JOURNAL OF BACTFRIOLOGY, May 1981, p. 676-683 Vol. 146, No. 20021-9193/81/050676-08$02.00/0

Toxicity of Leucine-Containing Peptides in Escherichia coliCaused by Circumvention of Leucine Transport Regulation

H. TAVORI, Y. KIMMEL, AND Z. BARAK*Department ofBiology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Received 18 November 1980/Accepted 9 March 1981

A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu,Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibitedthe growth of a prototrophic strain of Escherichia coli K-12 at concentrationsbetween 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. Whenpeptide transport was impaired by mutations, strains became resistant to therespective LCP. Inhibition of growth occurred immediately after the addition ofLCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteriabegan to grow at the normal rate 70 min after Gly-Leu became undetectable.Disappearance of the peptide corresponded with the appearance of free leucineand glycine in the medium. The concentration of leucine inside the LCP-treatedbacteria was higher than that in the leucine-treated and the control cultures. Wesuggest that entry ofLCP into the cells via peptide transport systems circumventsthe regulation of leucine transport, thereby causing abnormnally high concentra-tions of leucine inside the cells. This accumulation of leucine interferes with thebiosynthesis of isoleucine and inhibits the growth of the bacteria.

There are three transport systems for L-leu-cine in Escherichia coli K-12, two of which arecommon to isoleucine and valine: LIV-I, themajor component ofleucine entry accounting for80 to 90% of leucine transport, and LIV-II, ac-counting for 5 to 10%. The third one, the Lsystem (10 to 15%), is specific for leucine alone(8, 16). Leucine might also enter E. coli cells asa component of a peptide via one of the threepeptide transport systems known in this bacte-rium (1, 13): DPP, a dipeptide permease whichtransports all dipeptides containing L-aminoacids; OPP, a general oligopeptide permeasetransporting most oligopeptides (regardless oftheir amino acid composition), and OPP-I, aspecialized tripeptide permease transportingonly certain peptides, such as trileucine, trime-thionine, and trithreonine (2, 11).High concentrations of leucine cause repres-

sion of all the three systems capable of trans-porting this amino acid in its free form (14-16).Leucine also is known to play a role in theregulation processes of the flv pathway, whichis responsible for the biosynthesis of isoleucineand valine (Fig. 1). Thus, leucine represses theenzyme acetohydroxy acid synthetase III (6) andparticipates in the multivalent repression of theflv operons (7, 19). Also, high concentrations ofleucine inhibit the enzymes threonine deami-nase, (4, 21) and acetohydroxy acid synthetase(3) in crude extracts of E. coli K-12.

Previous data presented in 1951 by Simmondset al. (17) demonstrated that when leucine-con-taining peptides (LCP) are present in high con-centrations (2.4 to 4.8 mM of leucine in thepeptide), they are toxic to leucine auxotrophs orprototrophs of E. coli K-12. Since none of thedegradation products ofLCP was able to inhibitgrowth of these strains, these workers concludedthat the peptides as such were responsible forthe observed inhibition of growth. The toxiceffect of lysyl-leucine on prototrophic strains ofE. coli K-12 was also reported (12). No expla-nation was offered in any of these reports con-cerning the mode of toxicity of LCP in E. coliK-12. An attempt to understand the mechanismof inhibition exhibited by glycyl-leucine (GL),one of the most potent LCP tested, was carriedout by Vonder-Haar and Umbarger (21). Theypostulated that this dipeptide resembles isoleu-cine and mimics its inhibitory effect on threo-nine deaminase. They have demonstrated thatthe toxicity caused by GL is reversed by isoleu-cine added to the medium. Their explanation, iftrue, applies to GL only and not to any othertoxic LCP.

In this study we show that toxicity of LCP inE. coli K-12 is due to accumulation of theirdegradation product, leucine, to abnormallyhigh intracellular concentrations which interferewith the biosynthesis of isoleucine. (A prelimi-nary report on part of this study was presented

676

TOXICITY OF LCP IN E. COLI K-12 677

rbs ? ivDNA (83rrmn) - -a,t

IOu ivDNA (2min) ix H I

TRBTAT

a-Ketoisocaproate wLeucine

I IPMD3hlsopropylmolate

t ISOPyruvote a-lsoprylmoaltet aiPMS

tate-.a-a Dihydroxy-.-a-KetoisovteVineisovderote TRB

Pyruvote AHAS 1,1,1 IR DH

TRB-Acetohyy' q,,8-Dihydroxy -a- Keto-,8-methyl sokeucinebutyrate 3-metylrote voWrate

Threonine a--Vetobutyrate

FIG. 1. Biosynthetic pathways for leucine, isoleucine, and valine in E. coli K-12 (19). Enzymes areabbreviated as follows: TD, threonine deaminase; AHAS, a-acetohydroxy acid synthase; IR, a-acetohydroxyacid isomeroreductase; DH, dihydroxyacid dehydratase; TRB, transaminase B; TA, one or more transami-nases (controversial); IMPS, a-isopropylmalate synthase; ISO, isopropylmalate isomerase; IPMD, ,8-isopro-pylmalate dehydrogenase; TAT, tyrosine aminotransferase. The genes responsible for these enzymes are alsodiagrammed. The arrows above the genes indicate the probable direction of transcription.

at the Annual Meeting of the Israeli Microbio-logical Society [Z. Barak, H. Tavori, and N.Gollop, Isr. J. Med., in press]).

MATERIALS AND METHODSChemicals. All of the peptides tested contain L-

amino acids only. The peptides Phe-Leu (PL), Gly-Leu (GL), Leu-Leu-Leu (Leu3), Leu-Gly-Gly (LGG),Val-Gly (VG), Val-Gly-Gly (VGG), Trp-Phe (TP), andTrp-Gly (TG) were purchased from Sigma ChemicalCo. The peptides Pro-Leu (PrL), Ala-Leu (AL), Ala-Leu-Lys (ALL), Leu-Phe-Ala (LPA), and Om-Orn-Orn-Orn-Orn 4HCl (Orn3) were gifts from the Depart-ment of Biophysics, Weizmann Institute of Science,Rehovot, Israel. All peptides were found pure by high-voltage paper electrophoresis and by thin-layer chro-matography as described below. Amino acids werepurchased from Sigma Chemical Co. All other chemi-cals and solvents were of analytical grade.

Media. The minimal medium used was VB (20).Cysteine and tryptophan were added to final concen-trations of 20 and 5 ,ug/ml, respectively. Other aminoacids, when required for growth, were added at 20,ug/ml. The rich medium used was LB (9). Media weresterilized by autoclaving for 20 min. Peptide solutionswere sterilized by filtration through sterile membranefilters (0.45Mm), purchased from Schleicher & SchuellCo., and added aseptically to the sterile medium to thefinal concentrations mentioned below.

Bacterial strains. The strains used in this inves-tigation are listed in Table 1. Except for E. coli B, theyare derivatives of E. coli K-12.Growth conditions. Media were inoculated with

1% (vol/vol) of fresh late-log culture. The bacteriawere grown with shaking (150 rpm) at 37C in Kletttubes containing 5 ml of medium. Bacterial growthwas measured by the change in optical density of theculture as a function of time in a Klett-Summerson

colorimeter, using a 660-nm filter. One Klett unitcorresponds to 6 x 106 bacteria per ml.Paper electrophoresis. Separation of peptides

and amino acids was carried out by flat-plate electro-phoresis (Savant Instruments Inc.) on Whatman no.3 paper. For purity tests, samples of 50 to 100 Ag of apeptide were applied to the paper. Peptides wereelectrophoresed with a potential gradient of 3,000 Vfor either 2 h in pyridine-acetate buffer, pH 3.5 (0.22%and 2.2% [vol/vol], respectively), or 1.5 h in pH 2buffer (2.7% [vol/vol] formic acid).

Thin-layer chromatography. Ascending chro-matography was carried out either on DC-AlufolienKieselgel (0.25-mm thick) or on DC-Fertig-PlatternKieselgel 60 (0.25-mm thick) from E Merck AG; nosignificant differences could be detected between thetwo methods. Running solvents used for purity testswere butanol-acetic acid-water (4:1:1 [vol/vol]) and n-propanol-water (7:3 [vol/vol]). The latter solvent wasused for detection of GL and its degradation productsin the medium of a GL-treated culture.Development of spots. Papers or thin-layer plates

were dried for 2 h at 60C. For development of spots,papers or plates were dipped in 0.2% (wt/vol) ninhy-drine in acetone solution and heated for 20 min at80C. Determination of spots was carried out withreference to known markers.Measurement of intracellular amino acid

pools. The amino acid pools were measured by theprocedure described by Quay et al. (14), with slightmodifications. Cells were harvested at room tempera-ture by filtering 35-ml cultures through membranefilters (0.45 Mm). The pellet was suspended in 1 ml of3% sulfosalicylic acid with 0.1 Mmol of norleucine as aninternal marker. The suspension was incubated for 10min at 100C and separated by centrifugation (10 min;5,000 x g). The supematant was collected, and theamino acid content was determined with a Beckmanamino acid analyzer. The recovery of amino acids by

