An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. II. Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease

The double mutant of the lactose permease containing Val177/Asn319 exhibits proton leakiness by two pathways (see Brooker, R. J. (1991) J. Biol Chem. 266, 4131-4138). One type of H+ leakiness involves the uncoupled influx of H+ (leak A pathway) while a second type involves the coupled influx of H+ and galactosides in conjunction with uncoupled galactoside efflux (leak B pathway). In the current study, 14 independent lactose permease mutants were isolated from the Val177/Asn319 parent which were resistant to thiodigalactoside growth inhibition but retained the ability to transport maltose. All of these mutants contained a third mutation (besides Val177/Asn319) at one of two sites. Eight of the mutants had Ile303 changed to Phe, while six of the mutants had Tyr236 changed to Asn or His. Each type of triple mutant was characterized with regard to sugar transport, H+ leakiness, and sugar specificity. Like the parental strain, all three types of triple mutant showed moderate rates of downhill lactose transport and were defective in the uphill accumulation of sugars. However, with regard to proton leakiness, the triple mutants fell into two distinct categories. The mutant containing Phe303 was generally less H+ leaky than the parent either via the leak A or leak B pathway. In contrast, the triple mutants containing position 236 substitutions (Asn or His) were actually more H+ leaky via the leak A pathway and exhibited similar H+ leakiness via the leak B pathway at high thiodigalactoside concentrations. The ability of the position 236 mutants to grow better than the parent in the presence of low concentrations of thiodigalactoside appears to be due to a decrease in affinity for this particular sugar rather than a generalized defect in H+ leakiness. Finally, the triple mutants showed a sugar specificity profile which was different from either the Val177/Asn319 parent, the single Val177 mutant, or the wild-type strain. These results are discussed with regard to the effects of mutations on both the sugar and H+ transport pathways.     

Characterization of lactose carrier mutants which transport maltose

Brooker and Wilson (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963) previously isolated lactose carrier mutants which were able to transport maltose. All of the mutants were found to be single amino acid substitutions for alanine 177 or for tyrosine 236. In the present study, we have examined the ability of these mutants to transport maltose, lactose, o-nitrophenyl-beta-D-galactopyranoside, methyl-beta-D-thiogalactopyranoside, and H+. Both the position 177 and 236 mutants have enhanced rates of maltose transport and exhibit apparent Km values for maltose which are substantially less than that of the wild-type strain. The position 177 mutants transport lactose and other galactosides at a normal rate and with normal affinity during downhill transport and show counterflow transport rates which are faster than the wild-type strain. Interestingly, these mutants are markedly defective in accumulating substrates against a concentration gradient, yet retain a normal H+:galactoside stoichiometry. The position 236 mutants appear to be defective in the downhill, uphill, and counterflow transport of galactosides but exhibit a normal H+:galactoside stoichiometry.     

Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose

In the current study, lactose permease mutants were isolated which exhibited an enhanced recognition for maltose (an alpha-glucoside) but a diminished recognition for thiodigalactoside, TDG (a beta-galactoside). Maltose/TDGR mutants were obtained from four different parental strains encoding either a wild-type permease (pTE18), a mutant lactose permease which recognizes maltose (pB15) or mutant lactose permeases which recognize maltose but are resistant to inhibition by cellobiose (pTG and pBA). A total of 27 independent mutants were isolated: 12 from pTE18, 10 from pB15, 3 from pTG, and 2 from pBA. DNA sequencing of the 27 mutants revealed that the mutants contain single base pair substitutions within the lac Y gene which result in single amino acid substitutions within the lactose permease. All of the mutants obtained from pTE18, pTG, and pBA involved a change of Tyr-236 to histidine, phenylalanine, or asparagine. From pB15, three different types of mutants were obtained: Tyr-236 to histidine, Ile-303 to phenylalanine, or His-322 to asparagine. When assayed for [14C]maltose transport, the maltose/TDGR mutants were seen to transport maltose significantly faster than the wild type. Furthermore, although TDG was shown to inhibit the uptake of maltose in the four parental strains, all of the mutant strains exhibited a dramatic resistance to TDG inhibition. Most of the maltose/TDGR mutants were also shown to be very defective in the transport of lactose. However, certain mutants (i.e., Asn-322) exhibited moderate lactose transport activity. Finally, it was observed that all of the mutant strains were unable to facilitate the uphill accumulation of beta-methylthiogalactopyranoside. The locations of the amino acid substitutions are discussed with regard to their possible role in sugar recognition.     

