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.     

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.     

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.     

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.     

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A three- to fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminase or transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two- to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase.     

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.     

Escherichia coli K-12 Mutants Altered in the Transport Systems for Oligo- and Dipeptides

Two mutants of Escherichia coli K-12, defective in the oligopeptide and dipeptide transport system, are described. A mutant defective in the oligopeptide transport system (opp-1) was isolated as resistant to the inhibitory action of triornithine; this mutant is also resistant to glycylglycylvaline and does not concentrate (14)C-glycylglycylglycine, although it is still as sensitive as the parental strain to glycylvaline and valine. Starting from the opp-1 strain, a mutant defective also in the dipeptide transport system (dpp-1) was isolated; this mutant is resistant to the inhibitory action of glycylvaline, valylleucine, and leucylvaline and does not concentrate (14)C-glycylglycine, although it is still as sensitive as the parental strain to valine. The apparent kinetic constants for oligopeptide and dipeptide transport were measured. The opp marker is co-transducible with trp at 27 min on the E. coli genetic map. The dpp locus is separated from opp and is located between proC (10 min) and opp.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.     

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.     

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae

1. The influence of varying amounts of amino acids on the uptake of threonine, isoleucine, valine and leucine and their degradation to higher alcohols was investigated using a mutant strain of Saccharomyces cerevisiae, mating type a, genetic markers ade2, hom2, thr4, ilv2, leu1.
2. The cell mass is increased by increasing concentrations of threonine, isoleucine, valine and leucine, the latter two resulting in a higher dry weight. The amino acids are completely utilised at low concentrations. At higher contents up to 20% of the amino acids remain in the medium. The uptake of threonine, isoleucine, valine and leucine depends on the relative amounts of the concentrations of these amino acids in the medium. A greater amount of an amino acid is taken up if its concentration is comparatively higher than those of the other amino acids. There is a competition between the amino acids for the uptake into the cells.

Higher amounts of intracellular isoleucine and leucine are converted to 2-and 3-methylbutanol when compared with the degradation of valine and threonine to isobutanol and n-propanol-1, isoleucine and leucine up to 90%, valine up to 24% and threonine up to 20%. There is a competition between the four amino acids for their degradation to the corresponding higher alcohols. This behaviour confirms the earlier assumption of a degradation of the four amino acids by unspecific enzymes.     

Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth.     

Isoleucine and valine metabolism in Escherichia coli. XIX. Inhibition of isoleucine biosynthesis by glycyl-leucine

The inhibition of growth of the K-12 strain of Escherichia coli by glycyl-l-leucine observed originally by Simmonds and co-workers was investigated. The inhibition was reversed by isoleucine and those precursors of isoleucine beyond threonine in the biosynthetic pathway. Threonine reversed the inhibition poorly. With heavy cell suspensions, the inhibition was transient: the onset of growth followed the disappearance of the dipeptide from the medium and the appearance of glycine and leucine. Glycyl-leucine was shown to be an inhibitor of threonine deaminase (EC 4.2.1.16 l-threonine hydro-lyase [deaminating]). One kind of glycyl-leucine-resistant mutant had a threonine deaminase that was resistant to isoleucine and glycyl-leucine inhibition. The pattern of glycyl-leucine inhibition is compared with those of inhibition by isoleucine and by the weaker inhibitors leucine and valine.     

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

Homoserine is transported by a single system that it shares with alanine, isoleucine, leucine, phenylalanine, threonine, valine and perhaps cysteine, methionine, serine, and tyrosine. We investigated the regulation of this transport system and found that alanine, isoleucine, leucine, methionine, and valine each repress the homoserine-transporting system. From the concentration resulting in 50% repression of this transport system and the maximal amount of repression, we ranked the amino acids according to their effectiveness in repressing homoserine transport (in decreasing order): leucine>methionine>alanine>valine>isoleucine. The exponential rate of decrease in transport capacity after leucine addition equals the exponential growth rate of the culture, and protein synthesis is necessary for the derepression seen when leucine is removed. Threonine, in addition to using the above system, is transported by a second system shared with serine. We present further evidence for this serine-threonine transport system and show that it is not regulated like the homoserine-transporting system.     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Isoleucine Auxotrophy Due to Feedback Hypersensitivity of Biosynthetic Threonine Deaminase

Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose.

In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport.     

Growth Inhibition in Thiobacillus neapolitanus by Histidine, Methionine, Phenylalanine, and Threonine

Thiobacillus neapolitanus, a strict chemoautotroph, is sensitive to the addition of 10(-4)m methionine, histidine, threonine, or phenylalanine to the thiosulfate medium on which it grows. When histidine, threonine, or phenylalanine are added at the time of inoculation, spontaneous mutants tolerant to the three amino acids are selected. These mutants appear to result from a single genetic change; of 18 independently isolated histidine-tolerant mutants, all are also tolerant to phenylalanine and threonine. The uptake of (14)C-phenylalanine into exponentially growing cells of one such mutant is negligible in contrast with the uptake observed in the phenylalanine-sensitive parent. The addition of methionine to the medium slows growth, but spontaneous mutants are not selected. Inhibition of growth by these amino acids is observed only under conditions of amino acid imbalance; the addition of an equimolar mixture of 16 amino acids, in which each component is present at a concentration of 10(-3)m, causes no inhibition. Histidine and threonine inhibition may be released by equimolar amounts of any one of seven amino acids: serine, alanine, glycine, leucine, valine, tryptophan, or tyrosine; histidine inhibition is also released by isoleucine, and threonine inhibition by methionine. None of the inhibiting amino acids inhibits oxidation of thiosulfate in cell suspensions. A group of hexoses, pentoses, and Krebs cycle intermediates were tested for inhibition of growth or release of inhibition by histidine, phenylalanine, or threonine, but no effects, either inhibition or relief of inhibition, were found.     

Multivalent Repression of Isoleucine-Valine Biosynthesis in Saccharomyces cerevisiae

Regulation of the biosynthesis of four of the five enzymes of the isoleucine-valine pathway was studied in Saccharomyces cerevisiae. A method is described for limiting the growth of a leucine auxotroph by using valine as a competitor for the permease. Limitation for isoleucine and valine was accomplished by the use of peptides containing these amino acids conjugated with glycine as nutritional supplements for auxotrophs. The enzymes were repressed on synthetic medium containing isoleucine, valine, and leucine, as well as on broth supplemented with these amino acids. Limitation for any of the three branched-chain amino acids led to derepression of the isoleucine-valine biosynthetic pathway. Maximal derepression ranged from 3-fold for threonine deaminase to approximately 10-fold for acetohydroxyacid synthase. (Two of the enzymes, acetohydroxyacid synthase and dihydroxyacid dehydrase, may be controlled by a mechanism different from that regulating threonine deaminase.) Possible molecular mechanisms for multivalent repression are discussed.