IVOL. 146, 1981

TABLE 1. E. coli strains

Strain Genotype Relevant phenotypea Origin Source ReferenceB Valr; Val3r; VGr; Z. Barak, Department

LPAr; PL'; of Biology, Ben-PrL', ALr; Leu+ Gurion University,

Beer-Sheva, Israel

I cysB trpE9829 Trp-; Orn3s; Val36; MY 517 M. D. Yudkin, 18LPA8; Leu3; Department ofGL8; PL8; VGa; Microbiology, OxfordTGu; TPu University, Oxford,

England

I-TOR cysB trpE9829 Trp-; Orn3r; Val3r; I Spontaneous mutant, Y. Kimmel and Z.Opp LPAr; Leu35; resistant to Orn3 Barak, submit-

GLU; PL8; VG8, ted for publi-TGu; TPu cation

HA cysB Trp-; Orn3r; Val3r; I-TOR Spontaneous mutant Kimmel andTipE9829 LPAr; IAU3r; resistant to Leu3 Barak, submit-opp opp, GL;s; PL; VG5; ted for publi-

TGu; TPU cation

CSH-7 Val'; GU; PL8; Z. Barak, Department 10Leu+ of Biology, Ben-

Gurion Universitya Peptides are designed by the symbols mentioned in the text; r, resistance; s, sensitivity; u, utilizable as

amino acid source.

this method was estimated by that of the internalmarker. The amount of leucine, liberated from cleav-age of GL during the whole procedure, was estimatedby introducing 0.1 jmol of the dipeptide together withnorleucine into the reaction mixture at the sulfosali-cylic acid step.

RESULTS

Toxicity of LCP to prototrophic strainsof E. coli K-12. As shown in Table 2, all of theeight LCP tested (PL, GL, AL, PrL, Leu3, LGG,LPA, and ALL) inhibited the growth of strain Iat concentrations ranging from 0.05 to 0.29 mM.The inhibition of growth is expressed by anincrease in the lag period. At later times, afterinhibition has been overcome, the rate ofgrowthof the LCP-treated cultures is similar to that ofthe control. The degree of inhibition is ex-pressed, therefore, in Table 2 as A lag, i.e., thedifference in minutes in attaining growth of 10Klett units between the control culture and aculture treated with a peptide. Leucine in equi-molar or higher concentrations (up to 1 mM) didnot inhibit growth of strain I. Growth of anotherstrain of E. coli K-12, prototrophic for leucine(CSH-7), was also inhibited by the LCP, GL andPL (Table 2). However, E. coli B was not in-hibited by LCP (AL, PL, PrL, and LPA), evenat concentrations 10-fold higher than those thatinhibit E. coli K-12 strains (Table 2). With allLCP tested, the inhibition was prevented when

0.4 mM of isoleucine was added (Table 2).Effect of the ratio of LCP/cell number on

tosicity. The effect of LCP on the growth of E.coli K-12 strain I was measured with PL, GL,and LGG. As demonstrated in Fig. 2 for PL, thedegree of inhibition, as represented by the du-ration of the lag period, is proportional to theconcentration of the LCP added to the mediumat a concentration range of 3 to 30 Ig/ml. Higherconcentrations of PL (30, 40, and 50 jig/ml)caused the same degree of inhibition (A lag =1,450 min). The bacteria growing after this longlag were probably PL-resistant mutants. Theminimal inhibitory concentrations of PL, GL,and LGG in strain I were 0.25, 0.265, and 0.49,umol/107 celLs, respectively.

