JOURNAL OF BACTERIOLOGY, June 1975, p. 994-1000Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 3Printed in U.S.A.

Separate Regulation of Transport and Biosynthesis of Leucine,Isoleucine, and Valine in Bacteria

S. C. QUAY, D. L. OXENDER,* S. TSUYUMU,1 AND H. E. UMBARGERDepartment of Biological Chemistry, 7he University of Michigan, Ann Arbor, Michigan 48104,* and

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received for publication 6 January 1975

Since both transport activity and the leucine biosynthetic enzymes are re-pressed by growth on leucine, the regulation of leucine, isoleucine, and valinebiosynthetic enzymes was examined in Escherichia coli K-12 strain E0312, aconstitutively derepressed branched-chain amino acid transport mutant, to de-termine if the transport derepression affected the biosynthetic enzymes. Neitherthe ilvB 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 derepressionor repression compared to the parental strain. A number of strains with altera-tions in the regulation of the branched-chain amino acid biosynthetic enzymeswere examined for the regulation of the shock-sensitive transport system for theseamino acids (LIV-I). When transport activity was examined in strains with mu-tations leading to derepression of the ilvB, ilvADE, and leuABCD gene clusters,the regulation of the LIV-I transport system was found to be normal. The regula-tion of transport in an E. coli strain B/r with a deletion of the entire leucine bio-synthetic operon was normal, indicating none of the gene products of this operonare required for regulation of transport. Salmonella typhimurium LT2 strainleu-500, a single-site mutation affecting both promotor-like and operator-likefunction of the leuABCD gene cluster, also had normal regulation of the LIV-Itransport system. All of the strains contained leucine-specific transport activity,which was also repressed by growth in media containing leucine, isoleucine andvaline. The concentrated shock fluids from these strains grown in minimal me-dium or with excess leucine, isoleucine, and valine were examined for proteinswith leucine-binding activity, and the levels of these proteins were found to beregulated normally. It appears that the branched-chain amino acid transport sys-tems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhi-murium strain LT2 are not regulated together by a cis-dominant type of mecha-nism, although both systems may have components in common.

The intracellular pool of the branched-chainamino acids in bacteria is increased by theaction of biosynthetic enzymes and specifictransport systems and is decreased by proteinsynthesis. The regulation of biosyntheticenzymes for leucine, isoleucine, and valine hasbeen extensively investigated by Umbarger andco-workers (20). The level of the isoleucine andvaline enzymes specified by the ilvADE genecluster is under multivalent regulation (4),whereas the leucine biosynthetic enzymes of theleuABCD operon are regulated by leucine or aderivative but not isoleucine or valine (1). Thelevels of branched-chain aminoacyl-transfer

I Present address: Faculty of Agriculture, Shizuoka Uni-versity, 836 Ohya, Shizuoka, Japan.

ribonucleic acid synthetases also have beenshown to be regulated by the intracellularlevel of the branched-chain amino acids (orby derivatives of the branched-chain aminoacids; 9-11). In addition, transport activityby a shock-sensitive leucine-isoleucine-valine(LIV-I) system and by a leucine-specifictransport system is repressed by growth inmedium containing leucine (6, 13, 15, 19).The purpose of this communication is to

determine if transport activity and biosyntheticactivity for the branched-chain amino acids areregulated in a concerted manner. Theregulation of transport and biosyntheticenzymes was examined in strains of Escherichiacoli and Salmonella typhimurium with

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mutations in either the regulation of transportor of biosynthesis.

(A preliminary report of portions of this workwas Iresented at the 65th Meeting of theAmerican Society of Biological Chemists,Minneapolis, Minn., 2-7 June 1974 [S. C. Quay,M. Rahmanian, and D. L. Oxender, Fed. Proc.33:1394, 1974].)

MATERIALS AND METHODS

Bacterial strains, media, and chemicals. The E.coli and S. typhimurium strains used in these ex-periments are listed in Table 1. All E. coli strainsexcept E. coli B/r strain ELK4 were derived fromstrain K-12, and both S. typhimurium strains werederived from strain LT2. For transport experiments or

for experiments in which binding proteins wereisolated, the cells were grown in the minimal saltsmedium E of Vogel and Bonner (21) supplementedwith 20 mM D-glucose and other additions asindicated in the text. The minimal medium used forthe determination of the levels of the biosyntheticenzymes is that described by Davis and Mingioli (3),modified as described previously (18). All chemicalswere reagent grade, purchased from commercialsources.

Preparation of cell extracts and enzyme assays.Growth of cultures and the preparation of cell extractswere described previously (22). Enzyme assays wereperformed immediately. Enzymes in the pathwaysleading to valine, isoleucine, and leucine were assayedby the following published procedures: acetohydroxyacid synthetase (AHAS; acetolactate synthetase,EC 4.1.3.18) (17); 3-isopropylmalate dehydrogenase(IPMD; 2-hydroxy-4-methyl-3-carboxyvalerate-nico-tinamide adenine dinucleotide oxidoreductase, EC1.1.1.85) (2). Protein was determined by the methodof Lowry et al. (8) using bovine serum albumin as astandard.

Assay of transport and binding activity. Fortransport assays, 10-ml cultures of bacteria growinglogarithmically were harvested by centrifugation andwashed twice with cold 10 mM potassium phosphate,pH 7.2, and 0.1 mM MgSO4. Transport activities weredetermined as described previously (15), except thatpH 7.2 was used. One unit of activity represents 1Amol of substrate taken up in 1 min. Specific activi-ties are expressed as units per gram of cell (wetweight). The concentration of cells was determinedturbidimetrically using a Klett-Summerson colorim-eter with a number 66 filter.

For the isolation of proteins with leucine-bindingactivity, 1-liter cultures were grown at 37 C inFernbach flasks to logarithmic growth phase,

TABLE 1. List of strains

Strain Genotype Relevant distinctive Source or referencescharacteristics

E. coliE0301 leu,trp Parent of E0300 series R. L. SomervilleE0303 trp From strain E0301 by

transductionE0311 leu,trp Constitutively derepressed From strain E0301 by

branched-chain amino acid selection for D-leucinetransport and branched-chain, utilization; (16)amino acid-binding proteins

E0312 trp From strain E0311 bytransduction

SP31 glyA,proC Parent of strains.CU86, CU87, CU88 R. L. Somerville; (14)CU5 met Parent of CU5000 series Purdue culture collectionCU5001 met,azIA2,az1B4,azl-6 Constitutively repressed ilvADE; S. Dwyer; (14)

constitutively derepressed ilvBand leuABCD

CU5002 met,azlA1,azlB3,az1-5 Constitutively derepressed ilvB, S. Dwyer; (14)ilvADE, and leuABCD

CU86 proC,azlAl Low-level azlr; non-excreters of By transduction ofSP31leucine with CU5002; (14)

CU87 proC,azlB3 High-level azlr; non-excreters of By transduction of SP31leucine with CU5002; (14)

CU88 glyA,proC,azl-5 Highly derepressed and heavy By transduction of SP31leucine excreter with CU5002; (14)

ELK4 pyrAl,ara-leullOl,str Deletion mutant from araABOC E. L. Kline; (23)and including leuDCBAOgenes

S. typhimuriumLT2 Wild type Laboratory stockleu-500 leu-500 Mutation affecting both pro- Graf and Burns (5)

moter- and operator-like func-tions of the leuABCD operon

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harvested, and subjected to cold osmotic shockaccording to the procedure of Neu and Heppel (12).The shock fluid was concentrated approximately10-fold by ultrafiltration with an Amicon PM10membrane and then dialyzed in the cold with 10 mMphosphate, pH 7.2, and 0.1 mM MgSO4. Bindingactivities were measured by equilibrium dialysis (15)and are expressed as micromoles of leucine bound per

gram of protein. The protein determinations were

made using the method of Lowry et al. (8).

