MICROBIOLOGICAL REVIEWS, Mar. 1979, p. 42-58 Vol. 43, No. 1

014&0749/79/03-0042/17$02.00/0

Growth Inhibition as a Consequence of Antagonism BetweenRelated Amino Acids: Effect of Valine in Escherichia coli

K-12MAURILIO DE FELICE,'* MARK LEVINTHAL,2 MAURIZIO IACCARINO,' AND JOHN GUARDIOLA

International Institute of Genetics and Biophysics, National Research Council, 80125 Naples, Italy,l* andDepartment of Biological Sciences, Purdue University, West Lafayette, Indiana 47907'

INRTRODUCTION ....................................... 42MCECHANISM OF VALINE INHIBIMTON ..................... ......... 43SITE OF INHIBITION: PRODUCTION OF THlE ACETOHYDROXY ACIDS .... 45Acetohydroxy Acid Synthase (AHAS) Isoenzymes and the Problem of ilvGGene Expression .4.................................6..45

.Physiological Role of the AHAS I and AHAS HI Isoenzymes ... 47VALNE-RESISTANT MUTANTS. 48TransportMutants ......... .......... .. 49Mutants Altered in the Inhibitory Process .... 51(i) Structural modifications ofAHAS I 6.........51(ii)- Regulatory mutations affecting AHAS I 51(iii) Modifications of AHAS III.1....51(iv) Regulatory mutations promoting the expression of AHAS II.... 51(v) Other mutations.6...51

CONCLUSIONS 52I T17ERATURE CI D .. . 6.............. ... .... ..... ... 54

INTRODUCTION

Growth of a number of microorganisms isinhibited in minimal medium containing somenatural amino acids; such inhibition is usuallyreversed by other amino acids. Gladstone (42)found that in Bacillus anthracis either valine orleucine alone inhibits growth, but no inhibitionis observed when both are present. He alsoshowed that an analogous, though less marked,relationship exists between serine and threonine.Similar observations were reported in the sub-sequent years by several authors. Tatum (103)showed that Escherichia coli K-12 fails to growin minimal medium containing valine and thatisoleucine counters this inhibition. Beerstecherand Shive (9) reported that growth of an E. colistrain is inhibited by tyrosine and that phenyl-alanine reverses this effect. Antagonistic phe-nomena among amino acids were also noticed instudies with B. subtilis (93), Streptococcus bovis(114), and some lactic acid bacteria (15, 67). Acomprehensive formal analysis ofthese phenom-ena was performed by Rowley (94, 95) on a greatnumber of E. coli strains. In Table 1 we showthe pattern of amino acid inhibition, and inTable 2 we show a summary of the examples ofcomplete prevention of amino acid inhibition byother amino acids described by Rowley.More recent reports demonstrate that reA

mutants of E. coli, in experiments of downshiftfrom nutrient to minimal medium, are more

sensitive than wild-type strains to growth inhi-bition by amino acids (3, 4, 111, 112). This is atleast in part a consequence of the impairedexpression of biosynthetic enzymes in relA mu-tants (38, 40, 70, 99, 100, 119).Growth inhibition by natural amino acids is

not limited to procaryotes: it has been observedalso for a wide range of plants and plant tissuesand cells in culture (la, 10, 13, 14, 17, 51, 68, 96).In many of these cases reversion of the inhibitionby other amino acids has been reported. Forexample, valine inhibits growth of barley em-bryos (68) and tobacco in vitro cultures of tissue,cells, and protoplasts (13, 14, 96); as in E. coliK-12, in these plant materials the effect is re-versed by isoleucine.These findings show that growth inhibition by

amino acids may occur as a physiological con-sequence of a competition at the level of eitherbiosynthesis or utilization among amino acidshaving structural, biosynthetic, and regulatoryrelationships.Among the numerous cases observed, only the

effect of valine in E. coli K-12 has been studiedin detail, probably because this inhibition issevere, occurs in the most commonly studiedmicroorganisms, and makes possible the isola-tion of valine-resistant (Val') mutants, whichprovides a precious tool for genetic studies onseveral metabolic processes. The aim of thisarticle is to analyze the extensive literature ex-isting on this subject, which has never been

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GROWTH INHIBITION BY NATURAL AMINO ACIDS

TABLE 1. Growth inhibition by amino acids among356 E. coli strainsa

No. ofAmino acid tested strains

inhibited

a-Aminobutyric acid 4.................4Aspartic acid .......................... 13Cystine 40Histidine ....... .................. 2Lysine ............... .......... 3Methionine ........................... 4Norleucine ................. 333Norvaline............................. 261Serine.............................. 42Threonine ........................ 2Tyrosine........... 2Valine................................ 3

aAdapted from reference 94.

TABLE 2. Prevention ofamino acid growthinhibition by other amino acids in various E. coli

strains'No. ofstrains Inhibition by: Prevented by:tested

1 Aspartic acid Lysine1 Aspartic acid Valine8 Cystine Methionine3 Lysine Methionine1 Methionine Leucine or lysine

12 Norleucine Methionine5 Norvaline Leucine or methionine4 Serine Glycine1 Valine Isoleucine or leucine1 Valine Isoleucine or leucine or

methionine

nAdapted from reference 94.

reviewed in this framework. We intend to showthat the work on the phenomenon of valineinhibition has made possible the elucidation ofa typical process of antagonism between physi-ologically related metabolites. We also wish toemphasize that this work has substantially con-tributed to the understanding of active transportand regulation of biosynthesis ofbranched-chainamino acids, which represents, for reasons whichwill be focused on in this article, a unique modelsystem in microbial molecular biology. From theideas emerging from these studies we will derive,in Conclusions, a general interpretation ofgrowth inhibition by natural amino acids.

