia coli

InEscherichia coli, several terminal reductases catalyze the reduction of S- and N-oxide compounds. We have used mutants missing either the constitutive dimethylsulfoxide (DMSO) reductase,dmsABC, and/or the inducible trimethylamine N-oxide (TMAO) reductase,torA, to define the roles of each reductase. These studies indicated that the constitutive DMSO reductase can sustain growth on DMSO, TMAO, methionine sulfoxide (MetSO), and other N-oxide compounds. Only one inducible TMAO reductase is expressed inE. coli, and this enzyme sustains growth on TMAO but not DMSO or MetSO. Characterization of atorA ^–, dms^–double mutant revealed that adenosine N-oxide (ANO) reductase is specifically required for anaerobic respiration on ANO in this mutant.     

Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme

Dehydroquinate synthase has been purified 9000-fold from Escherichia coli K-12 (strain MM294). The synthase is encoded by the aroB gene, which is carried by plasmid pLC29-47 from the Carbon-Clarke library. Construction of an appropriate host bearing pLC29-47 results in a strain that produces 20 times more enzyme than strain MM294. Subcloning of the aroB gene behind a tac promoter results in E. coli transformants that produce 1000 times more enzyme than MM294: the synthase constitutes 5% of the soluble protein of the cell. A laborious isolation from 50 g of wild-type E. coli cells yields 80 micrograms of impure enzyme, whereas 50 g of cells containing the subcloned gene yields 150 mg of homogeneous enzyme in a two-column purification. Dehydroquinate synthase is a monomeric protein of Mr 40 000-44 000. The chromosomal enzyme from E. coli K-12, the cloned enzyme encoded by the plasmid pLC29-47, and the subcloned inducible enzyme encoded by pJB14 all comigrate on polyacrylamide gel electrophoresis under denaturing conditions.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with alpha-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl -4-hydroxyquinone N-oxide. CuSO4 inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates leads to cytochromes 548,552 leads to cytochromes 554,557 leads to trimethylamine-N-oxide reductase leads to TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and/or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.     

The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans

Dimethylsulphoxide (DMSO) and trimethylamine oxide (TMAO) sustained anaerobic growth of Proteus vulgaris with the non-fermentable substrate lactate. Cytoplasmic membrane vesicles energized by electron transfer from formate to DMSO displayed anaerobic uptake of serine, which was hindered by metabolic inhibitors known to destroy the proton motive force. This showed that DMSO reduction was coupled with a chemiosmotic mechanism of energy conversion; similar data for TMAO respiration have been presented previously. All biochemical tests applied indicated that the oxides were reduced by the same reductase system. The DMSO and TMAO reductase activities showed the same mobility on ion-exchange chromatography, and polyacrylamide disc gel electrophoresis (pH 8.9), gradient gel electrophoresis, and gel isoelectric focusing; mol. wt. and pI determined were 95,000 and 4.6, respectively. DMSO inhibited reduction of [¹⁴C]TMAO in vesicles. The reductase was inducible to a certain extent; both oxides being equally efficient as inducers. TMAO was reduced at a higher rate than DMSO, explaining faster growth of cells and increased uptake of serine in vesicles with TMAO as electron acceptor. Comparative studies with Escherichia coli also gave evidence for common TMAO and DMSO reductase systems.Abbreviations TMAO trimethylamine oxide - DMSO dimethylsulphoxide     

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.     

Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.     

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity

Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected. An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations. Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E. coli cells were transformed with plasmids coding for the enzyme and the particular peptides. Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex. It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay.