Regulation of Peptide Transport in Escherichia coli: Induction of the trp-linked Operon Encoding the Oligopeptide Permease

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1 JOURNAL OF BACTERIOLOGY, Feb. 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 165, No. 2 Regulation of Peptide Transport in Escherichia coli: Induction of the trp-linked Operon Encoding the Oligopeptide Permease JOHN C. ANDREWS, TIMOTHY C. BLEVINS, AND STEVEN A. SHORT* Department of Microbiology, Wellcome Research Laboratories, Research Triangle Park, North Carolina Received 6 August 1985/Accepted 5 November 1985 Growth of Escherichia coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-[U-'4Cjalanyl-L-alanyl-L- alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppa-encoded periplasmic binding protein. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium utilize small oligopeptides as both carbon and nitrogen sources for growth. For these bacteria to grow on oligopeptides in the absence of periplasmic or extracellular peptidases (5, 18), the peptides must penetrate the permeability barriers posed by both the outer and cytoplasmic membranes. Data obtained with E. coli have shown that the composition of the outer membrane channels formed by the OmpF and OmpC porins imparts selectivity to the permeation of peptides through the outer membrane and that an E. coli outer membrane lacking both the ompf and the ompc gene products is peptide impermeable (1). Tripeptide passage through the second permeability barrier, the cytoplasmic membrane, requires the presence of specific, genetically defined transport systems. In E. coli tripeptide transport requires the protein products encoded by the genes of the trp-linked opp operon and the oppe locus (1, 12), whereas in S. typhimurium, tripeptide accumulation is mediated by uptake systems encoded by the opp operon and the tppb locus (6, 9). In both E. coli and S. typhimurium, the trp-linked opp operon is polycistronic with the first operon gene, oppa, encoding a periplasmic binding protein (1, 7, 8). The identity and function of the protein products of the other opp operon genes remain to be clearly defined. In contrast to the similarity found for the oligopeptide permease specified by the trp-linked opp operon of both E. coli and S. typhimurium, not only do the oppe locus of E. coli and the tppb locus of S. typhimurium appear, at present, to be genus specific, but also E. coli OppE- mutants and S. typhimurium TppB- mutants have unique tripeptide transport phenotypes. In E. coli, a mutation in oppe renders the strain resistant to certain toxic tripeptides and unable to utilize others as amino acid sources and influences the transport of tripeptides via the trp-linked system when the tripeptide substrate is present at low extracellular concentrations (1). The S. typhimurium tppb-encoded peptide transport system, on the other hand, lacks specificity with respect to the tripeptides that it will transport and appears to function independently of the trp-linked oligopeptide permease (9). Although E. coli and S. typhimurium are capable of utilizing a wide variety of peptides as growth supplements, * Corresponding author. 428 these enteric bacteria accomplish this common goal somewhat differently through their elaboration of both similar and unique peptide transport systems. Recently, synthesis of the S. typhimurium trp-linked oligopeptide permease was shown to be constitutive, but expression of the second S. typhimurium peptide transport system, the tppb-encoded system, was found to be regulated positively by the product of the ompb locus (previously designated as tppa) and to be inducible by leucine or by anaerobiosis (9). Data obtained in this laboratory from the selection of E. coli peptide transport mutants and from our investigation into the regulation of E. coli peptide transport suggested that expression of the genes encoding the E. coli peptide transport proteins was different from that reported for S. typhimurium (9). The results presented in this study demonstrate that active peptide transport via the E. coli trp-linked oligopeptide permease is regulated. In bacteria growing in minimal medium, synthesis of the peptide transport proteins encoded by the trp-linked opp operon occurs at a basal level, but, upon addition of leucine to the culture, expression of the opp operon-encoded transport