Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine-repressible transport activities.

The characteristics of a mutant (hrbA) of Escherichia coli K-12 that is defective in a leucine-nonrepressible transport system, the LIV-3 system, for branched-chain amino acids were described previously (I. Yamato et al., J. Bacteriol 138:24-32, 1979). New mutants requiring a high concentration of isoleucine for growth were isolated from strain B763 (hrbA ileA) after mutagenesis with ethyl methane sulfonate. These mutants had a defect of the leucine-repressible transport activities for branched-chain amino acids of the parental strain. One of these mutants, strain B7634, had defects of two independent genetic loci (hrbBC and hrbD). The genes hrbBC were mapped at min 76 near malT, and the gene hrbD mapped at min 77 near xyl on the E. coli genetic map. The substrate specificity, kinetic properties, and source of coupling energy of the transport system coded for by each of these genes were studied using cytoplasmic membrane vesicles and intact cells. The results identified three transport systems with characteristic features other than the LIV-3 system. The hrbB and hrbC systems are responsible for the uptake activites of the LIV-2 system, with a high Km value, and the LIV-1 system, with a low Km value, respectively. Both activities are repressed by leucine and inhibited by threonine and the b(--) isomer of 2-aminobicycloheptyl-2-carboxylic acid. They both utilize adenosine 5'-triphosphate as coupling energy and are not detected in cytoplasmic membrane vesicles. The hrbD system is responsible for the LIV-4 system, with a high Km value. Its activity is repressed by leucine and partially inhibited by threonine. It is detected in cytoplasmic membrane vesicles with a proton motive force as the driving energy.     

Branched-Chain Amino Acid Transport in Cytoplasmic Membranes of Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273

Membrane vesicles of Leuconostoc mesenteroides subsp. dextranicum fused with proteoliposomes prepared from Escherichia coli phospholipids containing beef heart cytochrome c oxidase were used to study the transport of branched-chain amino acids in a strain isolated from a raw milk cheese. At a medium pH of 6.0, oxidation of an electron donor system comprising ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and horse heart cytochrome c resulted in a membrane potential (Deltapsi) of -60 mV, a pH gradient of -36 mV, and an l-leucine accumulation of 76-fold (Deltamu(Leu)/F = 108 mV). Leucine uptake in hybrid membranes in which a Deltapsi, DeltapH, sodium ion gradient, or a combination of these was imposed artificially revealed that both components of the proton motive force (Deltap) could drive leucine uptake but that a chemical sodium gradient could not. Kinetic analysis of leucine (valine) transport indicated three secondary transport systems with K(t) values of 1.7 (0.8) mM, 4.3 (5.9) muM, and 65 (29) nM, respectively. l-Leucine transport via the high-affinity leucine transport system (K(t) = 4.3 muM) was competitively inhibited by l-valine and l-isoleucine (K(i) and K(t) values were similar), demonstrating that the transport system translocates branched-chain amino acids. Similar studies with these hybrid membranes indicated the presence of high-affinity secondary transport systems for 10 other amino acids.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.     

Mutations Affecting the Different Transport Systems for Isoleucine, Leucine, and Valine in Escherichia coli K-12

