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.     

Difference in sodium requirement of branched chain amino acid carrier between Pseudomonas aeruginosa PAO and PML strains is due to substitution of an amino acid at position 292

Sodium dependence of leucine transport, a measure of the Na+-coupled leucine-isoleucine-valine II (LIV-II) transport system in Pseudomonas aeruginosa, was compared between two wild-type strains, PAO and PML. The leucine transport activity was saturated at 0.1 mM NaCl for PAO and at 5.0 mM for PML. From kinetics experiments, the apparent Km value for Na+ with respect to leucine transport was estimated to be 3 microM for PAO and 95 microM for PML. The Km value for leucine was 6 microM for PAO and 13 microM for PML. The LIV-II carrier gene (braB) of PML was isolated for comparison of its amino acid sequence with that of the PAO carrier cloned previously. The Km values for Na+ and leucine of the cloned LIV-II carriers of PAO and PML expressed in LIV-II defective mutants were similar to those in wild-type strains. Determination of the nucleotide and deduced amino acid sequences of the LIV-II carrier gene of PML showed an amino acid difference at position 292 between the PAO and PML carriers. The amino acid was threonine for PAO and alanine for PML. These results indicate that the substitution of the amino acid at position 292 of the LIV-II carrier causes a difference in the sodium requirement of the carriers of the PAO and PML strains.     

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.     

Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO.

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.     

Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system.

A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue.     

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.     

Purification and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa.

A binding protein for branched-chain amino acids was purified to a homogeneous state from shock fluid of Pseudomonas aeruginosa PML14. It was a monomeric protein with an apparent molecular weight of 4.3 x 10(4) or 4.0 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration, respectively. The isoelectric point was determined to be pH 4.1 by electrofocusing. Amino acid analysis of the protein showed that aspartic acid, glutamic acid, glycine, and alanine were major components and that the protein contained only one residue each of tryptophan and cysteine per molecule. The binding protein contained no sugar. The binding activity of the protein was specific for the branched-chain amino acids. The protein also bound alanine and threonine with lower affinity. The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 microM, respectively. Mutants defective in the production of the binding protein were identified among the mutants deficient in a transport system for branched-chain amino acids (LIV-I). The revertants from these mutants to LIV-I-positive phenotype simultaneously recovered normal levels of the binding protein. These findings suggest strongly the association of the binding protein with the LIV-I transport 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.     

Multiplicity of leucine transport systems in Escherichia coli K-12

The major component of leucine uptake in Escherichia coli K-12 is a common system for l-leucine, l-isoleucine, and l-valine (LIV-I) with a Michaelis constant (K(m)) value of 0.2 muM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l-alanine, l-threonine, and possibly l-serine. A minor component of l-leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K(m) value of 2 muM and a relatively low V(max) can be observed in E. coli. The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K(m) system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.     

Genetic analysis of the Pseudomonas aeruginosa PAO high-affinity branched-chain amino acid transport system by use of plasmids carrying the bra genes.

About 30 mutants of Pseudomonas aeruginosa PAO defective in the high-affinity branched-chain amino acid transport system (LIV-I) were isolated by the selection for resistance to 4-aza-DL-leucine, a toxic leucine analog for LIV-I. All of the mutants were complemented by plasmid pKTH24, harboring the braC gene, which encodes the branched-chain amino acid-binding protein, and the four open reading frames named braD, braE, braF, and braG (T. Hoshino and K. Kose, J. Bacteriol. 172:5531-5539, 1990). We identified five cistrons corresponding to these bra genes by complementation analysis with various derivatives of pKTH24, confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system. We also found mutations that seem likely to be mutations in a promoter region for the bra genes and those with polarity in the intercistronic region between braC and braD. Analysis with an omega interposon showed that the bra genes are organized as an operon and are cotranscribed in the order braC-braD-braE-braF-braG from a promoter located in the 5'-flanking region of the braC gene.     

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.     

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.     

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.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.     

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.