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.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

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.     

Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli.

A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.     

Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.     

Mutants of Salmonella typhimurium defective in transport of branched-chain amino acids

Five mutants, CD15, CD31, CE4, CE5, and CE14, defective in the transport of branched-chain amino acids were isolated as glycyl-l-isoleucine and glycyl-l-valine requirers from an isoleucine-valine-requiring mutant, KA931, of Salmonella typhimurium LT2. Although these transport mutants do not grow in minimal medium supplemented with isoleucine (10 mug/ml) and valine (20 mug/ml), where the parent strain, KA931, grows normally, they do when the supplements are increased 10-fold in concentration, and the growth rate becomes comparable to that of the parent strain. When the apparent K(m) and V(max) for uptake of isoleucine, valine, and leucine in the transport mutants were compared to those of KA931, the K(m) was generally lower in the mutants, and the V(max) for isoleucine and leucine decreased to one-fourth to one-seventh and for valine one-eighth to one-fifteenth of that in the parent. In all mutants except CE5, the transport of methionine, arginine, threonine, and glycine was normal. The transport of threonine in CE5 appeared to be slightly impaired. In addition to the original mutation in the ilvC locus, the transport mutant has a mutation at the ilvT locus, which is closely linked to proC and may be located on the side of proC proximal to purE. About 26-fold difference in values of the co-transduction is noted in reciprocal transductions between KA502 and CD15 strains.     

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

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 EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, 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 iluB, iluADE, 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-dominate type of mechanism, although both systems may have components in common.     

Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system.

The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a Km of 0.3 microM for L-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0.3 to 1.1%) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent Km values for methionine of 24 microM and approximately 1.8 mM. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.     

Auxotrophy accounts for nodulation defect of most Sinorhizobium meliloti mutants in the branched-chain amino acid biosynthesis pathway

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.     

Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

The regulation of synthesis of the valine-alanine-alpha-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of alpha-acetohydroxy acid synthase activity, causing alpha-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Proton-symport of L-valine in plasma membrane vesicles isolated from leaves of the wild-type and the Val(r)-2 mutant of Nicotiana tabacum L

Transport of amino acids across the plasma membranes of various cell types is a key process in controlling the nitrogen balance of leaves. We studied the transport of the neutral amino acid L-valine into plasma membrane vesicles obtained by aqueous polymer two-phase partitioning of a microsomal fraction isolated from leaves of the wild-type and the Val(r)-2 mutant of tobacco (Nicotiana tabacum L.). Initial influxes were determined after the imposition of a pH-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. The initial magnitudes of the imposed gradients were DeltapH=2 and Deltapsi=-68 mV. In vesicles from the wild-type, the DeltapH-dependent valine influx could be analysed into a high-affinity (Km approximately 20 microM) and a low-affinity (Km approximately 3 mM) component. The influx of valine by the low-affinity system was stimulated about twofold, and that by the high-affinity system more than sixfold by the imposition of Deltapsi. This strong stimulation of the high-affinity system may indicate that it transports 2H+/amino acid. In the Val(r)-2 mutant the high-affinity component appeared to be completely absent.     

Isolation of mutants lacking branched-chain amino acid transaminase.

Variants of the Chinese hamster ovary cell have been isolated which can no longer grow when valine, leucine, or isoleucine is replaced in the culture medium by its respective alpha-keto acid: alpha-ketoisovaleric acid, alpha-ketoisocaproic acid, or alpha-keto-beta-methylvaleric acid. These variants lack branched-chain amino acid transaminase activity. Evidence is presented indicating these variants to be single gene mutants. Genetic evidence is also presented confirming previous biochemical evidence that a single enzyme carries out transaminase functions on valine, leucine, and isoleucine. The branched-chain transaminase-deficient (trans-) mutants can be reverted to wild-type behavior by treatment with mutagenic agents. These mutants promise to be useful in exploring regulatory mechanisms in biochemical, genetic, and cancer research.     

Derepressed leucine transport activity in Escherichia coli

Transport activity and synthesis of binding protein for the amino acids leucine, isoleucine and valine in E. coli are coordinately controlled by the level of leucine in the growth medium. Spontaneous mutants (dlu) which can utilize D-leucine as a source of L-leucine show derepressed transport activity for the three-branched chain amino acids. The increased transport activity is a result of an increase in the binding protein for these amino acids. Azaleucine-resistant mutants have been isolated which have a defect in leucine transport but normal levels of the binding protein for leucine.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

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.     

Genetic mapping of bra genes affecting branched-chain amino acid transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO mutants defective in the transport systems for branched-chain amino acids were isolated and characterized. Two mutations in strains selected for trifluoroleucine resistance, braA300 and braB307, were mapped in the met-9020-dcu-9108 and the nar-9011-puuC10 region, respectively. The mutation loci in strains selected for azaleucine resistance, braC310 and bra-311 through bra-314, were all located near the fla genes, with an order of region I fla-bra-region II fla. Strains with braA300 showed a marked reduction in the high-affinity branched-chain amino acid transport system (LIV-I) and a considerable decrease in the lower-affinity system (LIV-II). Strains with braB307 were found to be defective in the LIV-II system. Strains selected for azaleucine resistance were all defective only in the LIV-I system and fell into three phenotypically distinct classes. Strains with braC310 produced a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-BP) altered in binding ability, indicating that the braC gene is the structural one for the LIVAT-BP. Strains with bra-311 or bra-312 showed a complete loss of production of the LIVAT-BP. Strains with bra-313 or bra-314 produced normal levels of functional LIVAT-BP, suggesting that these mutations are located in a gene(s) other than braC.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.