Functional role of arginine 302 within the lactose permease of Escherichia coli.

Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302. Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition. To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis. All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose. Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport. The wild-type strain exhibited a Km for lactose transport of 0.30 mM and a Vmax of 267 nmol of lactose/min.mg of protein. The Ser-302, His-302, and Leu-302 were observed to have Km values of 0.18, 2.3, and 2.8 mM, and Vmax values of 11.6, 56.4, and 22.0 nmol of lactose/min.mg of protein, respectively. In uphill transport assays, all three mutants were unable to accumulate beta-methyl-D-thiogalactoside. However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient. During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside. In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose. However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport. Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak. In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses. The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak.     

Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient.

The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.     

An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport

The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.     

Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose

In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.     

Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate. Probes for the energy barrier to uncoupled transport

The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.     

Antibacterial activity of phosphono dipeptides based on 1-amino-1-methylethanephosphonic acid

Phosphono dipeptides containing 1-amino-1-methylethanephosphonic acid (phosphonic acid analogue of alpha-methylalanine, MeAlaP) and glycine, alanine, valine, leucine phenylalanine, proline, methionine or lysine as N- terminal component were synthesized in order to determine their antibacterial properties. Peptides containing alanine, leucine, valine phenylalanine and methionine showed marked in vitro activity, especially against Escherichia coli and Serratia marcescens strains. There were, however, generally less potent than the respective phosphono dipeptides based on 1-aminoethanephosphonic acid (phosphonic acid analogue of alanine, AlaP). The possible mechanism of action of the peptides of MeAlaP involves their active transport into the bacterial cell, followed by intracellular release of MeAlaP, which most likely inhibits alanine racemase, a key enzyme in peptidoglycan biosynthesis. Studies on the uptake of AlaMeAlaP and LeuMeAlaP by Escherichia coli mutants defective in the oligopeptide permease suggest that these peptides are not transported by the oligopeptide transport system.     

Amino acid substitution in the lactose carrier protein with the use of amber suppressors.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.     

Role of conserved residues in hydrophilic loop 8-9 of the lactose permease.

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.     

Characterization of AAT1: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae

A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified. These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine). This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested. Leucine uptake through the leucine-specific permease is inhibited to less than 35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions. aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement.     

Effect of cypermethrin on the free amino acids pool in an organophosphorus-insecticide-resistant and a susceptible strain of Tribolium castaneum

1. Proline was found to be the major component of CTC-12 (44%) and FSS II (45%) strain.2. The cypermethrin treatment resulted in an increase in most of the amino acids of sixth instar larvae and all amino acids of adult beetles of CTC 12 strain.3. In the susceptible strain (FSS II), however, the tyrosine, phenylalanine and arginine increased, whereas serine, proline, glycine, alanine, valine, isoleucine, leucine and lysine were decreased significantly in the sixth instar larvae.4. In the FSS II adult beetles, only aspartic acid increased, while other amino acids either decreased (threonine, proline, glycine, alanine, valine, methionine, isoleucine, tyrososine, lysine, arginine) or remained unaffected (serine, glutamic acid, leucine, phenylalanine, histidine).     

Qualitative Characterization of Some Peptides Inducing Morphogenesis in the Nematode- Trapping Fungus Arthrobotrys oligospora

Some peptides inducing capture organ formation in Arthrobotrys oligospora were characterized with regard to their amino acid contents. The N-terminal, C-terminal and total amino acids were determined as their dinitrophenyl-derivatives by thin layer chromatography on silica gel in two different solvents. The amino acid composition was further confirmed by gas-liquid chromatography. The peptides investigated had a high proportion of non-polar and aromatic residues. Thus, leucine, isoleucine, valine, proline, and tyrosine were present in all the peptides. In addition, phenyl-alanine, glycine, and alanine occurred in some preparations. Tyrosine, valine, and phenylalanine were found in N-terminal position, and leucine or isoleucine were C-terminals.     

Receptors for chemotaxis in Bacillus subtilis.