RESULTS

Regulation of biosynthetic enzymes instrains derepressed for transport. Theregulation of leucine, isoleucine, and valinebiosynthetic enzymes was examined in strainE0312 and its parental strain, E0303. The ilvBgene product, AHAS, and the leuB geneproduct, IPMD, were used to estimate the levelof derepression of the isoleucine and valine andof the leucine biosynthetic pathways, re-spectively. Strain E0312 has been shown pre-viously to have a two- to threefold increaseover that of its parent in transport of thebranched-chain amino acids by a

shock-sensitive transport system (LIV-I) and acorresponding increase in branched-chain,amino acid-binding protein activity (15, 16).Furthermore, these increased levels of transportand binding activities in strain E0312 are notrepressed by growth in media containing leucineas they are in the parental strain E0303. Theactivities of AHAS and IPMD in strains E0312and E0303 and their Leu- derivatives are givenin Table 2. Strains E0303 and E0312 show verysimilar enzyme activities when grown inminimal medium. Furthermore, both strainsshow significant repression of branched-chainamino acid biosynthetic enzymes when grownon leucine, isoleucine, and valine. The Leu-derivatives of these strains permitted theexamination of derepression under leucinelimitation. With excess branched-chain aminoacids, strain E0301 has repressed levels ofAHAS and IPMD which derepress nearly10-fold and three-fold, respectively, whenleucine is limiting in batch cultures with excess

valine and isoleucine. Under similar ex-

perimental conditions, however, strain E0311shows a modest derepression of AHAS activityand no change in the IPMD level. These dataare consistent with those obtained with theleucine prototrophic strains, if we infer that theincreased efficiency of transport in strain E0311has delayed the onset of the "leucine-limiting"signal during the latter experiments. Onexamination of Fig. 1, which represents thegrowth curves of strains E0301 and E0311 on

excess or on limiting leucine, one can see that

TABLE 2. Regulation of branched-chain amino acidbiosynthetic enzymes in strains derepressed for

branched-chain amino acid transport

EnzymeStrain Growth activitiesb

conditionsaAHAS IPMD

E0303 MM 0.085 0.019MM + supplement 0.035 0.006

E0312 MM 0.067 0.014MM + supplement 0.008 0.004

E0301 MM + supplement 0.046 0.007Limiting leucine 0.457 0.020

(batch)Limiting leucine 0.130 0.010

(chemostat)

E0311 MM + supplement 0.014 0.006Limiting leucine 0.042 0.006

(batch)Limiting leucine 0.053 0.009

(chemostat)

a Growth was on minimal salts medium with 0.4%glucose and 50 ug of tryptophan per ml. Supplementwas 0.4 mM leucine, 0.4 mM isoleucine, and 1 mMvaline. Limiting leucine for batch cultures was 0.02mM. The chemostat growth was maintained for 22 to24 h. MM, Minimal medium.

° Enzyme activities are expressed as micromoles ofproduct produced per minute per milligram of pro-tein.

the period of time during which strain E0301demonstrates a leucine-limiting change ingrowth rate is much longer than in strainE0311. Strain E0311 grows at an unlimitedrate, regardless of the presence of isoleucine andvaline, until the end of growth when theconcentration of leucine is near depletion. Thus,the period of the leucine-limiting signal instrain E0311 was very short indeed.When strain E0311 was grown on limiting

leucine in a chemostat, the derepression ofIPMD was equal to that in strain E0301 (Table2). In addition strain E0311 showed a slightlygreater derepression of AHAS activity in thechemostat than was shown during growth in abatch culture.Regulation of transport activity in strains

altered in the regulation of biosynthesis. Thetransport of 1.5 ,M L-leucine in various strainswith mutations in the regulation of isoleucineand valine and of leucine biosynthesis wasexamined in strains grown in minimal mediumand in the same medium supplemented withexcess leucine, isoleucine, and valine. StrainCU5001, obtained from strain CU5 by selec-tion for DL-4-azaleucine resistance (Azlr), is