MECHANISM OF VALINE INHIBMONIn 1946 Tatum (103) observed that valine and

its keto acid precursor a-ketoisovaleric acid in-hibit the growth of E. coli K-12 and that isoleu-cine counters the inhibition. This effect was

confirmed and further investigated by Bonner

(12). Rowley (94) reported that growth restric-tion of E. coli K-12 by valine is bacteriostatic. Afurther investigation was accomplished byGlover in 1962 (43). This author reported that,whereas E. coli B is insensitive to, valine (up to10 mg/mi), E. coli K-12 is inhibited-at anyconcentration above 0.4,tg/ml. He also observedthat leucine (20 ,ug/ml) does not affect the re-sponse to valine (40 ,ug/ml), whereas isoleucineantagonizes the inhibition when it is present atapproximately 3% of the valine concentration.Finally, Glover reported that reversion can alsobe accomplished by some isoleucine precursorsbut not by some valine precursors.The ability of isoleucine and some of its pre-

cursors to antagonize the growth inhibition ex-erted by valine led the early investigators tobelieve that the effect of valine is to restrict thebiosynthesis of isoleucine.Adelberg and Umbarger (2) showed that the

presence of valine restricts the accumulation ofa-ketoisovalerate in a strain which is unable totransforn this intermediate into valine. There-fore, they concluded that valine inhibits its ownbiosynthesis. These results stimulated the ideathat growth inhibition occurs at the level of abiosynthetic reaction common to both isoleucineand valine, and Umbarger and Brown (107) pro-posed that the common enzyme inhibited byvaline might be isomeroreductase (EC 1.1.1.86).A few years later, more insight was gained intothe details of the biosynthesis of isoleucine andvaline (Fig. 1). Umbarger and Brown (108) stud-ied the formation of the first five-carbon precur-sor of valine, a-acetolactate, and found that thissynthesis is inhibited by valine. A short timelater, in the same laboratory, a microbiologicalassay for the first six-carbon precursor of isoleu-cine, a-aceto-a-hydroxybutyrate, was devised(58, 59). The availability of this method made itpossible to perform a comparative analysis ofthe synthesis of the acetohydroxy acid precur-sors of isoleucine and valine. Leavitt and Um-barger (59, 60) found that (i) the syntheses ofa-acetolactate and a-aceto-a-hydroxybutyrateare characterized, in vitro, by the same kineticparameters and by the same patterns of sensitiv-ity (in E. coli K-12) or resistance (in E. coli Wand in a valine-resistant E. coli K-12 mutant) tovaline inhibition; (ii) the rate of a-acetolactatesynthesis is reduced when the reaction mixturecontains, in addition to pyruvate, a-ketobutyr-ate, and therefore a-aceto-a-hydroxybutyrate isalso produced; (iii) the intracellular concentra-tion of valine is higher in E. coliW and in a Val'E. coli K-12 mutant than in wild-type E. coli K-12, whereas there is no difference in valine up-take by the two organisms. From these data, it

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44 DE FELICE ET AL.

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GROWTH INHIBITION BY NATURAL AMINO ACIDS 45

was possible to conclude that the synthesis ofthe two acetohydroxy acids is catalyzed by a

common enzyme activity, that this activity isthe target of valine inhibition, and that valineregulates its intracellular pool through feedbackcontrol of the first step of its own biosynthesis.The work of several investigators has confirmedthe findings of Umbarger and his collaboratorsand has substantiated their model later (28, 33,36, 39, 82, 90, 105).Cohen (20) reported that in E. coli K-12 ex-

ogenous valine is incorporated into proteins inthe place of other amino acids, thus producingfaulty proteins. Further investigation (105) sug-gests that this phenomenon does not account forthe growth inhibition. Other authors have shown(73) that valine inhibits the synthesis of isoleu-cyl-transfer ribonucleic acid (tRNA) by forminga complex with isoleucyl-tRNA synthetase. Valrmutants altered in isoleucyl-tRNA synthetasehave not been described, which indicates thatthis effect is not the primary cause of valine

inhibition. However, we cannot exclude thatthese phenomena might contribute to the sever-

ity of valine action. Also, the a-ketoisovalerateinhibition of isoleucine synthesis from a-keto-fl-methylvalerate (12, 104; see Fig. 1) could con-

tribute to valine inhibition.At present, the idea that valine inhibition is

mainly the consequence of a block of a-aceto-a-hydroxybutyrate synthesis is commonly ac-

cepted. However, the biochemical details of themechanism of inhibition still need to be clarified.A biochemical assay is available for the synthesisof a-acetolactate (101), whereas a-aceto-a-hy-droxybutyrate synthesis is only studied bymeans of a microbiological method (58, 59). Forthis reason, most of the work on acetohydroxyacid synthase (AHAS; EC 4.1.3.18) activity hasbeen performed on the first of these two reac-

tions, and no evidence has been presented as towhether essential differences exist in the kineticsof the two reactions. Furthermore, the totalAHAS activity is composed of isoenzymes (seebelow), which are difficult to separate physicallyand to purify, and, therefore, most biochemicalstudies have been performed on crude extracts.Relevant information is mainly based on geneticmanipulations.

SITE OF INHIBITION: PRODUCTION OFTHE ACETOHYDROXY ACIDS

The biosynthesis of isoleucine, leucine, andvaline (Fig. 1) occurs through a branched path-way in which three steps are common to eachend product. Mutants lacking threonine deami-nase (EC 4.2.1.16) or isoleucine transaminaseactivity are auxotrophic for isoleucine (Ile-).

Mutants lacking valine transaminase activityshould be auxotrophic only for valine, but theirgrowth also requires isoleucine and leucine, be-cause of the block of acetohydroxy acid synthesisdetermined by valine inhibition of the totalAHAS activity. Mutants lacking isomeroreduc-tase and dehydrase (EC 4.2.1.9) activity are aux-otrophic for both isoleucine and valine (Ilv-).The presence of leucine is not required forgrowth of such strains, because a-ketoisovaler-ate, the first leucine precursor, is produced bytransamination of valine. Mutants lackingAHAS activity are, for the same reason, auxo-trophic for isoleucine and valine.The AHAS reactions represent the first bio-

synthetic step common to the three products.They are catalyzed by isoenzymes and are thetarget of end-product inhibition by valine. Thesum of these properties determines a uniquepattem, which gives to this step a central role inthe carbon flow through the whole pathway.The biochemical features of the synthesis of

the acetohydroxy acid precursors of isoleucine,leucine, and valine in E. coli K-12 have beenstudied by Umbarger and his collaborators, aswell as by other investigators (58-60, 88 [andreferences quoted therein], 108, 109). The reac-tions consist of a condensation of active acetal-dehyde (derived from pyruvate) with either a-ketobutyrate or pyruvate to yield a-aceto-a-hy-droxybutyrate (a precursor of isoleucine) or a-acetolactate (a precursor of leucine and valine),respectively (Fig. 1).

In the last few years, an extensive genetic andbiochemical analysis of the properties of thisstep, mainly perforned in our laboratories, hasprovided the possibility of uncovering essentialbiological features of the phenomenon of valineinhibition and of gaining some insight into ilvregulation. In this section, we will review thisinformation.

AHAS Isoenzymes and the Problem of ilvGGene Expression

The property of E. coli K-12 of sensitivity togrowth inhibition by valine is unique among thecommonly studied Enterobacteriaceae; this isdue to the severity of valine inhibition on thetotal AHAS activity in E. coli K-12 as comparedwith other organisms (60).Work from different laboratories showed that

in Salmonella typhimurium and E. coliW thereare two AHAS activities, one sensitive (Val8)and one resistant to valine, the latter beingabsent in E. coli K-12 (11, 36, 74). It was there-fore suggested that the Val8 phenotype of E. coliK-12 is due to the absence of the Valr activity.Recently, it has been shown that three distinct

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46 DE FELICE ET' AL.