Effect of LCP on growth of mutants de-ficient in peptide transport systems. Toprove that toxicity of LCP requires their uptakeinto the bacterial cells, we tested the effect ofLCP on the growth of mutants of strain I defi-cient in their peptide transport systems. TheOpp- mutant I-TOR, a mutant defective in itsgeneral oligopeptide transport system, was notsensitive to the tripeptide LPA (0.28 mM), butLeu3 (0.28 mM) which is known to utilize theOPP-I specialized peptide transport system,caused the expected toxicity (Table 3). A mutantdefective in both OPP and OPP-I (strain HA)was not inhibited by either LPA or Leu3 (Table3). Inhibitory effects were exerted on both mu-

678 TAVORI ET AL. J. BACTERIOL.

TOXICITY OF LCP IN E. COLI K-12 679

TABLE 2. Effect ofLCP on growth of severalprototrophic E. coli strains

Strain Agent tested Concn (mM) tlag xGenerationLCP alone LCP + lleb tie (mi)

E. coli K-12 ALL 0.29 300 0 90strain I LPA 0.28 680 0 90

LPA 0.071 400 0 90LeU3 0.28 600 -50 90Leu3 0.05 150 0 90LGG 0.17 500 -20 90PL 0.072 600 0 90GL 0.079 600 0 90AL 0.074 640 -40 90PrL 0.065 400 0 90

E. coli K-12 PL 0.072 180 0 70strain CSH-7 GL 0.53 600 0 70

E. coliB LPA 0.28 0 60PL 0.72 0 60PrL 0.74 0 60AL 0.74 0 60L-leucine 0.40 0 60L-valine 0.40 0 60

a A lag is defined as the difference in minutes in attaining growth of 10 KU between the control culture anda culture with a respective concentration of a peptide.

b The concentration of isoleucine added to the media was 0.4 mM.

.C 800Eo 600/

C 400

200

2 4 6 8 10

pg Phe - Leu / 106 BacteriaFIG. 2. Inhibition of growth as a function of the

ratio of PL to bacteria. The data presented werecollected from two sets ofexperiments. In experiment1 (0) different concentrations ofPL (5, 7.5, 10, 12, 15,20, 30, 40 and 50 pug/ml) were added to culturesinoculated with late-log culture to give 5 x 106 bac-teria per ml. In experiment 2 (0) the peptide concen-tration added was always 20 pg/ml, whereas thebacterial inocula varied to give the following concen-trations: 5 x 106; 10 x 106; 20 x 106; 40 x 106, and 80x 106/ml.

tants by PL (0.072 mM) and GL (0.079 mM).These inhibitory effects are probably due to theability of PL and GL to enter the bacteria via

the dipeptide permease, DPP. Inhibitions werenot expressed when isoleucine was added to therestrictive medium. It can be concluded thattoxicity of LCP requires normal uptake of thepeptides.Immediate effects of LCP on exponen-

tially growing cultures of strain L To allowdetection ofimmediate effects ofLCP on growth,peptides were added to exponentially growingcultures (10-15 Klett units, 660 nm) of strain I.Bacterial cells were grown in VB medium fortwo generations before the addition of 0.125mMPL (Fig. 3), 0.3 mM GL (Fig. 4), or 0.3 mM LPAor LGG (data not shown). In all cases examined,the growth of strain I was inhibited immediatelyafter the peptide was added, although exponen-tial growth continued at a very slow rate. Gen-eration times were 290 min for the PL-treatedculture and 230 min for the GL-treated culture,compared with 70 and 60 min respectively, forthe controls. As expected, addition of 0.4 mMisoleucine to the LCP-inhibited culture resultedin an immediate relief of toxicity exhibited by areturn to the normal growth rate (Fig. 3).

Inhibition of growth was maintained as longas GL was present, but growth at uninhibitedrates began 70 min after GL could not be de-tected in the medium (Fig. 4). As the dipeptidedisappeared, the intensity of the spots repre-senting leucine and glycine increased. Despitethe increase in bacterial concentration, no sig-nificant decrease in the amount of leucine could

VOL. 146, 1981

TABLE 3. Effect ofLCP on growth ofmutants defective in their peptide transport systemsA lag (min)a GenerationStrain Peptide tested Concn (mM)

blag (min)LCP alone LCP + lleb time (mi)

I-TOR LPA 0.28 -40 -80 70Leu3 0.28 600 -30 70Leu3 0.05 150 Not tested 70GL 0.079 400 0 80GL 0.27 860 0 80GL 0.81 >1440 0 80PL 0.072 120 0 75

HA LeU3 0.28 0 0 100LPA 0.28 0 -30 100PL 0.072 130 0 100GL 0.079 450 -15 100

a See Table 2, footnote a.b See Table 2, footnote b.

E

80

40.

5

TIME (hours)

FIG. 3. Effect ofPL on exponentially growing cells

and reversal of inhibition by isoleucine. Experimen-

tal conditions are described in the text. Strain I was

grown in parallel tubes, (@) and2 (0), forperiodA.