system is dramatically increased. Moreover, results obtained with OppE- E. coli strains indicate that the decrease in the rate of peptide translocation produced by an oppe mutation does not result from a reduction in the expression of the trp-linked opp operon. The interpretation of the genetic and biochemical data presented in this communication is consistent with and extended by results obtained from the study of opp-lac operon fusions described in the accompanying paper (2). MATERIALS AND METHODS Bacteria and bacteriophages. The E. coli strains used in this study are listed in Table 1. Media and growth conditions. All E. coli strains were cultured in a minimal medium containing Vogel and Bonner (23) minimal salts, 0.2% glucose, and the appropriate supplements of amino acids and vitamins. Solid media contained 1.5% agar. Top layer agar contained 0.8% agar and 0.8% NaCl. Unless otherwise indicated, leucine was added to the media at a concentration of 50,ug/ml to induce expression of the trp-linked opp operon. Strains containing TnJO were grown on media containing 15,g of tetracycline per ml. Genetic manipulations. Lysates of P1 kc were prepared as described by Rosner (21). Transductions were performed as

2 VOL. 165, 1986 REGULATION OF PEPTIDE TRANSPORT IN E. COLI 429 TABLE 1. E. coli strains Strain Genotype Source or comments SS320 F- lac122 lacz pro-48 met-90 trpa trpr his-85 rpsl azi-9 gyra X- P1S (1) SS3213 As SS320, but opp_la (1) SS3222 AS SS320, but opp-2a (1) SS3233 AS SS320, but opp-3a oppel (1) SS3240 As SS320, but zji-2::tnjo oppel (1) SS3242 AS SS320, but zji-2::tnjo This laboratory SS4200 AS SS4201, but zji-2::tnjo oppel SS P1 (SS3240) SS4201 As W2915, but leu+ W P1(SS320) SS5012 AS SS320, but A(trp-tdk) This laboratory SS5211 AS SS320, but ilv::tnjo SS320 + P1 (GT103) SS5212 AS SS320, but oppel ilv::tnlo From SS3240 cured of TnlO, P1 transduction from GT10 W2915 F- thr-j thi-j proa2 lacyl galk2 mtl-l xyl-j ara-14 supe44 leub6 B. Bachmann GT103 F- thi pro rho-120 lacz::isims319 ilv::tnjo D. Calhoun UT5028 F+ thi serb zji-j::tnjo C. Lark acomplementation studies (1) demonstrated that the opp-j mutation affects expression of the entire trp-linked opp operon, that the opp-2 mutation affects only oppd expression, and that the opp-3 mutation is complemented only by an OppA- F'123 derivative. Thus, the Opp phenotype of a strain having an opp-j mutation is Opp(ABCD)-, that of a strain harboring an opp-2 mutation is Opp(ABC)+D-, and the phenotype of a strain carrying an opp-3 mutation is OppA+(BCD)-. described by Miller (15). The loss of TnJO was selected for as described by Maloy and Nunn (14). Response to toxic or nutritional peptides. The ability of a toxic peptide to inhibit bacterial growth was quantitated by determining the diameter of the inhibitory zone surrounding a filter paper disk saturated with the toxic peptide as described previously (1). The ability of peptides to support growth of an amino acid auxotroph was quantitated by measuring the zone of growth surrounding a disk saturated with 0.1,umol of the nutritional peptide. Transport of radiolabeled Ala3. The accumulation of radiolabeled tri-l-alanine (Ala3) was measured at 37 C as described previously (1). The uptake reaction was initiated by the addition of L-[U-14C]alanyl-L-alanyl-L-alanine ([14C]- Ala3; specific activity, 168 Ci/mol) to a final concentration of 20 p.m and terminated at the times indicated in Fig. 1 by washing the entire reaction mixture onto nitrocellulose membrane filters with 5 ml of 0.1 M LiCl. The filters were dissolved in 5 ml of ScintiVerse II scintillation fluid (Fisher Scientific Co., Pittsburgh, Pa.), and the radioactivity of each sample was determined. Preparation and analysis of osmotic shock fluids. The proteins present in the bacterial periplasmic space were labeled and isolated by osmotic shock as described by Emr and Bassford (4). In these experiments, the synthesis of the maltose-binding protein and the OppA binding protein were induced by the addition of 0.2% maltose and 100,ug of L-leucine per ml, respectively, to the medium. The lyophilized shock fluids were dissolved in Laemmli sample buffer (19), and the trichloroacetic acid-precipitable radioactivity of each extract was determined. Aliquots of each shock fluid sample containing 25,000 dpm of 14C were adjusted to a final volume of 10 pul and loaded