Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.     

The anaerobic oxidation of dihydroorotate by Escherichia coli K-12.

The oxidation of dihydroorotate under anaerobic conditions has been examined using various mutant strains of Escherichia coli K-12. This oxidation in cells grown anaerobically in a glucose minimal medium is linked via menaquinone to the fumarate reductase enzyme coded for by the frd gene and is independent of the cytochromes. The same dihydroorotate dehydrogenase protein functions in both the anaerobic and aerobic oxidation of dihydroorotate. Ferricyanide can act as an artificial electron acceptor for dihydroorotate dehydrogenase and the dihydroorotate-menaquinone-ferricyanide reductase activity can be solubilised by 2 M guanidine-HCl with little loss of activity.     

Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.     

Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase.

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV). The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop.     

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.     

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.     

Proteins of the Inner Membrane of Escherichia coli: Changes in Composition Associated with Anaerobic Growth and Fumarate Reductase Amber Mutation

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.     

Enzyme Complex Which Couples Glycerol-3-Phosphate Dehydrogenation to Fumarate Reduction in Escherichia coli

Anaerobic growth of Escherichia coli on glycerol as carbon source and fumarate as hydrogen acceptor results in the induction of both anaerobic sn-glycerol-3-phosphate (G3P) dehydrogenase and fumarate reductase. In previous studies, the G3P dehydrogenase was measured by the G3P-dependent reduction of a tetrazolium dye mediated by phenazine methosulfate in the presence of flavine mononucleotide and flavine adenine dinucleotide, and fumarate reductase was measured by the fumarate-dependent oxidation of reduced flavine mononucleotide. Results from the present study indicate that these two enzymes actually constitute a functional complex which is sedimentable at 200,000 x g in 2 h and which can catalyze the dehydrogenation of G3P at the expense of fumarate without any added cofactor. This coupling activity is not constituted by simply mixing an extract containing anaerobic G3P dehydrogenase with another extract containing fumarate reductase. Additional evidence for an organized complex is provided by the high degree of sensitivity of the coupled reaction to low concentrations of the detergents Triton X-100 and Emasol 1130 in comparison with the individual activities of the two component enzymes measured with the aid of artificial cofactors.     

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.     

Anaerobic growth of Escherichia coli on formate by reduction of nitrate, fumarate, and trimethylamine N-oxide.

Anaerobic growth of E. coli, strain K-10, depending on formate oxidation by nitrate, fumarate, and trimethylamine N-oxide was followed in a medium containing peptone. The presence of formate and peptone was indispensable for growth with fumarate and trimethylamine N-oxide reduction. While there was no growth in the absence of acceptor, growth was observed in the absence of formate by nitrate reduction though not as much as under aerobic conditions. Per mole consumed formate equimolar succinate or trimethylamine was formed, but 1.2 mole of nitrate was produced, probably depending partly on peptone oxidation. The molar growth yield on formate was found to be 6.5, 7.6, and 7.0 g cells/mole depending on the reduction of nitrate, fumarate, and trimethylamine N-oxide, respectively, suggesting the formation of one mole ATP coupled to the anaerobic electron transfers from formate.     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.     

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Geobacter sulfurreducens strain PCA oxidized acetate to CO2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases NADP+ and NAD+, respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and strictly CoA-dependent; a low activity was also observed with NADP+. Succinate dehydrogenase and fumarate ductase both were membrane-bound. Succinate oxidation was coupled to NADP+ reduction whereas fumarate reduction was coupled to NADPH and NADH Coupling of succinate oxidation to NADP+ or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.     

Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.