AHAS activities, one of which is Valr, can beseparated chromatographically from E. coli 9723extracts (117). An analysis in E. coli K-12 (26,28, 33, 36, 46, 49) has demonstrated that thisstrain contains the structural genes for threeAHAS activities, designated AHAS I, AHAS II,and AHAS III isoenzymes (see Fig. 1). AHAS Iand AHAS III are both inhibited by valine (49),whereas AHAS II is resistant to it (36). However,AHAS II is not expressed (33) except in thepresence of cis-dominant mutations in either theilvO or the ilvR locus (Fig. 1). Such mutants,because of the expression of AHAS II, are insen-sitive to growth inhibition by valine (36; C.Squires, M. Levinthal, M. Levinthal, P. Abres-cia, and M. De Felice, submitted for publica-tion). Thus, the peculiar sensitivity of E. coli K-12 to valine inhibition is a consequence of aspecific regulatory pattern ofthe ilv gene expres-sion, resulting in lack of expression of the ilvGgene.An analysis of the properties of the AHAS

isoenzymes in extracts of E. coli K-12 strains,each containing one isoenzyme, has been per-formed (32; Squires et al., submitted for publi-cation). The data summarized in Table 3 showthat the three enzymes are significantly differentin many respects.The ilvB gene codes for a polypeptide re-

quired for AHAS I activity. A mutation in thisgene causes lack of AHAS I activity (32, 49) andconfers an Ilv- phenotype in an ilvH ilvI back-ground (46). A temperature-sensitive mutationin ilvB causes the appearance of a temperature-sensitive AHAS I activity and confers a temper-ature-sensitive Ilv- phenotype in an ilvH ilvIbackground (49). The location of ilvB on the E.coli K-12 linkage map reported in Fig. 1 is basedon genetic evidence existing in the literature (46,49, 92). However, more recent results suggest

TABLE 3. Comparison of the biochemical propertiesof the AHAS isoenzymes of E. coli K-12a

Affinity (K., iLM):Inhibition by

Affnit(K,, M): valine(%Enzyme Flavin ad- pH op-timum

Pyruvate enine di- 50 AM 1.5 mMyua nucleo-

tide

AHAS I 1,300 0.16 10 85 7.3AHAS II 2,900 30 -0 -0 8.6AHAS III 7,600 -0 70 85 8.9

e The data reported are summarized from reference 32 andfrom Squires et al., (submitted for publication). They derivefrom studies on crude extracts of cells of various strains, eachcontaining one isoenzyme. For AHAS II, we report valuesderived from experiments in which an ilv0603 strain was used.These values were identical to those obtained with an ilvR264strain.

that ilvB is located at 81 min betweenpyrE andbgl (T. Newman and M. Levinthal, unpublisheddata). No other genes in addition to ilvB areknown to code for a polypeptide involved inAHAS I activity. Expression of the ilvB gene isunder the control of a cis-dominant regulatoryelement, iluP (91, 92), closely linked to it (Fig.1).AHAS III is the product of two structural

genes, ilvH and ilvI, both closely linked to theleucine operon (85% cotransduction frequency).The ilvI gene product is a polypeptide subunitresponsible for the catalytic activity of AHASIII: amber and temperature-sensitive mutationsin this gene have been studied (26). The ilvHgene product is a polypeptide subunit distinctfrom the ilvI product and responsible for thevaline sensitivity of AHAS III, as suggested bystudies on ilvH missense and amber mutations(28). Evidence that the itvH and ilvI gene prod-ucts are distinct polypeptides derives from an invitro complementation analysis (26). The detailsof the interaction between the two subunits areunknown. Recessive mutations in a nonadjacentlocus cause derepression of AHAS III (M. V.Ursini and M. De Felice, unpublished data).The hypothesis that major regulatory rela-

tionships occur between ilvH ilvI and the leuoperon in E. coli K-12 has been considered inthe past. Recent data perrnit us to rule out thispossibility. In fact, the two gene clusters areseparated by a minimum of 1,000 base pairs ofunrelated deoxyribonucleic acid and are tran-scribed from opposite strands (S. Wessler, M.De Felice, and J. M. Calvo, unpublished data).Furthermore, although regulation of both geneclusters is controlled by leucine, the two mech-anisms of expression are very different. In leu-cine-containing minimal medium the ilvH ilvIgene expression is completely blocked (30),whereas expression of the leu operon is onlyrepressed two- to threefold (24, 25). A limitationfor leucine causes a two- to threefold derepres-sion of AHAS III (F. Lacara, C. Squires, and M.De Felice, unpublished data), whereas in thesame conditions the leucine biosynthetic en-zymes are derepressed about 50-fold (25).

S. typhimurium may synthesize an enzymeequivalent to the E. coli K-12 AHAS III isoen-zyme: a restriction fragment of E. coli K-12deoxyribonucleic acid containing information forAHAS III hybridizes with an S. typhimuriumdeoxyribonucleic acid fragment located veryclose to the leu operon, apparently in the sameposition in which the E. coli K-12 ilvH ilvI genesare located (Wessler et al., unpublished data).For AHAS II, as for AHAS I, only one struc-

tural gene, ilvG, has been reported. The function

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GROWT'H INHIBIT'ION BY NAT'URAL AMINO ACII)S 47

of this gene has been elucidated through thestudy of amber and temperature-sensitive mu-tations (36, 46, 49). As mentioned above, in wild-type E. coli K-12, this gene is unexpressed. Webelieve that the regulatory block to ilvG geneexpression, which is strictly related to themolecular aspects of the control of theilvREJGDAO cluster, is the central problem inilv regulation, and therefore we will discuss it indetail.Favre et al. showed, by means ofa three-factor

cross, that the ilvG605 mutation is located be-tween ilvE and ilvD (36). Shortly thereafter,Umbarger and his collaborators, from studies onthe polarity of mutations determined by Muphage insertion, proposed that ilvG lies betweenrbs and ilvR (98). Because of these controversialconclusions, in Fig. 1 we report ilvG in paren-theses, as a marker that has not been definitivelymapped. In vivo and in vitro studies are inprogress in several laboratories, from which con-clusive information on this problem and an ex-planation for the discrepancies existing in theliterature should be derived. However, the ideaspresented herein are independent from the exactlocation of ilvG. The ilvREJGDAO cluster isnot referred to as an "operon" (56) in this review,because it has several features which are atypicalin the framework of the common view of anoperon. Two regulatory regions, ilvO and ilUvR,are present on each side of the cluster, and cis-dominant mutations have been found in bothregions, (22, 36, 61, 90, 91, 92; Squires et al.,submitted for publication). The polarity appearsto be from ilvE to ilvA (19, 98). Therefore, theilvR locus could bear the properties of an oper-ator, whereas the ilvO locus, downstream fromthe point of transcription initiation, could beinvolved in some novel regulatory functions. Forexample, ilvO could contain information for sta-bility or translatability of messenger RNA orcould affect the secondary structure and subse-quent translatability of messenger RNA (63).divO could also contain information for a cis-acting protein (35), as has been proposed (98).However, it cannot be excluded that- theilvREJGDAO cluster is a genetic unit regulatedin an unusual way, in which both ilvO and ilvRwould be new types of regulatory elements. Ad-ditional features of the ilvREJGDAO clusterare: (i) the ilvA gene product, threonine deami-nase, is part of the regulatory mechanism (45,61, 62); (ii) the effect of mutations in the rhogene suggests the presence of an attenuator (1,98, 115); (iii) the control is at least in part tran-scriptional (18, 64, 113); (iv) gene expression ismultivalently controlled by isoleucine, leucine,and valine (110).

Expression of the ilvG gene, and thereforeappearance of the Valr AHAS II isoenzyme,occurs only when a derepressed synthesis of theilvEDA gene products is promoted by ilvR orilvO mutations (36; Squires et al., submitted forpublication) and not when the same extent ofderepression is caused by corepressor starvation(34) or by a rho mutation (1). At the presenttime, there is no explanation for this phenome-non.The Valr AHAS activity of S. typhimurium

(see above) appears to have a role similar to thatof AHAS III of E. coli K-12. Its structural gene,ilvG, has been reported to be located on the S.typhimurium chromsome in a position analo-gous to the E. coli K-12 ilvG gene (75).An extensive discussion on the regulation of

ilv gene expression is reported in a specific re-view (55).Physiological Role of the AHAS I and

AHAS Im IsoenzymesMultiple enzymes for the catalysis of the first

reaction occur in several diverging biosyntheticpathways (16). The biosynthesis of both theaspartate family (21) and the aromatic group(66) of amino acids in E. coli K-12 is character-ized by a correlation between isoenzymes andterminal products, each isoenzyme being specif-ically inhibited by one end product. Therefore,carbon flow through the pathway cannot becompletely inhibited unless all the end productsare present. For the branched-chain amino acids,the fit common biosynthetic step is catalyzed,in wild-type E. coli K-12, by two isoenzymes,AHAS I and AHAS III (Fig. 1). Experimentsperformed with extracts of two isogenic strains,each containing only one isoenzyme, show thatthey are distinct from both a genetic and abiochemical point of view (Table 3). However,both enzymes are severely inhibited by valine(85% inhibition at 1.5 mM valine for both incrude extracts; see Table 3), moderately in-hibited by isoleucine (40% inhibition at 3.0 mMisoleucine for both), and insensitive to leucine(30, 32). Therefore, the presence of AHAS I andAHAS III does not prevent growth inhibition ofE. coli K-12 by valine. In fact, the appearance ofa third isoenzyme (AHAS II) is required tocircumvent the problem of valine sensitivity.The similar sensitivity to valine of AHAS I andAHAS III in vitro suggests that they shouldhave a physiological role different fromn thatillustrated in the cases of the aspartate family(21) and the aromatic group (66) of amino acids.Recent observations, based on the different

affinities of AHAS I and AHAS III for theirsubstrates, suggest that AHAS I is specialized

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48 DE FELICE ET AL.

for the biosynthesis of leucine and valine(through a-acetolactate) and that AHAS III isspecialized for the biosynthesis of isoleucine(through a-aceto-a-hydroxybutyrate), althougheach enzyme is able to synthesize both precur-sors with different efficiencies (32; M. De Felice,unpublished data). It is also interesting to notethat synthesis of AHAS I and AHAS III occursthrough different regulatory patterns, the for-mer being multivalently controlled by leucineand valine (31) and the latter being regulated byleucine alone (30). Isoleucine is not directly in-volved in the control of synthesis of either en-zyme. However, this amino acid controls,through end-product inhibition of threonine de-aminase (106), the synthesis of a-ketobutyrate,which is both a substrate for isoleucine synthesisand an inhibitor of leucine and valine synthesis(32). This allows isoleucine to affect the expres-sion of both AHAS I and AHAS III by indirectlyaffecting the pool size of leucine and/or valine.We believe that this pattern of specialized activ-ity and different regulation ofsynthesis ofAHASI and AHAS III allows the cell to exert a finecontrol of carbon flow through the pathway forbranched-chain amino acid biosynthesis in agreat variety of physiological situations, such asconditions determining unbalanced intracellularconcentrations of intermediates or products.This control would confer to the pathway aregulatory elasticity which could have deter-mined a selective advantage for the existence ofthe two isoenzymes.

Additional regulatory features of the AHASisoenzymes of E. coli K-12 have been investi-gated. Freundlich (38) reports that normalexpression of the total AHAS activity requiresthe reA gene and that cyclic adenosine 3',5'-monophosphate can replace this requirement.There is also evidence that expression of theAHAS activity is sensitive to catabolite repres-sion (116); this effect appears to be specificallyrelated to AHAS I (M. Freundlich, personalcommunication).The AHAS isoenzymes affect the synthesis of

lysine from meso-diaminopimelate in a negativeway. In fact, mutants lacking one or more AHASsynthesize lysine at an abnormally high rate.This deprives the cell of meso-diaminopimelateand, as a consequence, decreases the synthesisofpeptidoglycan, the macromolecule responsiblefor the cell shape and integrity, in which meso-diaminopimelate is an essential component.Therefore, such mutants show morphologicaldefects which are' suppressible by the introduc-tion of mutations in the lysA gene, the structuralgene for meso-diaminopimelate decarboxylase(29). The molecular details of these cross-effects

are unknown. A symmetrical finding is reportedin the case of aspartokinase III, one of the threeisoenzymes catalyzing the first step in the bio-synthesis of the aspartate family of amino acids,which acts as a positive effector of meso-diami-nopimelate decarboxylase synthesis (77). In con-clusion, these two sets of isoenzymes generateregulatory connections between metabolic proc-esses, suggesting that their presence might notonly be informational for specific pathways, butalso provide a more general contribution to thecell economy.