onto a sodium dodecyl sulfatepolyacrylamide gel for analysis. Separation of the 14C-labeled proteins extracted by the osmotic shock procedure was performed with 0.75-mm by 15-cm slab gels and the running buffer of Thomas and Kornberg (22). The slab gels consisted of a 14-cm 16.5% separating gel (200:1, acrylamide-bisacrylamide) overlaid with a 1-cm 4.5% stacking gel (36:1, acrylamidebisacrylamide). Electrophoresis of the proteins through the stacking gel was carried out at a constant voltage of 50 V and through the separating gel at a constant power setting of 5 W/gel. After electrophoresis, the gels were soaked in Enlightning (New England Nuclear Corp., Boston, Mass.), dried, and exposed to Kodak XAR5 X-ray film for 16 to 20 h at -70 C. Analytical procedures. The protein concentration of all samples was measured by the method of Lowry et al. (13). Chemicals. Orn3 was obtained from Bachem AG, Bubendorf, Switzerland. Other peptides used in this study were obtained from Bachem AG; Research Plus, Inc., Bayonne, N.J.; Vega Biochemicals, Tuscon, Ariz.; and Bachem Inc., Torrence, Calif. All peptides obtained from commercial sources were analyzed as to their purity before being used in this study. The [14C]Ala3 was synthesized and purified by V. L. Styles and S. A. Short. RESULTS Influence of leucine on the E. coli peptide transport systems. The growth of wild-type E. coli K-12 in minimal media is strongly inhibited by valine-containing tripeptides due to the inhibition of acetohydroxyacid synthase by valine liberated from the transported tripeptide (1, 11). In contrast, the growth of E. coli K-12 strains harboring and oppe mutation is unaffected by the presence of valyl tripeptides in the growth medium (1). This resistance of OppE- strains to tripeptides containing valine results from the inability of these mutants to actively transport the toxic valyl tripeptides (1). These results suggested that OppE- mutants could be selected directly by plating a mutagenized E. coli culture on minimal agar medium containing a valyl tripeptide and leucine. The addition of leucine to the selection medium would prevent or significantly reduce reentry, via the branch chain amino acid transport system (20), of valine that was cleaved intracellularly from the valyl tripeptide and excreted into the medium by OppE+ cells. When selection for OppEmutants was carried out as described above, no mutants were obtained. Therefore, the experiment was reconstructed with the OppE- mutant SS3240 (Table 1). As predicted, growth of this mutant on minimal medium containing Val3 was identical to the growth observed for wild-type E. coli on the same medium lacking Val3. However, when SS3240 was plated on Val3 medium containing leucine, this strain did not grow, suggesting that, under these coditions, Val3 was being concentrated to toxic levels by OppE- cells. That this was indeed the case is demonstrated by the data presented in Table 2. In the absence of leucine, SS3240 was completely resistant to Val3, but, upon addition of leucine to the medium, the Val3 sensitivity of this strain was indistinguish-

3 430 ANDREWS ET AL. J. BACTERIOL. TABLE 2. Influence of leucine on the toxicity of valine-containing peptides" Zone of growth inhibition (mm, diameter) Stiain Opp Phenotype Val-Val-Val Pro-Val-Asp Pro-Val-Gly Val-Asp Val-Gly Pro-Val _ SS320 Wild type SS3213 (ABCD)-E ND ND ND ND ND ND SS3222 (ABC)+D-E ND ND ND ND ND ND SS5012 A(ABCD)E SS3240 (ABCD) + E SS3233 A+(BCD)-E a Sensitivity of each strain to the toxic valyl peptides was determined as described in the text. The - and + symbols below each peptide indicate the absence or presende of 50,ug of L-leucine per ml in the medium. The amount of each peptide tested was 100,ug of Val3 and 50,ug each of Pro-Val-Asp, Pro-Val-Gly, Val-Asp, Val-Gly, and Pro-Val. The numbers represent the diameters of the clear zones surrounding each peptide-containing 6.5-mm disk. A value of 0 indicates that growth was not inhibited by the peptide tested. ND, Not done. able from that observed for the wild-type strain, SS320. The leucine enhancement of growth inhibition produced by valyl tripeptides was not a phenotype unique to SS3240 or E. coli strains harboring an oppel mutation. In the absence of leucine, the growth of SS320 was inhibited by Thr-Val-Leu, Phe-Ser-Val, V41-Gly-Gly, Val-Leu-Ser (data not shown), and Val3, but was