the global gene expression response of escherichia coli to l-phenylalanine

Journal of Biotechnology 115 (2005) 221–237

The global gene expression response ofEscherichia colitol-phenylalanine

T. Polena,∗,1, M. Kramerb,1, J. Bongaertsb, M. Wubboltsb, V.F. Wendischa

a Institut fur Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germanyb DSM Biotech GmbH, Karl-Heinz-Beckurts-Straße 13, D-52428 Jülich, Germany

Received 15 March 2004; received in revised form 29 July 2004; accepted 19 August 2004

Abstract

We investigated the global gene expression changes ofEscherichia colidue to the presence of different concentrations ofphenylalanine or shikimate in the growth medium. The response to 0.5 g l−1 phenylalanine primarily reflected a perturbed aromaticamino acid metabolism, in particular due to TyrR-mediated regulation. The addition of 5 g l−1 phenylalanine reduced the growthrate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbonavailability. Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophanrelieved growth inhibition by phenylalanine. In contrast to the wild-type, atyrRdisruption strain showed increased expression ofpckAand oftktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested.T ive strainsm©

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he absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defectight perform better in phenylalanine production.2004 Elsevier B.V. All rights reserved.

eywords: Escherichia coli; Microarray; Genomewide expression analysis; Phenylalanine; Shikimate; TyrR

. Introduction

The aromatic amino acids phenylalanine, tyrosinend tryptophan can be transported into theEscherichiaoli cell (Fig. 1). The carrier AroP mediates uptake

∗ Corresponding author. Tel.: +49 2461 613967;ax: +49 2461 612710.

E-mail address:[email protected] (T. Polen).1 T. Polen and M. Kramer contributed equally to this study.

of all three aromatic amino acids (Chye et al., 1986Honore and Cole, 1990). In addition,E. colican transport phenylalanine into the cell by the PheP sys(Pi et al., 1991), tyrosine by TyrP (Wookey and Pittard, 1988; Wookey et al., 1984) and tryptophan eitheby Mtr (Heatwole and Somerville, 1991) or by TnaB(Sarsero et al., 1991; Yanofsky et al., 1991). E. coliis able to degrade tryptophan, but neither tyrosinephenylalanine serves as a carbon or nitrogen so(McFall and Newman, 1996).

168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2004.08.017

222 T. Polen et al. / Journal of Biotechnology 115 (2005) 221–237

In E. coli, the aromatic amino acids phenylala-nine, tyrosine and tryptophan are synthesized from theprecursor metabolites phosphoenolpyruvate (PEP) anderythrose-4-phosphate (E4P), which are generated inglycolysis and the pentose phosphate pathway, respec-tively. Aromatic amino acid biosynthesis proceeds viathe intermediates shikimate and chorismate (Pittard,1996a,b; Fig. 1). For phenylalanine synthesis, cho-rismate is converted in the reactions of chorismate

mutase-prephenate dehydratase (PheA) and tyrosineaminotransferase (TyrB,Pittard, 1996a,b), whereas ty-rosine is generated from chorismate by chorismatemutase-prephenate dehydrogenase (TyrA) and tyrosineaminotransferase (TyrB,Pittard, 1996a,b) (Fig. 1). Theterminal synthetic pathway leading from chorismate totryptophan involves the gene products of thetrpED-CBAoperon, anthranilate synthase, anthranilate phos-phoribosyltransferase, phosphoribosylanthranilate iso-

Fm

ig. 1. Common and terminal pathways of aromatic amino acid biosembers of the TyrR regulon. Feedback inhibition is indicated by�.

ynthesis inE. coli. Boxes indicate enzymes encoded by genes that are

T. Polen et al. / Journal of Biotechnology 115 (2005) 221–237 223

merase, indoleglycerolphosphate synthetase and tryp-tophan synthase (Pittard, 1996a,b) (Fig. 1). MoreoverinE. coli, chorismate is also a precursor for the biosyn-thesis of folate, ubiquinone, menaquinone and enter-obactin (Pittard, 1996a,b) (Fig. 1).

Several enzymes of the aromatic amino acidbiosynthetic pathway are subject to feedback reg-ulation. The initial condensation of PEP and E4Pis catalyzed by the 3-deoxy-arabinoheptulosonate-7-phosphate (DAHP) synthases AroF, AroG and AroH,which are feedback-inhibited by tyrosine, phenylala-nine and tryptophan, respectively (Pittard, 1996a,b)(Fig. 1). The flux towards chorismate appears notto be affected significantly by inhibition of either3-dehydroquinate synthase (AroB), dehydroquinatedehydratase (AroD), low-affinity shikimate kinase I(AroK), high-affinity shikimate kinase II (AroL), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase(AroA) or chorismate synthase (AroC) (Pittard,1996a,b). However, shikimate dehydrogenase (AroE)is inhibited by the central intermediate shikimate (Delland Frost, 1993). The initial reactions of phenylala-nine and tyrosine formation from chorismate by cho-rismate mutase-prephenate dehydratase (PheA) andchorismate mutase-prephenate dehydrogenase (TyrA),respectively, are inhibited by phenylalanine and tyro-sine, respectively (Pittard, 1996a,b) (Fig. 1). Trypto-phan inhibits the anthranilate synthase and anthranilatephosphoribosyl transferase activities, whereas indole-3-glycerol phosphate synthetase activity is inhibited bya