VALINE-RESISTANT MUTANTSThe failure of E. coli K-12 to grow in minimal

medium containing valine provides the possibil-ity of positively selecting Val' mutants. This toolhas been and still is widely used and has beenessential in the understanding of the mechanismof valine inhibition. Furthermore, it has alloweda noticeable elucidation of the active transportand regulation of the biosynthesis of thebranched-chain amino acids. This procedure isnow used also for cell cultures of tobacco (Ni-cotiana tabacum), whose growth is inhibited byvaline (14).Manten and Rowley (65) showed that Valr

mutants occur spontaneously at a frequency ofabout 5 x 10-7 in a range ofvaline concentrationsbetween 1 ,ug/ml and 1 mg/ml. The analysis ofa Valr mutant allowed these authors to recognizea genetic locus for valine resistance, which wasshown to be closely linked, by conjugation ex-periments, to the leu operon, located at 1.5 minon the E. coli K-12 linkage map (7). Othermutants were described a few years later (20, 59,60). The biochemical characterization of thesestrains contributed to the clarification of themechanism of inhibition, but no genetic analysiswas performed. In 1962 Glover published a paper(43) containing a systematic genetic analysis ofValr mutants. This author isolated 62 sponta-neous independent mutations causing resistanceto 40 ,jg of valine per ml and defined their mapposition by crossing F- strains with three Hfrstrains having different origins and containingthe mutations under study. The analysis wascompleted by P1 transductions. This work de-fined six genetic classes of Valr mutations. Theseclasses are: A, approximately located in the ilv-metE region (83 to 84 min) but unlinked by P1transduction to either of these two markers; B,mapping very close to the thr operon (O min),counterclockwise to it; C, mapping very close tothe leu operon (1.5 min), clockwise to it; D,mapping between the thr and leu operons; E andF, both located close to the ilv cluster and pos-sibly inside it, with a possibility of each being a

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mixture ofmore than one class. Class F is locatedcloser than class E to the ilv mutation used. Thelatter class could be located clockwise to thismutation. Glover also studied the level of valineresistance of each class. Mutations of classes Band D give resistance up to 80 ,ug of valine perml; mutations of class C give resistance up to 1mg of valine per ml; and mutations of classes A,E, and F give resistance up to 5 mg of valine perml.The work of Glover stimulated further inves-

tigations. In fact, the surprisingly large numberof different classes of mutants found by thisauthor convinced other workers of the possibil-ity of understanding the phenomenon of valineinhibition and its relationship to the physiologyof E. coli K-12 through genetic analysis. A greatnumber of new Valr mutants have been isolatedand characterized in the last 16 years. These fallinto two classes: transport mutants and mutantsaltered in the inhibitory process.

Transport MutantsValine exerts its inhibition of E. coli K-12

growth whether it is present in the medium asa free amino acid or as a component ofdipeptidesand oligopeptides. The latter compounds arecleaved inside the cell by peptidases, thus liber-ating valine (79, 102). Mutants resistant to all ofthese compounds are altered in the intracellularinhibitory process. Mutants resistant to valinebut sensitive to valine-containing dipeptides andoligopeptides are altered in the transport of thefree amino acid. Mutants resistant to valine-containing oligopeptides but sensitive to valineand to valine-containing dipeptides, if not defec-tive in the mechanism of peptide hydrolysis, arealtered in the active transport of oligopeptides.Finally, uptake mutants resistant to valine-con-taining dipeptides but sensitive to valine andvaline-containing oligopeptides should be al-tered in the transport of dipeptides. However,such mutants cannot be isolated, because theoligopeptide transport also involves uptake ofdipeptides. Therefore, mutants lacking dipep-tide transport can only be isolated in strainscontaining alterations in the oligopeptide trans-port. For details on this subject, the reader isreferred to specific reviews (53, 54, 79, 102).The use of the positive selection described

above has allowed a comprehensive analysis ofthe active transport of oligopeptides, dipeptides,and branched-chain amino acids. Several studiesperformed in our and in other laboratories havedemonstrated that these compounds permeatethe cell envelope by means of a multiplicity ofactive transport systems, each having a specificgenetic pattern and typical Michaelis-Menten

kinetics.Oligopeptide uptake occurs through a trans-

port system which is nonspecific, since it me-diates the permeation of several oligopeptidesand even dipeptides (78). A genetic locus foroligopeptide transport, opp, has been defined,and its position at 27 min on the E. coli K-12map has been determined (8, 27). Evidence fora multiplicity of oligopeptide transport systemshas been reported (71).Mutants altered in the opp gene still transport

dipeptides (8, 27). In fact, dipeptide incorpora-tion occurs not only through the oligopeptidetransport system, but also, and predominantly,through a dipeptide-specific transport system,which takes up a number of different dipeptides(27, 78). A genetic locus, dpp, responsible for theactivity of this system has been defined in E.coli K-12. Its map position is at 5 min, distinctfrom that of the opp locus (7, 27).The criteria described above have been suc-

cessfully used to isolate mutants altered in thetransport of valine (5, 48, 50). The analysis ofthese mutants has shown that valine uptakeoccurs through a multiplicity of transport sys-tems, most of which are common to the threebranched-chain amino acids. A summary ofsome properties of the different transport sys-tems for the branched-chain amino acids is re-ported in Table 4. Table 5 lists the geneticmarkers thus far described for the transport ofbranched-chain amino acids and summarizestheir relevant properties. Other authors haveused alternative designations for some of thesystems described in this paper (89, 118). Theresults reported by these workers are includedin Tables 4 and 5, and the alternative designa-tions are listed in Table 4. The isoleucine-leu-cine-valine transport systems are differentiatedon the basis of different patterns of affinity forsubstrates and inhibition by some compounds(Table 4). Some of the loci reported in Table 5are common to more than one transport system.The brnR gene product is a protein required foractivity of both high-affinity 1 and high-affinity2 transport systems (48). This protein appearsto play a role analogous to that observed for thehisP gene product in the case of histidine trans-port in S. typhimurium (37); namely, it appearsto represent a branching point to both high-affinity systems. In fact, whereas brnQ and brnSmutants only lack one transport system, highaffinity 1 and high affinity 2, respectively, brnRmutants lack both of them. The brnR gene couldalso be involved in the expression or activity ofthe very high affinity transport system, sinceone ofthe mutations described [brnR6(Am)] hasless than 10% of the activity of this system (48).