absolutely unaffected by Pro-Val-Asp or Pro-Val-Gly (Table 2). Sensitivity of SS320 to these latter two peptides was obtained, however, upon incorporation of leucine into the assay medium (Table 2). The Pro-Val-Asp and Pro-Val-Gly resistance displayed by SS320 in the absence of leucine did not result from a peptidase deficiency, since both Pro-Val-Asp and Pro-Val-Gly satisfy the proline requirement of SS320 in the absence of leucine and since this strain was equally sensitive to Val-Asp, Val-Gly, and Pro- Val in the absence or presence of leucine (Table 2). The differential sensitivity to valyl tripeptides described above for strains SS320 and SS3240 is consistent with the interpretation that the addition of leucine to E. coli resulted in elevated intracellular concentrations of these valyl peptides and thus their toxic hydrolysis product, valine, and that in the absence of leucine the intracellular valine concentration resulting from hydrolysis of Pro-Val-Asp and Pro-Val-Gly in SS320 or Val3 in SS3240 never achieved a toxic level. The influence of leucine on the sensitivity of E. coli to toxic peptides was not limited to tripeptides containing valine. Sensitivity of E. coli strains SS320 (wild-type) and SS3240 (oppe) to Orn3 was also affected by leucine. When assayed over a 20-fold Orn3 concentration range, the zone of growth inhibition produced by each Orn3 concentration was significantly larger in the presence of leucine than in the absence of this amino acid in the assay medium (Table 3). Moreover, by analogy to the results obtained with toxic tripeptides, leucine would be predicted to enhance the growth of E. coli on nutritional tripeptides. The data given in Table 4 for SS3240 (with Lys-His-Lys as a histidine source and with Pro-His-Leu, Pro-His-Phe, and Pro-His-Glu as either proline or histidine sources) and the ability of SS5211 (ilv::tnio, Table 1) to use Pro-Val-Asp or Pro-Val-Gly as valine sources only in the presence of leucine confirm this prediction. The data presented in Tables 2, 3, and 4 thus are probably reflections of the intracellular concentration of exogenously supplied tripeptides and as such support the hypothesis that the addition of leucine to E. coli results in an increase in the transport of extracellular tripeptides into the cell. It should be noted that the response of E. coli to leucine described above was not a property peculiar to strains derived from SS320, since identical results were obtained with the E. coli W2915 derivative strains, SS4200 and SS4201 (Table 1, data not shown). Response of Opp- mutants to leucine. E. coli SS3230 (opp-3 oppe) and its derivatives were found previously to be resistant to all toxic tripeptides and unable to utilize nutritional peptides as amino acid sources when assayed in the absence of leucine (1). The data shown in Tables 2 and 4 confirm this earlier observation and indicate that, even in the presence of leucine, the growth of a strain harboring an opp-3 mutation and an oppe mutation was unaffected by toxic tripeptides and not supported by nutritional peptides. Therefore, the E. coli peptide transport system responsible for the leucine effect documented above is apparently encoded by either the genes of the trp-linked opp operon or the gene(s) of the oppe locus. Since tripeptide accumulation by the OppE- mutant SS3240 was leucine responsive, the oligopeptide permease encoded by the trp-linked opp operon must be the mediator of the leucine effect. Those E. coli strains harboring a mutation in the trp-linked opp operon (SS3213, SS3222) or carrying a deletion of the entire opp operon (SS5012) were resistant to Orn3 (1) and valyl tripeptides except Val3 and unable to utilize n1umerous nutritional peptides as amino acid sources both in the presence and absence of leucine. Confirmation that the TABLE 3. Effect of leucine on Orn3 toxicity' Zone of growth inhibition (mm, diameter) with the following amt of Orn3 on disk (pg) Strain Opp phenotype ~++ + SS320 Wild type SS3240 (ABCD) -+E a Sensitivity of each strain to Orn3 was determined as described in the text. The - and + symbols below each peptide amount indicate the absence or presence of 50 Fg of L-leucine per ml in the medium. The numbers represent the diameters of the clear zones surrounding each peptide-containing 6.5-mm disk. A value of 0 indicates that growth was not inhibited by the peptide tested.

