Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

Abstract

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)

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

“…The flavin-binding domain of flavocytochrome b2 is closely related to several other 2-hydroxy acid dehydrogenases with different substrate specificities. These include the glycollate oxidase from spinach [18], lactate oxidase from Mycobacterium smegmatis [19], lactate dehydrogenase from E. coli [20], mandelate dehydrogenase from Pseudomonas putida [21] and long-chain hydroxy acid dehydrogenase from rat kidney [22]. The catalytically important residues are well conserved throughout this family but we have sought to determine the structural basis for substrate selectivity.…”

Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past

“…The sequence of a putative MCT from the archaebacterium Sulfolobus solfataricus (see Table 1) also shows similarity to the eukaryotic MCT family with 36 % similarity and 25 % identity with human MCT1 over the first 200 residues. Interestingly, this sequence is directly adjacent in the genome to a sequence encoding another putative membrane protein with homology (32 % identity, 58 % similarity) to the Escherichia coli lactate permease (lctP) [35]. However, lctP shows only 12 % similarity to the eukaryotic MCT family member sequences and thus it is unlikely that the mammalian plasma membrane MCTs evolved from the prokaryotic lactate permease.…”

A developmentally regulated gene encoding a repressor‐like protein is essential for sporulation in Streptomyces coelicolor A3(2)

“…The N-terminal region also showed some evidence of relatedness to the equivalent region of members of the DeoR family of transcriptional regulators. It has been inferred in several cases that the N-terminal domain conserved between members of the GntR family contains a helix-turn-helix DNA-binding motif (Yoshida et al, 1993;Schneider et al, 1993;Dong et al, 1993), and analysis for this motif using the Dodd and Egan (1990) weighted matrix method gave a marginally significant score for this region of WhiH (2.09 standard deviation units, on a scale on which a score of 2.5 units is said to imply near certainty of a helix-turn-helix if the protein is known to bind DNA; Dodd and Egan, 1990). Most of the GntR-like proteins, including those most similar to whiH, contained about 250 aa residues, although several, all falling into another subgroup, contained about 450 aa residues.…”