The dipeptide PL (0.125 mM) was added to tube 2 atthe beginning ofperiod B. Both cultures were diluted

twofold by the addition of 5 ml of the respective

medium, prewarmned to 37C, when entering periodC. A sample of 5 ml was withdrawn from culture 1,

whereas culture 2 was divided into two tubes of 5-ml

each, 2(0) and 3(0). Isoleucine (0.4 mM) was addedto tube 3 at period D. Reading at periods C and Dwere corrected for dilution.

be detected in the medium of the leucine-treated

culture, even after 240 min of incubation. a timeperiod sufficient to absorb completely equimolar

concentrations of GL.Accumulation of leucine inside E. coli

ceils durig incubation witWGL. The nega-tive regulation of leucine on its own transport

might control the internal pool of leucine inside

E. coli K-12 cells. Entry of leucine as part of apeptide is not regulated by either the peptide or

its degradation products, so if peptides are takenin and cleaved faster than leucine is metabolized,leucine should accumulate intracellularly to ab-normally high concentrations. To demonstratethis phenomenon, we determined the internalamino acid pools of three cultures of E. coli K-12 strain I: (i) a control culture grown exponen-tially in minimal VB medium, (ii) an exponen-tially growing culture to which leucine (0.3 mM)was added, and (iii) an inhibited culture to whichGL (0.3 mM) was added. Leucine and GL wereadded to separate exponentially growing cul-tures when the turbidity reached 10 Klett units(ffilter, 660 nm).Table 4 contains the acidic and the neutral

amino acid levels found in each of the threecultures. The important features of these datarelated to our discussion are: (i) the strikingincrease in intracellular leucine in the GL-treated culture compared with that found in theleucine-treated and control cultures, (ii) the in-tracellular isoleucine level decreased dramati-cally in the GL-treated culture, and (iii) theintracellular valine levels were not significantlylowered in the GL-treated culture.

DISCUSSIONVarious LCP were found to inhibit the growth

of several E. coli K-12 strains (12, 17; Table 2).This may indicate that toxicity of LCP is ageneral phenomenon in E. coli K-12.To demonstrate an inhibitory effect of LCP,

a normal uptake ofpeptides into cells is required,as shown by the use of mutants of strain Idefective in the oligopeptide transport systems.In principle, toxicity may be due to either theintact peptide or its cleavage compounds, liber-ated intracellularly. It does not seem likely,though, that the intact peptide exerts a toxiceffect, because the various LCP tested (12, 17;

aovoo TAVORI ET AL. J. BACTERIOL.

TOXICITY OF LCP IN E. COLI K-12 681

B

A

.

.I .*

I M LGL|C L GLC L G IC L GLIC L GL

Markers 0 60 120 2400 60 120 180 240 300 360

Time (min) Time (min)FIG. 4. Effect of GL on the growth of exponential culture of strain I with respect to the presence of the

peptide or its degradation products in the medium. (A) The growth of a control culture grown in VB (0) wascompared with that of cultures to which either 0.4 mM leucine (0) or 0.3 mM GL (A) was added at the timeindicated by the arrow; (B) shows chromatographic separation of the ninhydrin-positive compounds presentin the media of the three cultures mentioned in (A). Samples (15 1d) were withdrawn from each culture atintervals as follows: 0, 60, 120, and 240 min. Samples were separated by thin-layer chromatography withpropanol-H20 (7:3) as the running solvent and developed with ninhydrin. Markers are: (L) Leu, 0.78 pg; Gly-Leu GL, 0.86 jig; Ile (I) 0.72 jg; and (M) 15 jil of VB medium. Horizontal line at bottom indicates origin.

Table 2) are different in configuration and in therelative position of their leucines. The only com-mon element to all toxic LCP tested was thepresence of leucine. The toxicity of the LCPshould therefore be attributed to the leucinewhich is liberated from the peptide(s) inside thecells. Such a notion requires fast cleavage of theLCP intracellularly. This has been demon-strated indirectly by the utilization of LCP asthe sole source of leucine for growth of leucine-requiring strains of E. coli K-12 (2, 11, 17).Auxotrophs grew equally well with either freeleucine or LCP, when supplemented with lowequimolar concentrations. The appearance ofleucine in the medium which correlated with thedisappearance of GL (Fig. 4) required cleavageof the peptide, followed by efflux of leucine fromthe cells. These results present, therefore, a di-rect demonstration of cleavage of LCP intracel-lularly. The fast intracellular cleavage of GL issupported by the absence of even traces of thedipeptide in the bacterial pool, whereas the poolof leucine in the GL-treated culture was highcompared with the endogenous leucine found inthe control cells (Table 4).