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TABLE 4. Transport systems for the branched-chain amino acids in E. coli K-12Transport ~~~~~~~Relevant Alternative RfrneTransport Substrates K, (M) inibitor designation References

VHA Isoleucine 1.0 x lo-, Methionine, LIV-I 47, 48, 89, 118Leucine 2.0 x 10-8 threonine, andValine 1.0 x 10-7 alanine

HA-1 Isoleucine 2.0 x 10-6 LIV-II 47, 48, 89, 118Leucine 2.0 x 10-6Valine 2.0 x 10-6

HA-2 Isoleucine 2.0 x 10-6 Threonine LIV-I1 47, 48, 89, 118Leucine 2.0 x 10-6Valine 2.0 x 10-6

L-HA Leucine 2.0 x 10-7 Leucine- 41,47, 48, 89, 118specificsystem

I-LA Isoleucine 0.5 x 10-4 47, 48L-IA Leucine 0.5 x 10-4 47,48V-LA Valine 0.5 x 10-4 47, 48

aVHA, Very high affinity; HA-1 and HA-2, high affinity 1 and 2, respectively; L-HA, leucine-specific highaffinity; I-LA, L-LA, and V-LA, isoleucine-specific, leucine-specific, and valine-specific low affinity, respectively.

TABLE 5. Genetic loci for isoleucine, leucine, and valine transport in E. coli K-12

Transport system Map positionLCU8novda(ntp Gene Product Referencesinvolved' (unitsY'brnQ* HA-1 8 Unknown protein 48,50brnR* VHA, HA-1, and HA-2 8 Unknown protein 48brnS* HA-2 1 Unknown protein 48brnT VHA and I-LA 61 Unknown 48,53livH* VHA 74 Unknown 5livJ* VHA 74 LIV-binding protein (?)c 5livK* L-HA 74 L-binding protein (?)d 5livRE VHA 20 Regulatory locus 6lstR L-HA 20 Regulatory locus 6

a Genetic designations and map positions are as reported by Baclhmann et al. (7) with more recent additions.Loci marked with an asterisk are those to which Valr mutations have been assigned.

b See Table 4 for abbreviations.c Binding protein for isoleucine, leucine, and valine.d Leucine-specific binding protein.

A similar pleiotropic effect has been observedfor mutations in the brnT locus, indicating thatthis geneis required for the activity of both thevery 'high affinity and the isoleucine-specific,low-affinity transport systems (48, 53).Some loci are'listed in Table 5 as possible

structural genes for two binding proteins, onespecific for leucine and the other common to thethree branched-chain amino acids. A third bind-ing protein, sIpecific for isoleucine, has also beenfound, but no genetic elements affecting it havebeen described (41, 80, 89). These proteins canbe extracted by osmotic shock (52, 72) from E.coli K-12 cell and have been shown to be in-volved in active transport (76).

Multiplicity of transport systems has beenfound in several other cases (97). The regulationof isoleucine, leucine, and valine transport iscurrently under study, mainly by Oxender and

his collaborators. These authors report that reg-ulation of transport appears to be independentfrom regilation of the biosynthesis of theseamino acids (87). They also show that leucineplays a central'role in the control of activetransport. They suggest that isoleucine and va-line do'not intervene directly in the process butexert somq indirect effect, probably because oftheir role on the expression of the leucine bio-synthetic enzymes (85). Finally, they presentevidence that the rho factor (86), an intact leu-cyl-tRNA synthetase (83), and fully maturetRNA`Au (84) are required for normal regulationof transport.The evolutionary origin and physiological sig-

nificance of the multiplicity of transport sys-tems, which involves a great amount of geneticmaterial, are not yet understood. Since the lipidbilayer of the cell membrane seems to be im-

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permeable to many nutrients (see 53 for a dis-cussion), active transport could be absolutelynecessary for life. Therefore, multiplicity couldbe required to allow the cell to take up nutrientsin a wide range of concentrations and in thepresence of an excess of other nutrients or com-pounds negatively affecting the expression oractivity of specific transport systems. Further-more, transport systems could serve additionalroles besides uptake, such as excretion or func-tions related to the cell envelope properties.

Mutants Altered in the Inhibitory ProcessMutants resistant to valine and to valine-con-

taining peptides are altered in the intracellulartarget of valine inhibition. They will be group,edinto the following classes: (i) structural modifi-cations of AHAS I; (ii) regulatory mutationsaffecting AHAS I; (iii) modifications of AHASIII; (iv) regulatory mutations promoting theexpression of AHAS II; (v) other mutations.

(i) Structural modifications ofAHAS L. Amutation rendering total AHAS activity insen-sitive to valine inhibition and consequently con-ferring a Valr phenotype to strains containing itwas designated ilvB197 (81, 92). A specific studyof the effect of this mutation on AHAS I has notbeen performed. No other lesions of the sametype are known.

(ii) Regulatory mutations affectingAHASL The ilvP locus (Fig. 1) has been defined as anoperator for expression of the ilvB gene (91). Astrain containing a cis-dominant mutation inthis locus was isolated as resistant to 1 mM a-aminobutyric acid and was found to be alsoresistant to 0.2 mM valine. This phenotype ap-pears to be due to the presence of high levels oftQtal AHAS activity.

(iii) Modifications ofAlAS HI. As reportedabove, the genetic information for AHAS III iscoded for by two genes, ilvH and ilvI. Mutationsin ilvI cause absence of AHAS III activity andare responsible for an auxotrophy for isoleucineand valine in an ilvB background, even in thepresence of the wild-type allele of the ilvH gene(26). No Valr mutations altered in ilvI have beenfound. On the contrary, four ilvH mutants havebeen described, having a Valr phenotype, inwhich the AHAS III activity is Valr in vitro (28,57). Two additional mutations, iiv-660 and ilv-661 (23), are probably located in the ilvH gene.Another Valr mutation, unlinked to any ilv gene(the closest marker is argH), affects the sensi-tivity of AHAS III to valine, probably becauseit causes an enhanced production of an antago-nist of the ilvH subunit (G. Grimaldi and J.Guardiola, unpublished data).

(iv) Regulatory mutatiQns promoting theexpression of AHAS II. In 1964, Ramakrish-

nan and Adelberg described a cis-dominant mu-tation mapping between the ilvA and ilvC genesand causing- a derepressed synthesis of the ilvA,ilvD, and ilvE gene products (91, 92). At thetime, the existence in E. coli K-12 of the ilvGgene was not yet known, and therefore the rea-son for the Valr phenotype of that mutant wasunclear. In 1976, Favre et al. (36) described anewly isolated mutation (i1v0603) giving rise toa Valr phenotype and mapping between ilvA andilvC, thus confirming the existence of a cis-dom-inant regulatory locus in this position. They alsorecognized and characterized the ilvG gene,which permitted the explanation of the Valrphenotype of iiv0603 mutants. One more mu-tation causing a Valr phenotype, ilv-200(5 (61),maps between ilvA and ilvC and therefore hasbeen proposed to be located in the ilvO locus.On the other hand, Cohen and Jones (22)showed that two cis-dominant Valr 'mutationsoriginally isolated but not mapped by Ramak-rishnan and Adelberg (90) (now called ilvR268and ilvR269) are located on the rbs side of ilvE(Fig. 1). Almost simultaneously, the direction oftranscription being from ilvE to ilvA (instead ofthe reverse, which had been assumed for manyyears) was- proposed (19, 98). Smith et al. (98)introduced the designation ilvR for the region inwhich the two mutations mapped by Cohen andJones are located. Two additional cis-dominantmutations causing a Valt phenotype (itvR264and iivR651) and located in this region havebeen found recently (Squires et al., submittedfor publication).A comparison of the properties of AHAS in

mutants containing the iivR264 and i1v0603 mu-tations shows that the two lesions give riise toexpression of activities with identical features(reported in Table 3), which confirms that alter-ations in either ilvR or ilvO-promote the ap-pearance of a single enzyme, AHAS II, as aconsequence of expression of the ilvG gene.