4 VOL. 165, 1986 REGULATION OF PEPTIDE TRANSPORT IN E. COLI 431 TABLE 4. Inducibility of the E. cqli peptide transport systems" Size of growth zone (mm) on: Lys-His- Pro-His- Pro-His- Pro-His- Lys-Ala- Ala-Pro- Met-Met- Met-Leu- Strain Opp phenotype Lys Leu Phe Glu Pro Ala Met Phe _ + _ + _ + _ SS320 Wild type SS3222 (ABC)+D-E SS5012 A(ABCD)E SS3240 (ABCD) + E SS3233 A+(BCD)-E a The ability of each strain to use a tripeptide as the sole source of an essential amino acid was determined as described in Materials and Methods with 0.1 p.jnol of amino acid in peptide linkage applied to each disk. The - and + symbols below each tripeptide indicate the absence or presence of 50 p.g of L-leucine per ml in the mnedium. The numbers represent the diameters of the zones of growth surrounding each tripeptide-saturated disk. A value of 0 indicates that the strain was unable to grow on the tfipeptide applied to the disk. leucine enhancement of tripeptide transport observed in E. coli requires a wild-type trp-linked opp operon was obtained (i) by the selection of Orn3-resistant mutants of SS3240 (oppe) in the presence of leucine, (ii) by transduction of SS3233 (opp-3 oppe) to oppe+ with P1 lysates prepared on either UT5028 (zji-j::tnlo oppe+) or SS3242 (zji-2::tnjo oppe+), and (iii) by determination of the phenotype obtained after introduction of F'123 into SS5012 (AoppABCD). Analysis of the results obtained from these experiments demonstrated that (i) all secondary mutations selected in SS3240 that resulted in leucine-nonresponsive Orn3r Val3r mutants mapped by P1 transduction in the trp-linked opp operon, (ii) all Tcr, OppE+ transductants of SS3233 remained resistant to Orn3, Pro-Val-Asp, and Pro-Val-Gly in the presence or absence of leucine, and (iii) introduction of F-factor F'123, which has been shown to carry the trp-linked opp operon (3, 12), into SS5012 yielded only Orn3-sensitive exconjugants that were more sensitive to Pro-Val-Asp and Pro-Val-Gly in the presence than in the absence of leucine. The results obtained through genetic analysis of the leucine effect complement the phenotypic data described in the preceding section and together suppprt the hypothesis that leucine enhances tripeptide uptake by E. coli and that this enhancement is dependent on the presence of a wild-type trp-linked opp operon. Influence of leucine on ('4C]Ala3 transport. The accumulation of radiolabeled Ala3 by E. coli grown in the absence of leucine has been shown to proceed through the transport system encoded by the trp-linked opp operon and to be influenced by the gene product(s) encoded by the oppe locus (1). In the absence of leucine and at 20 F.M [14C]Ala3, the peptide transport system requiring a functional oppe locus accounted for the majority of the [14C]Ala3 uptake measured with wild-type E. coli SS320 (1). When [14C]Ala3 accumulation was measured with SS320 cells harvested from leucinecontaining medium, the initial rate of [14C]Ala3 uptake was increased ca. fourfold and the apparent steady-state level of tripeptide accumulation was elevated ca. twofold compared with these parameters of Ala3 transport measured with SS320 cells collected from medium lacking leucine (Fig. 1A). Similarily, growth of the OppE- mutant SS3240 (which expresses the trp-liriked opp tripeptide trahsport system) in medium containing leucine yielded cells in which [14C]Ala3 accumulation was significantly elevated relative to the uptake measured for cells of this strain grown in the absence of leucine (Fig. 1B). In control experiments, the transport of serine, glutamine, histidine, and proline was found to be identical for strain SS320 (or SS3240) that had been grown in the absence and presence of leucine (data not shown). Moreover, preincubation of SS320 or SS3240 cells collected from cultures grown in the absence of leucine with leucine in the transport assay mixtures did not result in increased [14C]Ala3 transport as was noted above for cells from leucine-grown cultures. In fact, the levels of [14C]Ala3 uptake obtained after leucine preincubation were indistinguishable from that shown in Fig. 1 for SS320 and SS3240 grown in the absence of leucine. These results indicate that the increased activity of the tripeptide transport systems described for leucine-grown E. coli was specific to peptide accumulation and that this increased transport activity required growth of the bacteria in leucine-containing medium. Regulation of oppa gene expression. If leucine is functioning to enhance peptide transport by elevating expression of the trp-linked opp operon as in7dicated in the preceding sections, then the amount of oppa gene product released by osmotic shock should be greater in the periplasmic protein fraction prepared from leucine-grown E. coli than from cells grown in the absence of leucine. Analysis of the periplasmic proteins labeled and extracted from strains SS320 (wild type), SS3240 (oppe), and SS5012 (AoppABCD) indicated that leucine increased synthesis of the oppa-encoded bind- E E Ua 0 0 E C 0 I- 0. c rx TIME (min) FIG. 1. Influence of leucine on Ala3 transport. The transport of ['4C]Ala3 was measured as described in the text with E. coli strains (A) SS320 (wild type) and (B) SS3240 (oppe). Ala3 accumulation was measured with cell suspensions prepared from cultures grown in the absence of leucine (0) or in the presence of 50,ug of leucine per ml (A).