At least three receptors for chemotaxis toward L-amino acids in Bacillus subtilis could be found with the aid of taxis competition experiments. They are called the asparagine receptor, which detects asparagine and glutamine, the isoleucine receptor, which detects isoleucine, leucine, valine, phenylalanine, serine, threonine, cysteine, and methionine, and the alanine receptor, which detects alanine and proline. Histidine and glycine could not be assigned to one of these receptors. Cysteine and methionine were found to be general inhibitors of chemotaxis and serine was found to be a general stimulator of chemotaxis. Some structural analogues of amino acids were tested for chemotactic activity. The chemotactic activity of B. subtilis is compared with that of Escherichia coli.     

Antibacterial activity of phosphono peptides based on 4-amino-4-phosphonobutyric acid

Abstract Phosphono dipeptides based on 4-amino-4-phosphonobutyric acid (phosphonic acid analogue of glutamic acid, GluP) were synthesized and evaluated for their antibacterial activity. Dipeptides containing N-terminal alanine, leucine, isoleucine, phenylalanine or lysine showed marked antibacterial activity against Escherichia coli , whilst those containing alanine, leucine, valine or proline were active against Serratia marcescens . AlaGluP and LeuGluP were nearly equipotent with the respective dipeptides based on 1-aminoethylphosphonic acid (phosphonic acid analogue of alanine). The structure-activity relationship, i.e. dependence of the activity of phosphono dipeptides on the nature of their N-terminal component, indicated that transport of the peptide through the bacterial cytoplasmic membrane constitutes a crucial step in its antibacterial activity.     

Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier. Enhanced specificity for sucrose and maltose

A mutant of the Escherichia coli lactose carrier has been selected (in an invertase-positive strain) based on its ability to grow on 6 mM sucrose in a manner dependent upon lactose carrier induction by isopropyl-1-thio-beta-D-galactopyranoside. The mutant was cloned, and DNA sequencing revealed a point mutation in lacY which changed alanine 177 to valine. The valine 177 mutation increased the transport rate for both [14C]sucrose and the maltose analog 4-nitrophenyl-alpha-maltoside. The potency for inhibition of beta-ONPG transport by several sugars containing the glucopyranosyl moiety (maltose, cellobiose, or palatinose) was increased significantly relative to the parental carrier. Similar experiments showed that the mutation did not affect the affinity for such commonly studied substrates as 4-nitrophenyl-alpha-D-galactopyranoside and beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside. These data indicate that gross structural alteration of the galactoside binding site cannot account for increased transport of sucrose and maltose by the valine 177 mutant. We conclude that effects of the valine 177 mutation are not limited strictly to changes in observed sugar affinity and that sugar-specific changes in turnover number may be an important determinant of the altered spectrum of sugar specificities exhibited by the Val-177 carrier. These phenomena may be related to the effect of this mutation on proton recognition (described in King, S.C., and Wilson, T.H. (1990) J. Biol. Chem. 265, 9645-9651).     

Characterization of the double mutant, Val-177/Asn-322, of the lactose permease

The double mutant, Val-177/Asn-322, was investigated with regard to its ability to transport H+ and galactosides. In downhill lactose transport assays, the wild-type strain had a Km value for lactose uptake of 0.9 mM and a Vmax of 0.65 mumol lactose/min.mg protein while the mutant had a significantly higher Km value of 1.9 mM but a similar Vmax of 0.49 mumol/min.mg protein. In spite of its moderate ability to transport lactose downhill, the Val-177/Asn-322 mutant exhibited the striking property of being completely defective in the uphill accumulation of lactose or methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport, however, showed that the mutant's defect in active accumulation is not due to a defect in the ability to transport H+ with lactose or methyl-beta-D-thiogalactopyranoside. The Val-177/Asn-322 mutant strain had a H+:lactose stoichiometry of 0.84 which was similar to that measured in the wild-type strain (0.68). These results are discussed with regard to the role His-322 plays in H+ transport, active accumulation of sugars, and sugar recognition.     

Studies on Amino Acid Sequence Determination from N-Terminal of Peptide by Methylthiohydantoin

Methylisothiocyanate reacts with the amino group of amino acid in alkaline solution to give methylthiocarbamyl amino acid. This is converted to the methylthiohydantoin derivative (MTH-amino acid) by subsequent acidification.

Gas chromatographical identification of 20 amino acids were examined by making MTH-amino acids. Among them, ten amino acids (glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, serine and threonine) were successfully identified. Aspartic acid and glutamic acid could be identified as the methyl esters of the MTH-amino acids.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.