(v) Other mutations. A- mutation causing aValr phenotype, iivF465, has been described andshown to be unlinked to any ilv gene (82). Thetotal AHAS activity is insensitive to valine instrains carrying this mutation. It is not knownwhether this mutation expresses an additionalVale AHAS or affects in some way the expressionor activity of one of the three isoenzymes. InTable 6 we report a list of the genetic lociinvolved in the process of valine inhibition.

Resistance to valne inhibition arises also as asecondary effect of alterations in the structuralgenes for leucyl-tRNA synthetase (leS) (M.laccarino, unpublished data) and for valyl-tRNAsynthetase (valS) (120). This phenotype couldbe due to an enhanced synthesis ofAHAS I and/or AHAS III indirectly caused by the mutations.

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52 DE FELICE ET AL. MICROBIOL. REV.

TABLE 6. Genetic loci involved in the process of valine inhibition in E. coli K-12Map

Locus' position Function Phenotype of mutants References(units)'

ilvB 83 Structural gene for AHAS I flv- in an ilvI background 46,91ilvF 54 Unknown Valr; appearance of a pH 6 AHAS 82

activity; total AHAS iS Val'ilvG 83 Structural gene for AHAS II Ilv- in an ilvO ilvB ilvI 33, 36

backgroundilvH 2 Structural gene for a regulatory Val'; appearance of a Val' form of 28

subunit of AHAS III AHAS IllilvI 2 Structural gene for a catalytic Ilv- in an ilvB background 26

subunit of AHAS IIIilvO 83 cis-Dominant regulatory effects Val'; appearance of the Valr 36, 61, 91, 92

for synthesis of the ilvEJDA AHAS IIgene products and forexpression of ilvG

iluP 83 cis-Dominant regulatory effect on Resistance to a-aminobutyric acid 91, 92ilvB expression and valine

ilvR 83 Same as for ilvO Same as for ilvO 22, 98; Squires et al.,submitted forpublication

aGenetic designations and map positions are adapted from Bachmann et al. (7).

It is worthwhile to interpret the collection ofGlover's Valr mutants (see above) in the light ofthe work accomplished in the last 16 years.Glover defined the existence of at least threeclasses of mutants, A, E, and F, in the ilv-metEregion. Presently, we know that mutations inthree different genes located in this region, ilvR,ilvO, and ilvP, give rise to a Valr phenotype andcould correspond to Glover's three classes orpart of them. Mutants in Glover's class B (map-ping close to the thr operon at 0 min) have notyet been described. It is unlikely that this classis equivalent to mutations in valS (95 min).Structural alterations in the ilvH and/or ilvIgenes or regulatory mutations affecting expres-sion of these genes could identify Glover's classC, mapping very close to the leu operon, clock-wise to it. Finally, Glover's class D mutants arelikely to be altered in the brnS gene. Unfortu-nately, the use of Glover's mutants for furtherinvestigations is not possible, since these mu-tants have been lost (S. W. Glover, personalcommunication).

Figure 2 shows a linkage map of E. coli K-12in which all the loci relevant to the phenomenonof valine inhibition are reported.

CONCLUSIONSThe occurrence of numerous cases of growth

inhibition by natural amino acids in procaryotesand eucaryotes and the reversion of such inhi-bition by other natural amino acids suggest theexistence of antagonism between metabolites inthe cell.

Valine inhibition in E. coli K-12, being themost dramatic of these phenomena in the mostcommonly studied microorganism, has been an-

alyzed in detail and provides a typical mecha-nism of antagonism between structurally relatedamino acids sharing common features of activetransport, biosynthesis, and physiological regu-lation. A large body of literature demonstratesthat the major cause of valine inhibition is theblock that this amino acid exerts on the synthe-sis of the acetohydroxy acid precursors of thebranched-chain amino acids, which generates arestriction of isoleucine biosynthesis. An analo-gous mechanism has been proposed for growthinhibition by valine in barley embryos (68) andin tobacco cell lines (14). Similarly, in somephysiological conditions tryptophan inhibitsgrowth of B. subtilis as a consequence of itsinhibitory effect on the activity of prephenatedehydratase, a phenylalanine biosynthetic en-zyme (93). It is likely that many other examplesof growth inhibition by natural compounds arethe result of similar connections in the cell'sprocesses of biosynthesis and utilization of re-lated metabolites.The occurrence of these inhibitory phenom-

ena indicates that they escape the limits of thecell's regulatory elasticity. We think that livingorganisms have not selected biological functionsto circumvent this antagonism because condi-tions of growth in the presence of a severe excessof one compound as compared to a related onedo not occur normally in natural environments.The studies on valine inhibition suggest that inthe extreme situations in which there is growthinhibition, mutations giving rise to a resistantphenotype occur spontaneously in several differ-ent genetic loci. Therefore, the problems causedby antagonism are minor consequences of a cel-lular organization in which structurally related

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4'

FIG. 2. E. coli K-12 linkage map (adapted from reference 7) showing the loci relevant to valine inhibition.Inside the circle are reported Glover's classes of Var mutants. Loci marked with an asterisk are those towhich Var mutations have been assigned.

compounds are also physiologically related,which presumably causes a major energy advan-tage.