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04 08 10 4 04 08L0 120 140

0.4-<

0.3 01r--

0 . 2

20 40 60 80 100 120 140 20 40 60 80 120 140

MINUTESFIG. 1. Growth curves of strains E0301 and E0311. Cells were grown on glucose-basal salts medium with 1mM

isoleucine, 0.8mM valine, and either 0.4 mM leucine (a) or 0.02mM leucine (0). Cell density was estimated bymeasuring absorbance at 660 nm in a Gilford model 240 spectrophotometer with a 1-cm lightpath. (A) strainE0311; (B) strain E0301.

constitutively derepressed for ilvB and theleucine biosynthetic enzymes (leuABCD). Inthis strain (CU5001) the ilvADE operon isconstitutively repressed. Strain CU5002contains a derepression pattern similar to thatfound in strain CU5001 with respect to ilvB andleuABCD, but is also derepressed in ilvADE(14; S. Dwyer, Ph.D. thesis, Purdue University,Lafayette, Ind., 1969). At least three mutationshave been identified with these phenotypes.Two loci, designated azlA (controlling low-levelazaleucine resistance) and aziB (controllinghigh-level resistance), have been found to beco-transducible with the nadB-glyA region ofthe E. coli chromosome (14). Strains withmutations in these loci do not exhibit excretionof leucine as observed in strains CU5001 andCU5002. A third locus, azl-5, was identified bydirect selection of azaleucine resistance in P1transduction from strain CU5002. The azl-5mutation (strain CU88) gives rise to highlyderepressed biosynthetic enzymes for leucineand correspondingly heavy leucine excretionexhibited by the parental strain CU5002. StrainSP31 (glyA,proC) was the recipient for all threeclasses of mutations obtained by transductionfrom strain CU5002 (14). The transport ofleucine in the two parental strains, CU5 andSP31, the azl' mutants, and their transductantsis given in Table 3. The parental strains SP31and CU5 have similar leucine transport activitywhen grown on minimal medium, and thisactivity is repressed when the strains are grownon leucine, isoleucine, and valine (Table 3).Strains CU5001 and CU5002 have transportactivities similar to their parental strain CU5.Two of the azaleucine-resistant loci from strainCU5002, azlA (CU86) and aziB (CU87), appearnot to be involved in the regulation of transportactivity. Strain CU88, which contains the thirdazlr mutation (azl-5), is highly derepressed for

TABLE 3. Regulation of branched-chain amino acidtransport systems in strains with mutations in theregulation of branched-chain amino acid biosynthesis

Total leucine Leucine-specificuptakea uptakeb

Strain Mini-Minimal Supple- mal Supple-mediumc mentedd me- mented

dium

CU5 0.68 0.24 0.07 0.01CU5001 0.68 0.24 0.06 0.01CU5002 0.81 0.26 0.05 0.02SP31 0.40 0.17CU86 0.47 0.25 0.04 <0.01CU87 0.48 0.24 0.04 <0.01CU88 0.18 0.12 0.04 0.01ELK4 0.85, t 0.15 0.07' 0.02LT2 0.41 (0.30') 0.17 0.02 <0.01leu-500 0.33' 0.16 0.02' <0.01

a Uptake assay contained 1.5 MM L-leucine. Resultsare the average of two to four separate experiments.Specific activity is expressed as micromoles of leucinetaken up per minute per gram of cells (wet weight).

Leucine-specific uptake was estimated by sub-tracting 0.75MgM leucine uptake with 50MM leucine asinhibitor, from 0.75 MM leucine uptake with 50 AMisoleucine as inhibitor.

c Minimal medium consisted of Vogel-Bonner salts,20 mM D-glucose, 0.2 mM proline, 0.3 mM glycine,and 0.07 mM methionine.

d Supplement was 0.4 mM leucine, 0.4 mM isoleu-cine, and 1 mM valine.

e Minimal medium consisted of Vogel-Bonner salts,20 mM 1-glucose, 0.2 mM arginine, and 0.4 mMuracil.

' Minimal medium included 10 MM L-leucine.

biosynthesis. This strain would be expected toshow low transport activity even in minimalmedium if the pool of leucine and othertransport repressors was increased as a result ofthis mutation. It is interesting that strain

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CU5002 with the azl-5 mutation in the presenceof azlA and azlB does not have the sametransport phenotype that is seen in strainCU88, which is a transductant with only theazl-5 mutation transferred to the SP31 strainbackground.Methionine has been shown to repress leu-

cine, isoleucine, and valine transport (6, 19).The Met- phenotype of strains CU5, CU5001,and CU5002 required growth of the strains onmethionine. Preliminary experiments were doneby transducing these strains to methionineindependence and testing the level of repressioncaused by the low level of methionine used forthese experiments. It was shown that 0.07 mMmethionine did not significantly repress leucinetransport.

Strain ELK4 contains an ara-leu deletionthat starts in the araA gene and includesaraABOC and leuDCBAO and the regionbetween these gene clusters (23). This strainpermitted the study of the regulation oftransport in the absence of gene products of theleucine biosynthetic operon. The activity oftransport appears normal in this strain grown inminimal medium, and the transport activityappears to be repressed normally by growth inmedium containing leucine, isoleucine, andvaline (Table 3).Leucine transport activity and its regulation

were next examined in S. typhimurium strainleu-500 and its parent, LT2. Graf and Burns (5)have shown that the leucine auxotrophy of S.typhimurium strain leu-500 is the result of apresumptive single-site mutation affecting bothpromotor-like and operator-like functions of theleucine operon. If proteins involved in transportin Salmonella are under the control of one of thegenes of the leucine operon, this mutation, likethe ara-leu deletion, should result in a change inthe regulation of transport activity. However,the leu-500 mutation results in neither a changein the level of transport activity nor a change inits regulation. Table 3 includes transport ac-tivity in wild-type strain LT2 grown both inminimal medium and in the leucine-containingminimal medium required for strain leu-500. Allof the above transport experiments were alsoperformed with 1.5 MM L-isoleucine; the resultswere qualitatively identical to those found forL-leucine.

Since excess leucine represses the leu-cine-specific system (15) as well as the LIV-Itransport system, the presence and regulation ofthe leucine-specific system were examined inthese strains. The results of experiments tomeasure the leucine-specific transport activityare included in Table 3. These experiments

indicate that the leucine-specific transportsystem is present in all strains and that thisactivity is repressed by growth in mediumcontaining leucine, isoleucine, and valine in theparental strains and in the strains withmutations involving branched-chain amino acidbiosynthesis.Regulation of branched-chain, amino

acid-binding proteins in strains altered inthe regulation of biosynthesis. A role for os-motic shock-sensitive, branched-chain, aminoacid-binding proteins in branched-chain aminoacid transport has been inferred from ki-netic, genetic, and biochemical studies (13,15, 16). Proteins with leucine-binding activitywere obtained by osmotic shock from the strainswith the azi mutations when the strains weregrown on minimal medium and on minimalmedium supplemented with leucine, isoleucine,and valine (Table 4). Strains CU5001 andCU5002 have leucine-binding activity which isnearly equal to the parental strain, CU5, andthis activity is repressed to the same extent bygrowth on excess leucine, isoleucine, and valine(Table 4). When the azlr loci are examinedindividually, it appears that the azlA and azlBmutations have very little effect on theregulation of proteins with leucine-bindingactivity, as was observed earlier for transportactivity. The leucine-binding proteins in strainCU88 (azl-5), like the transport systems, were

TABLE 4. Regulation of branched-chain, aminoacid-binding proteins in strains with mutations in theregulation of branched-chain amino acid biosynthesis

CU5CU5001CU5002CU86CU87CU88LT2leu-500

Wild typeazlA2,azlB4,azl-6azlA1,azlB3,azl-5azlAlazlB3azl-5Wild typeleu-500

Leucine-bindingactivitya

Minimal Supple-me- mented

diumb

0.690.620.710.760.630.260.690.53c

0.330.260.400.370.17

<0.010.360.42

aProteins with leucine-binding activity were ob-tained by osmotic shock as described in Materials andMethods. Leucine-binding activity was determined asdescribed in Materials and Methods and was assayedat 10 gM leucine and 4 C. The specific activity isexpressed as micromoles of leucine bound per gram ofprotein.

