In Escherichia coli, the let locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the kct locus contained three overlapping genes in the clockwise order of lktD (encoding a flavin mononucleotide-dependent dehydrogenase), IctR (encoding a putative regulator), and kctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A F(kctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal.Growth of Escherichia coli on L-lactate results in the induction of a flavin-linked L-lactate dehydrogenase (9,13,29), and this trait is associated with the Ict locus at min 80.8 on the chromosome map (3,19,31 Bacterial strains, phages, and plasmids. All strains used were E. coli K-12 derivatives. The genotypes and sources of the bacterial strains, phages, and plasmids are given in Table 1.Growth conditions. Overnight cultures were grown in LB medium (28). Minimal agar media (38) were supplemented with thiamine. When used, the following compounds were added to the media at the following concentrations unless otherwise specified: D-or L-lactate, 20 mM; D-xylose, 10 mM; CAA, 1%; thiamine, 2 ug/ml; kanamycin (kan), 40 ,ug/ml; and ampicillin, 100 ,ug/ml. Mannitol (1%)-MacConkey agar plates were used for screening the inheritance of the mtl allele.Cultures for enzyme and permease assays were grown in 300-ml flasks containing 10 ml of MOPS (0.1 M) mineral medium at pH 7.6 with appropriate supplements (15). Ample aeration was ensured by vigorous agitation, and cells were harvested when growth reached the mid-exponential phase.Enzyme and permease assays. For L-and D-lactate dehydrogenase assays, cells were harvested by centrifugation at 5,000 x g for 15 min and washed once in cold 10 mM potassium phosphate buffer (pH 7.0). The pellet was weighed and suspended in 4 volumes of the same buffer. The suspended cells were lysed for 1 min/ml in a model 60 W ultrasonic disintegrator (MSE) at 1.5 A while being chilled in a dry ice-ethanol bath. Lysates were cleared by centrifugation for 30 min at 10,000 x g. Enzyme assays were performed in a manner similar to that for glycerol-3-phosphate dehydrogenase by measuring the reduction of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide mediated by phenazine methosulfate (20)

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.

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.

Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.

No abstract