Uptake of isoleucine, leucine, and valine in Escherichia coli K-12 is due to several transport processes for which kinetic evidence has been reported elsewhere. A very-high-affinity transport process, a high-affinity transport process, and three different low-affinity transport processes were described. In this paper the existence of these transport processes is confirmed by the isolation and preliminary characterization of mutants altered in one or more of them. The very-high-affinity transport process is missing either in strains carrying the brnR6(am) mutation or in strains carrying the brn-8 mutation. This appears to be a pleiotropic effect since other transport systems are also missing. Mutant analysis shows that more than one transport system with high affinity is present. One of them, high-affinity 1, which needs the activity of a protein produced by the brnQ gene, transports isoleucine, leucine, and valine and is unaffected by threonine. The other, high-affinity 2, which needs the activity of a protein produced by the brnS gene, transports isoleucine, leucine, and valine; this uptake is inhibited by threonine which probably is a substrate. Another protein, produced by the brnR gene, is required for uptake through both high-affinity 1 and high-affinity 2 transport systems. The two systems therefore appear to work in parallel, brnR being a branching point. The brnQ gene is located close to phoA at 9.5 min on the chromosome of E. coli, the brnR gene is located close to lac at 9.0 min, and the brnS gene is close to pdxA at 1 min. A mutant lacking the low-affinity transport system for isoleucine was isolated from a strain in which the high-affinity system was missing because of a brnR mutation. This strain also required isoleucine for growth because of an ilvA mutation. The mutant lacking the low-affinity transport system was unable to grow on isoleucine but could grow on glycylisoleucine. This mutant had lost the low-affinity transport for isoleucine, whereas those for leucine and valine were unaffected. A pleiotropic consequence of this mutation (brn-8) was a complete absence of the very-high-affinity transport system due either to the alteration of a common gene product or to any kind of secondary interference which inhibits it. Mutants altered in isoleucine-leucine-valine transport were isolated by taking advantage of the inhibition that valine exerts on the K-12 strain of E. coli. Mutants resistant both to valine inhibition (Val(r)) and to glycylvaline inhibition are regulatory mutants. Val(r) mutants that are sensitive to glycylvaline inhibition are transport mutants. When the very-high-affinity transport process is repressed (for example by methionine) the frequency of transport mutants among Val(r) mutants is higher, and it is even higher if the high-affinity transport process is partially inhibited by leucine.     

Transport of Biosynthetic Intermediates: Homoserine and Threonine Uptake in Escherichia coli

Although amino acid transport has been extensively studied in bacteria during the past decade, little is known concerning the transport of those amino acids that are biosynthetic intermediates or have multiple fates within the cell. We have studied homoserine and threonine as examples of this phenomenon. Homoserine is transported by a single system which it shares with alanine, cysteine, isoleucine, leucine, phenylalanine, threonine, tyrosine, and valine. The evidence for this being the sole system for homoserine transport is (i) a linear double-reciprocal plot showing a homoserine K(m) of 9.6 x 10(-6) M, (ii) simultaneous reduction by 85% of homoserine and branched-chain amino acid uptake in a mutant selected for its inability to transport homoserine, and (iii) simultaneous reduction by 94% of the uptake of homoserine and the branched-chain amino acids by cells grown in millimolar leucine. Threonine, in addition to sharing the above system with homoserine, is transported by a second system shared with serine. The evidence for this second system consists of (i) incomplete inhibition of threonine uptake by any single amino acid, (ii) only 70% loss of threonine uptake in the mutant unable to transport homoserine, and (iii) only 40% reduction of threonine uptake when cells are grown in millimolar leucine. In this last case, the remaining threonine uptake can only be inhibited by serine and the inhibition is complete.     

Multiplicity of Isoleucine, Leucine, and Valine Transport Systems in Escherichia coli K-12

The kinetics of isoleucine, leucine, and valine transport in Escherichia coli K-12 has been analyzed as a function of substrate concentration. Such analysis permits an operational definition of several transport systems having different affinities for their substrates. The identification of these transport systems was made possible by experiments on specific mutants whose isolation and characterization is described elsewhere. The transport process with highest affinity was called the "very-high-affinity"process. Isoleucine, leucine, and valine are substrates of this transport process and their apparent K(m) values are either 10(-8), 2 x 10(-8), or 10(-7) M, respectively. Methionine, threonine, and alanine inhibit this transport process, probably because they are also substrates. The very-high-affinity transport process is absent when bacteria are grown in the presence of methionine, and this is due to a specific repression. Methionine and alanine were also found to affect the pool size of isoleucine and valine. Another transport process is the "high-affinity" process. Isoleucine, leucine, and valine are substrates of this transport process, and their apparent K(m) value is 2 x 10(-6) M for all three. Methionine and alanine cause very little or no inhibition, whereas threonine appears to be a weak inhibitor. Several structural analogues of the branched-chain amino acids inhibit the very-high-affinity or the high-affinity transport process in a specific way, and this confirms their existence as two separate entities. Three different "low-affinity" transport processes, each specific for either isoleucine or leucine or valine, show apparent K(m) values of 0.5 x 10(-4) M. These transport processes show a very high substrate specificity since no inhibitor was found among other amino acids or among many branched-chain amino acid precursors or analogues tried. The evolutionary significance of the observed redundancy of transport systems is discussed.     

Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.     

Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.     

Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system.

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.     

Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.

The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.     

The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner.

Lactococcin A is a bacteriocin produced by Lactococcus lactis. Its structural gene has recently been cloned and sequenced (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). Purified lactococcin A increased the permeability of the cytoplasmic membrane of L. lactis and dissipated the membrane potential. A significantly higher concentration of lactococcin A was needed to dissipate the membrane potential in an immune strain of L. lactis. Lactococcin A at low concentrations (0.029 microgram/mg of protein) inhibited secondary and phosphate-bond driven transport of amino acids in sensitive cells and caused efflux of preaccumulated amino acids. Accumulation of amino acids by immune cells was not affected by this concentration of lactococcin A. Lactococcin A also inhibited proton motive force-driven leucine uptake and leucine counterflow in membrane vesicles of the sensitive strain but not in membrane vesicles of the immune strain. These observations indicate that lactococcin A makes the membrane permeable for leucine in the presence or absence of a proton motive force and that the immunity factor(s) is membrane linked. Membrane vesicles of Clostridium acetobutylicum, Bacillus subtilis, and Escherichia coli were not affected by lactococcin A, nor were liposomes derived from phospholipids of L. lactis. These results indicate that lactococcin A acts on the cytoplasmic membrane and is very specific towards lactococci. The combined results obtained with cells, vesicles, and liposomes suggest that the specificity of lactococcin A may be mediated by a receptor protein associated with the cytoplasmic membrane.     

Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12.

Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction. Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map. The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems. Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels. The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.     

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.     

Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein.

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.     

Transport of sugars and amino acids in bacteria. XVIII. Properties of an isoleucine carrier in the cytoplasmic membrane vesicles of Escherichia coli.

The properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of "Kaback vesicles" prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force. Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.     

Regulation of branched-chain amino acid transport in Escherichia coli.

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 of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.     

Repression of Escherichia coli pyridine nucleotide transhydrogenase by leucine.

Addition of 0.1% casein hydrolysate to a minimal growth medium decreased membrane-bound transhydrogenase activity in Escherichia coli by about 80%. Of the amino acids added individually to the growth medium, only leucine and, to a lesser extent, methionine and alanine were effective, alpha-Ketoisocaproate- and leucine-containing peptides repressed the activity, and leucine also repressed activity in adenyl cyclase-deficient and relaxed strains. Derepression of transhydrogenase followed the removal of leucine from the growth medium and was sensitive to rifampin and chloramphenicol. A phosphoglucoisomerase-deficient strain that was forced to use the hexose monophosphate shunt exclusively had normal levels of transhydrogenase, which was repressed by leucine. Transhydrogenase activity doubled in mutants lacking either of the shunt dehydrogenases but was still repressed by leucine. In strains constitutive for the leucine biosynthetic operon, transhydrogenase was repressed by leucine but in strains livR and lst R, with leucine transport resistant to leucine repression, transhydrogenase was not repressed by leucine. These data suggest that transhydrogenase may have a function in the transport of branched-chain amino acids. In a hisT strain (which has altered leucyl-tRNA), transhydrogeanse was at a repressed level without the addition of leucine, suggesting that leucyl-tRNA may be involved in the regulation.