" Minimal medium and supplements were the sameas in Table 3.

c Minimal medium included 10 uM L-leucine.

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partially repressed in minimal medium andwere undetectable in strain CU88 grown underrepressing conditions. The leu-500 mutation inS. typhimurium did not affect the regulation ofleucine-binding activity when compared to theparental strain LT2. The close correspondenceof total leucine uptake activity from Table 3with the leucine-binding activity from Table 4is consistent with a role of osmoticshock-sensitive, leucine-binding proteins inbranched-chain amino acid transport.

DISCUSSIONWith'the observation that exogenous leucine

represses both leucine biosynthesis andbranched-chain amino acid transport, itbecomes of interest to determine the extent towhich these activities are regulated together.Since the chromosomal location of all structuraland regulatory loci responsible for leucine,isoleucine, and valine transport activity is notyet known (7), one possible model for concertedregulation involves placing one or more

transport genes either within the biosyntheticoperons or under the control of the "cis-domi-nant" regulatory regions which affect expres-

sion of the structural genes for the leucinebiosynthetic enzymes. This model wouldpredict that under conditions of derepression ofthe ilv or leu operon transport activity wouldalso be derepressed. The finding that E. coliK-12 strain CU5002, which is derepressed forthe leuABCD, ilvADE, and ilvB gene clusters,and E. coli B/r strain EKL4, which has a

complete deletion of the leu biosyntheticoperon, have normal regulation of transporttends to indicate this hypothesis is untenable.The normal branched-chain amino acid trans-port activity and regulation in S. typhimuriumLT2 strain leu-500 supports this conclusion.The transport regulation pattern in strains

ELK4 and leu-500 suggests that no geneproduct of the leucine operon is required foreither repression or derepression of leucine,isoleucine, and valine transport systems. Thenormal regulation of branched-chain aminoacid transport systems in E. coli K-12 strainCU372 (gal-, ilvDAC115, leu-455) with a

deletion of the entire ilvA gene and the absenceof complete ilvD and ilvC structural genes

indicates that this portion of the ilv operon isnot involved in the regulation of transport (S. C.Quay and D. L. Oxender, unpublished data).The observation that the mutations in strainsCU86 and CU87, which are unlinked to eitherthe ilv or leu operons, cause strong derepressionof biosynthesis in the absence of alterations inthe regulation of transport is also significant.

These results suggest that the regulation oftransport and the regulation of the biosyntheticenzymes are at least partially separate withrespect to what must reasonably be considered adiffusible ("trans-dominant") regulatoryelement.The lowered transport activity and leu-

cine-binding activity of strain CU88 (highlyderepressed for biosynthesis) even in minimalmedium, taken together with the finding thatexogenous leucine represses both transport andbiosynthesis, suggests that some component(s)(leucine or a derivative) may be common to thetwo regulatory systems. The opposite effects ofthe azl-5 mutation in strain CU88 on the level ofexpression of the leucine operon and thetransport system could then be a pleiotrophiceffect explicable solely in terms of excretion ofleucine; i.e., derepression of leuABCD causedby azl-5 and the concomitant accumulation ofleucine could result in lower levels of transportactivity. These results could also be explainedin terms of a direct effect of azl-5 on the exit ofleucine from the cell, causing a derepression ofthe leuABCD operon from the loss of intra-cellular leucine.The close parallel between the level of

