Fumarate Reductase Gene Research Articles

Cloning and sequencing of the gene encoding the soluble fumarate reductase from Saccharomyces cerevisiae.

A gene of the soluble fumarate reductase (FRDS) that binds FAD non-covalently was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides designed from partial amino acid sequences of highly purified enzyme. The nucleotide sequence of a 0.99-kb amplified product was found to be nearly identical to a partial sequence of an open reading frame (ORF) previously reported (EMBL database accession number S-30830). According to the sequence in the EMBL database, we cloned 1.7-kb fragment containing entire sequence of this ORF by PCR and found that this fragment contained a perfect match to the 0.99-kb sequence amplified with the degenerate primers. From these results, we concluded that this ORF is the FRDS gene. The amino acid sequences of the regions involved in the non-covalent binding of FAD and the active site, which are conserved among the flavoprotein subunits of membrane-bound fumarate reductase and succinate dehydrogenase, were found in FRDS. However, unlike the membrane-bound enzymes, FRDS did not contain the histidine residue that covalently binds the isoalloxazine ring of FAD at or near the corresponding position. FRDS showed high homology to the product of S. cerevisiae OSM1 gene which was reported to be required for growth in hypertonic media.

Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli.

In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC), fumarate reductase (frdABCD) and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate-dependent control, and apparently function as members of a two-component regulatory system. NarX is a presumed sensor-transmitter for nitrate and possibly molybdenum detection. The presumed response-regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate-dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate-dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate-dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor-transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate-dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor-transmitter proteins.

Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria

Operon fusion strains and mutants of Escherichia coli K-12 lacking the NADH-dependent nitrite reductase have been used to determine the regulation and physiological roles of two independent pathways for nitrite reduction to ammonia. Both the formate- and NADH-dependent pathways (Nrf and Nir, respectively) were totally repressed during aerobic growth, partially active during anaerobic growth in the absence of nitrite and further induced anaerobically by nitrite. Both were dependent upon a functional Fnr protein (a transcription activator of genes for anaerobic respiration). During anaerobic growth in the presence of nitrate, the Nir pathway was fully induced but Nrf was strongly repressed. Mutants defective in the NarL protein, which induces transcription of nitrate reductase genes but represses fumarate reductase genes in the presence of nitrate, were derepressed for Nrf activity during growth with nitrate, but the Nir enzyme was less active. The synthesis of Nrf components was also sensitive to glucose repression and weak activation by NarL during growth in the absence of nitrate. These data indicate that the Nir pathway provides a mechanism for detoxifying nitrite formed in the cytoplasm as a product of nitrate reduction. In contrast, the electrogenic reduction of nitrite by the Nrf pathway provides a secondary source of energy during anaerobic growth and is consequently repressed by the NarL protein when the thermodynamically more favourable electron acceptor, nitrate, is available. Two short DNA sequences, 5'-TACCAT-3' and 5'-CTCCTT-3', were found in the promoters of operons known to be activated or repressed by the NarL protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of positive redox potentials (>+400mV) on the expression of anaerobic respiratory enzymes in Escherichia coli

The expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (Eh) in the medium. Redox potentials up to +300 mV allowed full expression of fumarate reductase (frd) genes. Higher values resulted in decreased expression. The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential Eo' = +400 mV to 440 mV. At Eh values greater than +510 mV (generated anaerobically by hexacyanoferrate(III] the degree of repression was the same as that obtained by O2. Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases. Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of Eo' = +400 mV to 440 mV.

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.

Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12.

