A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli (original) (raw)

A study on L-threonine and L-serine uptake in Escherichia coli K-12

Frontiers in Microbiology

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the ...

Characterization of a novel L-serine transport system in Escherichia coli

Journal of Bacteriology, 1988

A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture. This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system. Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine. This system was induced by L-leucine, but not by L-serine. The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein. Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor. Uptake of H+ was induced by L-serine influx. These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.

Leucine binding protein and regulation of transport in E. coli

Journal of Supramolecular Structure, 1977

Leucine is transported into E. coli cells by high-affinity transport systems (LIV-I and leucine-specific systems) which are sensitive to osmotic shock and require periplasmic binding proteins. In addition leucine is transported by a low-affinity system (LIV-11) which is membrane bound and retained in membrane vesicle preparations. The LIV-I system serves for threonine and alanine in addition to the 3 branched-chain amino acids. The LIV-I1 system is more specific for leucine, isoleucine, and valine while the high-affinity leucine-specific system has the greatest specificity. A regulatory locus, livR at minute 2 2 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the binding proteins. Valineresistant strains have been selected to screen for transport mutants. High-affinity leucine transport mutants that have been identified include a LIV-binding protein mutant, livJ, a leucine-specific binding protein mutant livK and a nonbinding protein component of the LIV-I system, l i v l f. A fourth mutant, /ivP, appears to be required only for the low-affinity LIV-I1 system. The existence of this latter mutant indicates that LIV-I and LIV-11 are parallel transport systems. The 4 mutations concerned with high-affinity leucine transport form a closely linked cluster of genes on the E. coli chromosome at minute 74. systems suggests that an attenuator site may be operative in its regulation. This complex regulation appears t o require a modified leucyl-tRNA along with the transcription termination factor rho. Regulation of leucine transport is also defective in relaxed strains. in LIV-I activity suggesting a special role of this amino acid in the physiology of E. coli. It was shown that the rapid exchange of external leucine for intracellular isoleucine via the LIV-I system could create an isoleucine pseudoauxotrophy and account for the leucine sensitivity of E. coli. The results of recent studies on the regulation of the high-affinity transport Among the branched-chain amino acids only leucine produces regulatory changes

Regulation of branched-chain amino acid transport in Escherichia coli

Journal of Bacteriology, 1976

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression o...

Influence of threonine exporters on threonine production in Escherichia coli

Applied Microbiology and Biotechnology, 2002

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [ 14 C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Active transport of amino acids in Thiobacillus thioparus is a low-affinity process

Journal of Bacteriology, 1981

A method for the isolation of amino acid auxotrophs of Thiobacillus thioparus is described. Characterization of a leucine auxotroph indicated that leucine biosynthesis in T. thioparus was not different from that of heterotrophic bacteria. T. thioparus cells accumulated amino acids via an active mechanism. Kt values of amino acid transport were between 15 and 330 microM, and Vmax values were 200 to 350 pmol min-1 mg of protein-1. Amino acid transport was carried out by a limited number of systems, each responsible for the uptake of several amino acids. Amino acid auxotrophs of T. thioparus exhibited transport and growth properties similar to those of transport-deficient mutants of heterotrophs which lost the high-affinity, but retained the low-affinity, amino acid transport systems.

Regulation of amino acid transport in Thiobacillus thioparus

Journal of Bacteriology, 1981

Amino acid transport in amino acid auxotrophs of Thiobacillus thioparus was enhanced during growth on rate-limiting amino acid concentration. A pleiotropic mutation enhanced general amino acid transport as manifested by higher values of Vmax of amino acid transport. Affinity constants remained unaltered. Mutants with enhanced transport properties did not show changes in oxidation of thiosulfate, did not oxidize various organic compounds, and did not increase the heterotrophic potential of T. thioparus. The mutations for enhanced transport caused increased synthesis of amino acid transport system components. A method for genetic transformation of T. thioparus is described.

Regulation of high‐affinity leucine transport in escherichia coli

Journal of …, 1980

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, lowaffinity system (LIV-11). Expression of the high-affinity transport systems is altered by mutations in IivR and l s t R , genes for negatively acting regulatory elements, and by mutations in rho, the gene for transcription termination. All four genes for high-affinity leucine transport (livJ, livK, ZivH, and livC) are closely linked and have been cloned on a plasmid vector, pOXl. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that 2ivJ and livK are separated by approximately one kilobase and give a gene order of livJ-livK-livH. livJ and livK appear t o be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is inactivated by mutation or deletion. The results support the existence of separate promoters for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear t o be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucinespecific transport systems by increasing the expression of livJ and livK, genes for the LIV-specific and leucine-specific binding proteins, respectively.

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria

Journal of Bacteriology, 1975

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain E0312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the ilvB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), nor the leuB gene product, 3-isopropylmalate dehydrogenase (2hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxidoreductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the ilvB, ilvADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominant type of mechanism, although both systems may have components in common.