binding activities for leucine and the observedlevel of transport activity in a variety of strainsis consistent with earlier claims that thebinding proteins play a role in transport (13, 15,16) and also suggests that the binding proteinsare the limiting component in the transportprocess. From the close relationship betweentransport and binding proteins, one wouldexpect to find transport mutations that resultfrom alterations in the level or the properties ofthe binding proteins. Strain E0311 is aD-leucine-utilizing mutant that has increasedtransport activity resulting from derepressedbinding protein activity (16).The observation that strain E0311 was able

to grow to a higher density on limiting leucinethan its parent, strain E0301, is a good exampleof the selective advantage a derepression intransport activity can provide an auxotrophicstrain. Whether the increased transport abilityresults in an increased branched-chain aminoacid pool in these auxotrophic strains orwhether it simply maintains the level of the poolamino acids for a longer period of time cannotbe determined by these studies. The increasedtransport activity of strain E0311 when placedin a Leu+ background does make it more sensi-tive to toxic analogues of the branched-chainamino acids such as trifluoroleucine and azaleu-cine. The increased toxicity has provided a con-venient basis for selecting a large variety of

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analogue-resistant mutants that have defects inthe transport system involving the branched-chain, amino acid-binding proteins (Quay et al.,Fed. Proc. 33:1394, 1974).

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

grants GM11024 to D.L.O. and GM12522 to H.E.U. from theNational Institute of General Medical Sciences.We thank E. L. Kline for providing us with E. coli B/r

strain ELK4.

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1966. Expression of the leucine operon. J. Bacteriol.91:1570-1576.

2. Burns, R. O., H. E. Umbarger, and S. R. Gross. 1963. Thebiosynthesis of leucine. III. The conversion ofa-hydroxy-,-carboxy-isocaproate to a-ketoisocaproate.Biochemistry 2:1053-1058.

3. Davis, B. D., and E. S. Mingioli. 1950. Mutants ofEscherichia coli requiring methionine or vitamin B,,. J.Bacteriol. 60:17-28.

4. Freundlich, M., R. 0. Bums, and H. E. Umbarger. 1962.Control of isoleucine, valine and leucine biosynthesis. I.Multivalent repression. Proc. Natl. Acad. Sci. U.S.A.48:1804-1808.

5. Graf, L. H., Jr., and R. 0. Burns. 1973. The supX/leu-500mutations and expression of the leucine operon. Mol.Gen. Genet. 126:291-301.

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

7. Guardiola, J., M. DeFelice, T. Klopotowski, and M.Iaccarino. 1974. Mutations affecting the differenttransport systems for isoleucine, leucine, and valine inEscherichia coli K-12. J. Bacteriol. 117:393-405.

8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

9. McGinnis, E., and L. S. Williams. 1971. Regulation ofsynthesis of the aminoacyl-transfer ribonucleic acidsynthetases for the branched-chain amino acids inEscherichia coli. J. Bacteriol. 108:254-262.

10. McGinnis, E., A. C. Williams, and L. S. Williams. 1974.Derepression of synthesis of the aminoacyl-transferribonucleic acid synthetases for the branched-chain

amino acids of Escherichia coli. J. Bacteriol.119:554-559.

11. Nass, G., and F. C. Neidhardt. 1967. Regulation offormation of aminoacyl t-ribonucleic acid synthetasesin Escherichia coli. Biochim. Biophys. Acta134:347-359.

12. Neu, H. C., and L. A. Heppel. 1965. The release ofenzymes from Escherichia coli by osmotic shock andduring the formation of spheroplasts. J. Biol. Chem.240:3685-3692.

13. Penrose, W. R., G. E. Nichoalds, J. R. Piperno, and D. L.Oxender. 1968. Purification and properties of aleucine-binding protein from Escherichia coli. J. Biol.Chem. 243:5921-5928.

14. Pledger, W. J., and H. E. Umbarger. 1973. Isoleucine andvaline metabolism in Escherichia coli. XXI. Mutationsaffecting derepression and valine resistance. J.Bacteriol. 114:183-194.

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