In Escherichia coli, aerobiosis inhibits the synthesis of enzymes for anaerobic respiration (e.g., nitrate reductase and fumarate reductase) and for fermentation (e.g., formate-hydrogen lyase). Anaerobically, nitrate induces nitrate reductase synthesis and inhibits the formation of both fumarate reductase and formate-hydrogen lyase. Previous work has shown that narL+ is required for the effects of nitrate on synthesis of both nitrate reductase and fumarate reductase. Another gene, narK (whose function is unknown), has no observable effect on formation of these enzymes. We report here our studies on the role of nar genes in fumarate reductase and formate-hydrogen lyase gene expression. We observed that insertions in narX (also of unknown function) significantly relieved nitrate inhibition of fumarate reductase gene expression. This phenotype was distinct from that of narL insertions, which abolished this nitrate effect under certain growth conditions. In contrast, insertion mutations in narK and narGHJI (the structural genes for the nitrate reductase enzyme complex) significantly relieved nitrate inhibition of formate-hydrogen lyase gene expression. Insertions in narL had a lesser effect, and insertions in narX had no effect. We conclude that nitrate affects formate-hydrogen lyase synthesis by a pathway distinct from that for nitrate reductase and fumarate reductase.

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.

Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate.

The fumarate reductase enzyme complex allows Escherichia coli to grow anaerobically with fumarate as a terminal electron acceptor for oxidative phosphorylation when the preferred compounds oxygen and nitrate are not available. We used the pKO promoter test vectors to identify a single promoter for the frdABCD genes which encode fumarate reductase. Expression of galactokinase from the frd promoter-galK operon fusion plasmid was repressed by oxygen and by nitrate and was induced by fumarate, indicating that frd gene expression is regulated at the transcriptional level by these terminal electron acceptors. S1 nuclease analysis, using a single-stranded DNA probe from the frd promoter region and mRNA isolated from a fumarate reductase-induced culture, revealed that the frd mRNA transcript initiates with an adenine residue 93 bases prior to the start of frdA translation. No promoters internal to the frd genes were revealed with the plasmid promoter screening system. S1 nuclease analysis revealed that the frd mRNA terminates in a uridine-rich region centered at 46 bases after the last codon of frdD. A stem and loop structure previously described as the growth rate-dependent attenuator for the linked ampC gene precedes the frd mRNA terminus. This result confirms the proposal that the stem and loop structure serves the dual role of a frd terminator anaerobically and an ampC attenuator aerobically. The four frd genes encoding the subunits of the fumarate reductase complex thus comprise an operon which is regulated at the transcriptional level in response to the cellular availability of the alternate electron acceptors oxygen, nitrate, and fumarate.

Cloning and expression of fumarate reductase gene of Escherichia coli.

Mutants of Escherichia coli deficient in fumarate reductase activity and therefore unable to grow anaerobically with fumarate as an electron acceptor have been isolated. By F+-mediated conjugation and complementation with the mutant host, two E. coli: Col E1 recombinant DNA plasmids have been identified from the Clarke and Carbon Colony Bank which carry the structural genes for fumarate reductase. Bacteria harboring either of these plasmids express about ten times the normal level of fumarate reductase. Enzyme purified from the two sources, plasmid-carrying and plasmidless E. coli, have identical physical and kinetic properties indicating that both the 69 000 and 25 000 dalton polypeptides are amplified. Regulation of plasmid-encoded enzyme, like the chromosomally encoded enzyme, is dependent upon the presence of fumarate and anaerobiosis.

Genetic and physical characterization of lambda transducing phages (lambda frdA) containing the fumarate reductase gene of Escherichia coli K12.

Two types of fumarate reductase transducing phages, lambda frdA, carrying the wild-type frdA gene but differing in the orientation of a R.HindIII fragment of bacterial DNA were isolated from populations of recombinant transducing phages by their ability to complement the lesions of frdA mutants of E. coli. In lysogens, the cloned frdA gene was controlled by its own promoter and was fully responsive to normal regulatory stimuli. The lambda frdA phages would not complement the defects of succinate dehydrogenase (sdh) mutants. Genetic studies showed that the R.HindIII fragment contains ampA, the cis-acting regulatory locus for the chromosomal beta-lactamase gene ampC. No evidence for the presence of other markers was detected but the bacterial segment could be extended to produce plaque-forming phage derivatives containing the amp operon and a gene concerned with bacteriophage morphogenesis, groE(mop). A physical map of the 4.9 kb R.HindIII fragment was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization analysis. The frdA region contained a single asymmetric R.EcoRI target 3.33 kb from one end and the orientation of the physical map with respect to the E.coli linkage map was established.