Investigation of Escherichia coli Fumarate Reductase Subunit Function Using Transposon Tn5

1988

Molecular Microbiology

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Recombinant plasmids which carried portions of the Escherichia coli frd operon were constructed and their expression examined by in vivo complementation of E. coli MI1443. This strain lacked a chromosomal frd operon and was unable to grow anaerobically on glycerol and fumarate. Introduction of all four fumarate reductase subunits into E. coli MI1443 was essential for the restoration of growth. The FRD A, FRD B dimer (but neither subunit alone) was active in the benzyl viologen oxidase assay. Both FRD C and FRD D were required for membrane association of fumarate reductase and for the oxidation of reduced quinone analogues. Introduction into E. coli MI1443 of the frdABC and frdD genes on two separate plasmid vectors failed to restore anaerobic growth on glycerol and fumarate. Thus separation of the DNA coding for the FRD C and FRD D proteins affected the ability of fumarate reductase to assemble into a functional complex.

Section: Introductionmentioning

confidence: 81%

Section: Piasmid Construction and Transformationmentioning

confidence: 99%

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Fumarate reductase of Escherichia coli: an investigation of function and assembly using in vivo complementation

Condon

1988

Molecular Microbiology

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“…is the catalytic domain, which can exist alone in the cytoplasm (11) while the membrane bound anchors, FrdCD, are required for coupling to electron transport in vivo or to quinone analogues in vitro (11,17). The FrdAB dimer has a two-fold higher activity with benzyl viologen as donor than does the FrdABCD tetramer, while it lacks activity with quinone analogues (11).…”

Section: Discussionmentioning

confidence: 99%

Regulation ofin vitroexpression of theEscherichia coli frdoperon: alanine and Fnr represent positive and negative control elements

Latour

1988

Nucl Acids Res

The frdABCD operon of Escherichia coli encodes the anaerobically expressed terminal electron transport enzyme, fumarate reductase. Two mutually exclusive hairpin loop structures can occur in frdmRNA just downstream of the start of the frdA cistron. The mRNA sequence involved encodes a stretch of sequence rich in Ala and uses all four of the codons for this amino acid. In vitro expression of the frdABCD operon showed that as the level of plasmid DNA was increased from 150 fmol to 225 fmol, transcription of mRNA was suddenly elevated 6.5-fold, consistent with the concept of titrating out a repressor protein. Further studies showed that the concomitant 10.9-fold increase in translation of protein was heavily biased towards the proximal end of the operon, with little or no expression of FrdC or FrdD and a ratio of FrdA:FrdB of 2.6:1. Addition of Ala to the S-30 extract caused a 6.1-fold amplification of frd messenger transcription, a 17.6-fold increase in Frd protein translation, and a balancing of the subunit ratios to 1:1:1:1. The expression of the bla gene carried on the plasmid was not affected by DNA titration or the addition of Ala. When fnr DNA was added in equimolar ratio to frdDNA the amplification of fumarate reductase expression by Ala was abolished and the ratio of subunits produced showed a high degree of polarity with or without Ala.

“…Cultures were grown anaerobically in flasks filled to the top, using a peptone-glucose-fumarate medium (10 16,000 lb/in2. Fractionation of the lysate into membranes and cytoplasm was done as described previously (25).…”

mentioning

confidence: 99%

Dimethyl sulfoxide reductase of Escherichia coli: an investigation of function and assembly by use of in vivo complementation

Damaraju

1991

J Bacteriol

Dimethyl sulfoxide (DMSO) reductase of Escherichia coli is a membrane-bound, terminal anaerobic electron transfer enzyme composed of three nonidentical subunits. The DmsAB subunits are hydrophilic and are localized on the cytoplasmic side of the plasma membrane. DmsC is the membrane-intrinsic polypeptide, proposed to anchor the extrinsic subunits. We have constructed a number of strains lacking portions of the chromosomal dmsABC operon. These mutant strains failed to grow anaerobically on glycerol minimal medium with DMSO as the sole terminal oxidant but exhibited normal growth with nitrate, fumarate, and trimethylamine N-oxide, indicating that DMSO reductase is solely responsible for growth on DMSO. In vivo complementation of the mutant with plasmids carrying various dms genes, singly or in combination, revealed that the expression of all three subunits is essential to restore anaerobic growth. Expression of the DmsAB subunits without DmsC results in accumulation of the catalytically active dimer in the cytoplasm. The dimer is thermolabile and catalyzes the reduction of various substrates in the presence of artificial electron donors. Dimethylnaphthoquinol (an analog of the physiological electron donor menaquinone) was oxidized only by the holoenzyme. These results suggest that the membrane-intrinsic subunit is necessary for anchoring, stability, and electron transport. The C-terminal region of DmsB appears to interact with the anchor peptide and facilitates the membrane assembly of the catalytic dimer.Dimethyl sulfoxide (DMSO) reductase catalyzes the final step of an energy-transducing anaerobic electron transport chain in Escherichia coli (3, 4). The enzyme catalyzes the reduction of DMSO, trimethylamine N-oxide (TMAO), and a number of S-and N-oxide compounds (33) but is genetically distinct from other TMAO reductases in E. coli (5). The membrane-bound enzyme has been purified to homogeneity (33); the dms operon, coding for DMSO reductase, has been cloned (4), and its DNA sequence has been determined (2). It is an iron-sulfur molybdoenzyme composed of three nonidentical subunits in equimolar ratio. The large 87,350-Da DmsA subunit is homologous to a number of molybdopterincontaining proteins, binds this cofactor, and is involved in the catalytic function. A 23,070-Da DmsB subunit contains 16 cysteine residues arranged in four groups. These groups bear homology to the iron-sulfur center binding sequences of the (4Fe-4S) ferredoxins (2) and serve to coordinate the electron transfer centers (8). Hydrophobicity analysis of the DmsC subunit (30,789 Da), using the Kyte-Doolittle algorithm (14), indicates that it is composed of eight hydrophobic segments of appropriate length to cross the E. coli plasma membrane in an a-helical configuration, and we have proposed that this polypeptide anchors the hydrophilic DmsAB subunits to the cytoplasmic surface of the membrane (2, 25). However, the physiological role of DMSO reductase in anaerobic respiration remains obscure. The occurrence of multiple reductases in E. coli (35...