5 432 ANDREWS ET AL. ing protein (Fig. 2; compare lane A with B and lane D with E). In these experiments, identification of the oppa gene product was based on the fact that strain SS5012 (AoppABCD) (Fig. 2, lane C) failed to synthesize a periplasmic protein having a subunit molecular weight of 56,000 and on the size similarity between the E. coli OppA protein (Fig. 2, band 1) and the S. typhimurium OppA protein (6). The increased synthesis of OppA protein after the addition of leucine to the bacterial culture was not a general, nonspecific phenomenom common to all periplasmic proteins. Synthesis of the periplasmic maltose-binding protein (Fig. 2, band 3) was unaffected by inclusion of leucine in the growth medium (Table 5), whereas synthesis of the LIV-binding protein (Fig. 2, band 2), the product of the livj gene (16), was repressed upon the addition of leucine to the E. coli cultures as has been previously described (17, 19). The magnitude of the leucine-specific increase in OppA protein shown in Fig. 2 for strains SS320 and SS3240 was estimated by using the induced level of maltose-binding protein as an internal standard. The addition of leucine to each of these E. ccli cultures produced a 2.1- to 3.3-fold increase in the amount of OppA protein released by the osmotic shock procedure when compared with the amount of maltose-binding protein present in each periplasmic protein fraction (Table 5). These data also indicate that in the absence of leucine the abundance of the E. coli OppA protein was not significantly greater than the LIV-binding protein (Fig. 2, compare bands 1 and 2 of lanes A and D), and that under inducing conditions the amount of OppA protein synthesized and exported into the bacterial periplasm was comparable to the amount of A B C D E I - v..r 2 3-_-ow..:....:. ;.:3 67k 43k 30k 20k FIG. 2. Periplasmic proteins prepared from E. coli grown in the absence and presence of leucine. The E. coli periplasmic proteins were pulse-labeled with "'C-amino acids, extracted, and analyzed as described in the text. The protein profiles shown in this autoradiogram were obtained with SS320 (wild type) grown and labeled in the (A) absence and (B) presence of leucine, (C) SS5012 (AoppABCD) grown and labeled in the presence of leucine, and SS3240 (oppe) grown and labeled in the (D) absence and (E) presence of leucine. The positions of the E. coli OppA binding protein (1, Mr 56,000), the LIV-binding protein (2, Mr 39,000), and the maltose-binding protein (3, Mr 36,000) are indicated of the left side of this figure. The positions of the molecular weight standards bovine serum albumin (Mr 67,000), ovalbumin (Mr 43,000), carbonic anhydrase (Mr 30,000), and trypsin inhibitor (Mr 20,100), are indicated on the right. TABLE 5. J. B3ACTERIOL. Influence of leucine on the synthesis of the OppA binding proteina Leucine in Expt 1 (dpm) Expt 2 (dpm) Strain medium MBP OppA MBP OppA SS320-4,140 1,665 5,920 2, ,440 4,540 3,840 4,660 SS3240-4,240 1,400 6,080 2, ,600 3,010 4,810 4,550 a Data presented represent the radioactivity of the maltose-binding protein (MBP) and the OppA binding protein determined after solubilization of the area of each gel lane corresponding to these proteins. maltose-binding protein synthesized in the presence of maltose. DISCUSSION Growth of E. coli in medium containing leucine resulted in increased sensitivity to toxic peptides (Tables 2 and 3) as well as stimulating or enabling growth on numerous nutritional tripeptides (Table 4). For leucine to educe these responses with such a wide variety of both toxic and nutritional tripeptides, this amino acid must be affecting a common step in the utilization of tripeptides by E. coli. The biochemical and genetic data presented in this communication demonstrate that growth of E. coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell and that this leucineinduced increase in tripeptide transport requires expression of the trp-linked opp operon. The biochemical and genetic data presented in this study and in the accompanying communication (2) indicate that leucine is not functioning to induce the synthesis of a new, completely independent peptide transport system similar to the leucine-inducible thymine PP, transport system described in S. typhimurium (9). Rather, the data described in this study of E. coli peptide transport are consistent with the interpretation