Abnormal accumulation of leucine by E. coliK-12 was demonstrated in this study (Table 4).The internal concentration of leucine in the GL-treated culture was fivefold higher than that ofa culture incubated with free leucine at equi-molar concentration. This abnormal accumula-tion of leucine might be responsible for the ob-served toxicity of LCP and may best be ex-plained by avoidance of the natural negativecontrol of leucine on its transport into the cells.This assumption of ours is in accordance withthe findings ofQuay et al. (14) that livR mutants,which are constantly derepressed in Liv-I andLiv-II, accumulated leucine intracellularly totoxic abnormal concentrations. The temporarytoxicity of leucine, when added to cultures of E.coli K-12 grown in minimal media (15), can alsobe explained by an abnormal accumulation ofleucine, resulting from the complete derepres-sion of its transport systems known to occurunder these growth conditions. The finding thatleucine toxicity in each of the three describedsituations (introduction of LCP, introduction ofleucine to livR mutants, and introduction ofleucine to cultures grown in minimal media) is

E00Qo

:t_0

.4-

-

VOL. 146, 1981

682 TAVORI ET AL.

TABLE 4. Amino acid pools in Leu- and GL-treatedculturese

Amino Concn (nmol/10"' cells)acid Control Leu-treated GL-treated

Ala .......... 45 19.3 12.8Asp .......... 37 35.4 45.8Cys .......... 0.9

TOXICITY OF LCP IN E. COLI K-12 683

droxy acid synthase Ill isoenzyme of Escherichia coliK-12: regulation of synthesis by leucine. Biochem. Bio-phys. Res. Commun. 79:82-87.

7. De Felice, M., T. Newman, and M. Levinthal. 1978.Regulation of synthesis of the acetohydroxy acid syn-thase I isoenzyme in Escherichia coli K-12. Biochim.Biophys. Acta 541:1-8.

8. Guardiola, J., M. De Felice, T. Klopotowski, and M.laccarino. 1974. Multiplicity of isoleucine, leucine, andvaline transport systems in Escherichia coli K-12. J.Bacteriol. 117:382-392.

9. Miller, J. H. 1972. Formulas and recipes, p. 433. InExperiments in molecular genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, New York.

10. Miller, J. H. 1972. Strain list, p. 15. In Experiments inmolecular genetics. Cold Spring Harbor Laboratory,Cold Spring Harbor, New York.

11. Naider, F., and J. M. Becker. 1975. Multiplicity ofoligopeptide transport systems in Escherichia coli. J.Bacteriol. 122:1208-1215.

12. Payne, J. W., and C. Gilvarg. 1968. The role of terminalcarboxyl group in peptide transport in Escherichia coli.J. Biol. Chem. 243:335-340.

13. Payne, J. W., and C. Gilvarg. 1978. Transport of pep-tides in bacteria, p. 325-383. In B. P. Rosen (ed.),Bacterial transport. Marcel Deckker, New York.

14. Quay, S. C., T. E. Dick, and D. L. Oxender. 1977. Roleof transport systems in amino acid metabolism: leucinetoxicity and the branched chain amino acid transportsystems. J. Bacteriol. 129:1257-1265.

15. Quay, S. C., and D. L. Oxender. 1976. Regulation ofbranched chain amino acid transport in Escherichiacoli. J. Bacteriol. 127:1225-1238.

16. Rabmanian, M., D. R. Claus, and D. L Oxender. 1973.Multiplicity of leucine transport systems in Escherichiacoli K-12. J. Bacteriol. 116:1258-1266.

17. Simmonds, S., J. L Harris, and J. S. Fruton. 1951.Inhibition of bacterial growth by leucine peptides. J.Biol. Chem. 188:251-262.

18. Tully, M., and M. D. Yudkin. 1977. Fine structure map-ping and complementation analysis of the Escherichiacoli cys B gene. J. Bacteriol. 131:49-56.

19. Umbarger, H. E. 1978. Amino acid biosynthesis and itsregulation. Annu. Rev. Biochem. 47:533-606.

20. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinaseof Escherichia coli: partial purification and some prop-erties. J. Biol. Chem. 218:97-106.

21. Vonder Haar, R. A., and H. E. Umbarger. 1972. Iso-leucine and valine metabolism in Escherichia coli XIX.Inhibition of isoleucine biosynthesis by glycyl-leucine.J. Bacteriol. 112:142-147.

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