Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Abstract

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.

The succinate dehydrogenase assembly factor, SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria

“…Consistent with published data, WT E. coli could not grow anaerobically with glycerol or fumarate individually, but grew on glycerol and fumarate together (Fig. 1D) [24]. The deletion of sdhA did not impair anaerobic growth on glycerol and fumarate (Fig.…”

“…All aerobic cultures were grown in 10 ml of medium in 100 ml flasks. Anaerobic cultures were grown in 100 ml serum vials containing 80 ml glycerol-fumarate (GF) medium with 80 mM glycerol and 80 mM fumarate as previously described [24] with low shaking at 50 rpm. The medium was flushed for 2 min with Nitrogen gas and sealed with a rubber stopper and metal lid prior to sterilisation.…”

Differential roles of the Escherichia coli fumarases and fnr-dependent expression of fumarase B and aspartase

“…The rich medium was L broth [14] supplemented with glucose (1%), BCIG (20 /~g/ml), ampicillin (50-100 /Lg/ml) and chloramphenicol (20 btg/ml) as required. The minimal medium of Spencer and Guest [15] contained glucose (0.4%), glycerol (40 raM), fumarate (40 mM) or L-malate (40 mM) supplemented with methionine (40 /~g/ml) and ampicillin (20-100/~g/ml) where appropriate. Vitamin-free Casamino acids (0.5 g/l) was added to the glycerol plus fumarate or malate media (GF or GM) used for anaerobic growth.…”

Amplification and aerobic synthesis of fumarate reductase in ampicillin-resistant mutants of escherichia coli K-12

“…Escherichia coti contains two membrane-bound flavoproteins which catalyse the interconversion of succinate and fumarate, succinate dehydrogenase and fumarate reductase [1,2]. Fumarate reductase synthesis is repressed aerobically but derepressed during anaerobic growth, whereas succinate dehydrogenase is induced aerobically and repressed anaerobically [1].…”