In addition to leading to these general consid-erations, studies on valine inhibition have pro-vided insight into specific physiological prob-lems, such as regulation of gene expression andactive transport.We have presented the concept that valine

inhibits growth because in E. coli K-12 the ilvGgene, the structural gene for the Valr AHAS IIisoenzyme, is not expressed, although structur-ally unaltered, and therefore exogenous valineblocks the synthesis of isoleucine through feed-back inhibition ofAHAS I and AHAS III. Spon-taneous regulatory mutations in two loci adja-cent to ilvG promoting expression of this geneoccur and are selected in valine-containing min-imal medium. Since the structural gene for aValr AHAS isoenzyme appears to be normallyexpressed in other Enterobacteriaceae (11, 36,74, 117), E. coli could be envisioned as deriving

from a different strain (or vice versa) after somealteration in organization and expression of theilv genes. If we assume that having ilvG unex-pressed is not a fortuitous regulatory failure forE. coli K-12 and that having it expressed is nota useless function for S. typhimurium (and pre-sumably for E. coli 9723 and W), then we shouldconclude that the ilvG problem points to a majoraspect of evolutionary divergence among theseEnterobacteriaceae.The problem of ilvG expression is interesting

also in the framework of the structure and func-tion of the ilvREJGDAO gene cluster. Regula-tion of these genes cannot be explained in termsof the current ideas on gene expression. Wethink that this is very useful at a time in whichthe operon model (56) is being extended and putin a new context. We believe that the studies onthe regulation of the ilvREJGDAO cluster,through fine genetic analysis and in vitro workwith purified deoxyribonucleic acid, which arein progress in several laboratories will allow the

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54 DE FELICE ET AL.

establishment of new paradigms for biologicalregulatory mechanisms.The isolation of Valr mutants has been essen-

tial in revealing the existence, in E. coli K-12, oftwo normally expressed AHAS isoenzymes cat-alyzing the first biosynthetic step commnon tothe branched-chain amino acids. As we havediscussed above, these isoenzymes appear toplay an essential role in the control of carbonflow through the pathway and to be involved inmetabolic interactions between branched-chainamnino acid biosynthesis and lysine biosynthesis.Biochemical studies with purified preparationsand analysis of regulatory mutants, presently inprogress, should add more insight into the prop-erties of these isoenzymes and will probably leadto a more general understanding of the role ofisoenzymnes in diverging metabolic pathways.Work on the phenomenon of valine inhibition

has been very useful in the study of the processof peptide uptake. Furthermore, it has beenessential for the understanding of the genetics ofmultiplicity of active transport for branched-chain amino acids: this has proven to be aninteresting model system for energy-mediatedcell permeability and is now being extensivelystudied.

Valine inhibition has also been successfullyused for investigating the utilization of peptidesas a source of amino acids: Miller and Schwartz(69) have recently described E. coli K-12 mu-tants resistant to valine containing peptideswhich are deficient in several peptidases.

ADDENDUMThe E. coli K-12 linkage map of Bachmann et al.

(7) reported ilvB and ilvG as genes for subunits ofacetolactate synthase I and ilvH and ilvI as genes forsubunits of acetolactate synthase II. In the presentpaper we use a different terminology (see Fig. 1 andTable 6), in agreement with later experimental acqui-sitions and with a consolidated use in the most recentliterature.

LITERATURE CITED1. Abrescia, P., J. Guardiola, M. Foresti, A.

Lamberti, and M. Iccarino. 1979. Threoninedeaminase: autogenous regulator of the ilvgenes in Escherichia coli K-12. Mol. Gen. Ge-net., in press.

la. Acedo, G. N., and G. P. Red6i. 1976. Rtegula-tion of amino acid metabolism in Arabidopsis.Arabidopsis Inf. Serv. 13:120-124.

2. Adelberg, E. A. and H. E. Umbarger. 1953.Isoleucine and valine mietabolism in Esche-richia coli. V. a-Ketoisovaleric acid accumu-lation. J. Biol. Chem. 205:475-482.

3. Alfoldi, L., and E. Kerekes. 1964. Neutraliza-

tion of the amino acid sensitivity of RCre" Esch-erichia coli. Biochim. Biophys. Acta 91:155-157.

4. Alfoldi, L., G. S. Stent, M. Hoogs, and R. Hill.1963. Physiological effects of the RNA control(RC) gene in E. coli. Z. Vererbungsl. 94:285-302.

5. Anderson, J. J., and D. L. Oxender. 1977.Escherichia coli transport mutants lackingbinding protein and other components of thebranched-chain amino acid transport systems.J. Bacteriol. 130:384-392.

6. Anderson, J. J., S. C. Quay, and D. L. Oxen-der. 1976. Mapping of two loci affecting theregulation of branched-chain amino acid trans-port in Escherichia coli K-12. J. Bacteriol.126:80-90.

7. Bachmann, B. J., K. B. Low, and A. L. Tay-lor. 1976. Recalibrated linkage map of Esche-richia coli K-12. Bacteriol. Rev. 40:116-167.

8. Bakar, Z., and C. Gilvarg. 1974. Trionithine-resistant strains of Escherichia coli. Isolation,definition and genetic studies. J. Biol. Chem.249: 143-148.

9. Beerstecher, E., Jr., and W. Shive. 1947. Pre-vention of phenylalanine synthesis by tyrosine.J. Biol. Chem. 167:527-534.

10. Behrend, J., and R. I. Mateles. 1975. Nitrogenmetabolism in plant cell suspension cultures. I.Effect of amino acids on growth. Plant Physiol.56:584-589.

11. Blatt, J. M., W. J. Pledger, and H. E. Umbar-ger. 1972. Isoleucine and valine metabolism inEscherichia coli. XX. Multiple forms of ace-tohydroxy acid synthetase. Biochem. Biophys.Res. Commun. 48:444-450.

12. Bonner, D. 1946. Further studies of mutantstrains of Neurospora requiring isoleucine andvaline. J. Biol. Chem. 166:545-554.

13. Bourgin, J. P. 1976. Valine-induced inhibitionof growth of haploid tobacco protoplasts andits reversal by isoleucine. Z. Naturforsch. TeilC. 319:337-338.

14. Bourgin, J. P. 1978. Valine-resistant plants fromin vitro selected tobacco cells. Mol. Gen. Genet.161:225-230.

15. Brickson, W. L., L. M. Henderson, I. Solhjell,and C. A. Elvehjem. 1948. Antagonism ofamino acids in the growth of lactic acid bacte-ria. J. Biol. Chem. 176:517-528.

16. Brown, C. S., E. L. Kline, and H. E. Umbar-ger. 1975. Single reactions with multiple func-tions: multiple enzymes as one of the threepatterns in microorganism, p. 249-273. In C. L.Markert (ed.), Isoenzymes, vol. 2. AcademicPress Inc., New York.

17. Brown, H. T. 1908. Cultivation of barley em-bryos. Trans. Guinness Res. Lab. 1:288-290.

18. Childs, G. J., and M. Freundlich. 1975. Tran-scriptional control of the isoleucine-valine mes-senger RNA's in E. coli K-12. Mol. Gen. Genet.138:257-268.

19. Childs, G. J., H. Ohtsubo, E. Ohtsubo, F.Sonnenberg, and M. Freundlich. 1977. Re-

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