that growth of E. coli in leucine-containing medium results in increased expression of the trp-linked opp operon. The data obtained from the osmotic shock experiments (Fig. 2 and Table 5) confirm this conclusion and provide a direct demonstration of the leucine-mediated enhancement of opp gene expression. Moreover, the growth results described in Tables 2, 3, and 4 as well as the [14C]Ala3 transport results (Fig. 1) support this interpretation and can be readily understood in terms of an increase in the number of functional opp-encoded transport systems incorporated into the bacterial membrane. Apart from demonstrating the leucine inducibility of peptide transport in E. coli, an examination of both the capacity of each peptide transport mutant to utilize nutritional peptides (Table 4) and the sensitivity or resistance of each mutant to toxic peptides (Tables 2 and 3) has permitted a qualitative definition of substrate preference for the E. coli peptide transport systems. These data indicate that peptides such as Lys-His-Lys, Pro-His-Leu, Pro-His-Phe, Pro-His- Glu, Leu-Ala-Pro (data not shown), and Orn3 are transported predominantly via the system encoded by the trplinked opp operon; that Lys-Ala-Pro, Ala-Pro-Ala, Ala3 (1), and Val3 display a marked preference for the transport system absent in E. coli strains harboring an oppe mutation; and that Met3, Met-Leu-Phe, Met-Ala-Ser (1), Pro3, and Met-Met-Ala (data not shown) appear to lack any transport system preference for their entry into the bacterial cell. This

6 VOL. 165, 1986 qualitative classification of E. coli peptide transport system specificity is in agreement with previous results (1) and provides further evidence suggesting that not all tripeptides enter E. coli by each transport system with an equal efficiency. Previously, data were presented which suggested that in E. coli a relationship exists between mutations mapping at the oppe locus and the transport system specified by the genes of the trp-linked opp operon (1). The data obtained in this study with E. coli SS3240 (oppe) are in agreement with this proposal. Introduction of an oppe mutation into wildtype E. coli resulted in a strain which was unable to actively transport Orn3 to a toxic intracellular concentration (Table 3) and which had lost the ability to utilize oligopeptide permease preferred tripeptides as sources of required amino acids (Table 4) when grown in the absence of leucine. However, when an OppE- mutant such as SS3240 was grown with exogenous leucine, the influence that the oppe mutation had on peptide transport was significantly diminished. Since the genetic and biochemical evidence presented in this study indicate that the leucine responsiveness of E. coli peptide transport requires the presence of a wild-type trp-linked opp operon, then the effect that the oppe mutation has on E. coli peptide transport must result from an alteration in either the activity or the expression of the transport system encoded by the trp-linked opp operon. The oppe mutation does not affect expression of the oppa-encoded periplasmic binding protein (Fig. 2 and Table 5). In fact, the level of oppa gene product synthesis relative to the induced level of maltose-binding protein synthesis for the OppEmutant SS3240 was essentially identical to that found with the wild-type E. coli SS320 when measured with bacteria grown in the absence or presence of leucine. These results have been confirmed and are extended by the data obtained from an examination of opp operon regulation and expression with opp-lac operon fusions described in the accompanying paper (2). Although the data presented in this study have documented that a relationship exists between the gene(s) of the oppe locus and E. coli peptide transport, the identity of the mechanism through which the oppe gene product(s) influences the functioning of the E. coli peptide transport systems remains to be defined. However, one interpretation of the data presently available is that a mutation in the oppe locus significantly impairs the activity (i.e., turnover number or affinity) of the E. coli transport system encoded by the trp-linked opp operon. LITERATURE CITED 1. Andrews, J. C., and S. A. Short Genetic analysis of Escherichia coli oligopeptide transport mutants. J. Bacteriol. 161: Andrews, J. C., and S. A. 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