a cidb tiona on-t st nyl-t tten-u gere

atica inoa andTrte er-l e

promoter (Pittard, 1996a,b). Repression by TrpRspecifically requires tryptophan (or the analogue 5-methyl-tryptophan) as corepressor (Marmorstein andSigler, 1989) and repression factors are between3-fold for trpR and 300-fold fortrpEDCBA (Pittard,1996a,b). Tyrosine, but also phenylalanine and tryp-tophan, modulates transcriptional regulation by TyrR(Pittard, 1996a,b). Binding to specific DNA sequences(TyrR boxes), the TyrR protein can either activate orrepress transcription at several� 70 promoters (Cuiand Somerville, 1993; Yang et al., 1993). The TyrRregulon comprises its own genetyrRand at least six fur-ther genes or operons (aroLM, aroP, tyrP, aroF-tyrA,tyrB andaroG). In the presence of ATP and tyrosineor of ATP and high concentrations of phenylalanine,TyrR forms hexamers and represses expression oftyrP, aroF-tyrA, aroP and tyrB, whereas it exists asa dimer in the presence of tryptophan or low concen-trations of phenylalanine (Pittard, 1996a,b; Wilsonet al., 1995). Generally, TyrR-mediated repressioninvolves binding to a TyrR box overlapping with thepromoter and thus excluding RNA polymerase fromthe promoter (Pittard and Davidson, 1991). However,repression ofaroP is different as it involves bindingof TyrR to downstream TyrR boxes. In the presenceof either tyrosine or phenylalanine and to a lesserextent tryptophan, TyrR activates transcription of anon-productive promoter on the opposite strand of twoproductivearoP promoters and thus repressesaroPexpression (Yang et al., 2002). Expression oftyrP isr eakT ithti boxw sent(r oft ofe e oft -p Ra iseP

ndsd im-p sp rie

nthranilate (Pittard, 1996a,b).With the exception ofaroA, aroB, aroC, aroD, aroE

ndaroK, the genes involved in aromatic amino aiosynthesis are subject to regulation by attenuand/or by transcriptional regulation. Expression c

rol of thepheA, trpEDCBAandpheST, which encodehe two subunits of the essential enzyme phenylalaRNA synthetase, involves translation-mediated aation (Landick et al., 1985; Pittard, 1996a,b; Sprint al., 1985).

Transcriptional regulation of genes of the arommino acid pathway in response to aromatic amcids is mediated by two regulatory proteins, TrpRyrR. The tryptophan-activatedtrp repressor TrpRepresses transcription of its own genetrpRand of therpEDCBA, aroH,mtrandaroLMoperons (Khodurskyt al., 2000) by binding operator sequences ov

apping with or upstream of (aroL) the respectiv

epressed when TyrR binds both a strong and a wyrR box located upstream of and overlapping whe -35 region of thetyrPpromoter. Expression oftyrPs activated as TyrR only binds to the strong TyrRhen phenylalanine is present and tyrosine is ab

Pittard, 1996a,b). Activation of mtr transcriptionequires binding of TyrR to a TyrR box upstreamhe -35 region of themtr promoter in the presenceither tyrosine or phenylalanine and in the absenc

ryptophan (Pittard, 1996a,b). Besidesmtr, also exression ofaroLM is regulated by both TyrR and Trps repression ofaroLM by tyrosine-activated TyrRnhanced by tryptophan-activated TrpR (Lawley andittard, 1994).Production of aromatic amino acids and compou

erived from them is of considerable industrialortance (Bongaerts et al., 2001). Phenylalanine irimarily used for the production of the low-calo


sweetener aspartame, whereas tryptophan is used as afeed additive and tyrosine for the synthesis of the anti-Parkinson’s disease drugl-DOPA (Bongaerts et al.,2001). BesidesE. coli, Corynebacterium glutamicumis used for aromatic amino acid production (Bongaertset al., 2001; Eggeling et al., 2001). InE. coli, metabolicengineering to improve flux into the pentose phosphatepathway and thus raising the supply of E4P for aromaticamino acid production was achieved, e.g. by overex-pression oftktA and/ortal encoding the transketolaseand transaldolase, respectively, (Bongaerts et al., 2001;Draths et al., 1992) or by deletion of the phospho-glucoisomerase genepgi (Mascarenhas et al., 1991).Alternatively, PEP supply was increased by function-ally replacing the PTS by galactose permease or theZymomonas mobilisglucose facilitator (Flores et al.,2002; Kramer, 2000; Snoep et al., 1994) or by us-ing non-PTS carbohydrates (Patnaik et al., 1995). In-creased PEP recycling by overexpression of the PEPsynthetase gene (Patnaik and Liao, 1994) or decreasedPEP consumption by mutating the genes of pyruvatekinase or PEP carboxylase (Berry, 1996; Gosset etal., 1996; Grinter, 1998) increased the PEP supply,as well. Generally, aromatic amino acid producingE.coli strains overexpress alleles for feedback-resistantenzymes of the common pathway leading to choris-mate as well as in the terminal pathway leading eitherto phenylalanine, tryptophan or tyrosine (Bongaerts etal., 2001). The use ofE. colistrains deficient for eithershikimate dehydrogenase (AroE) or for shikimate ki-n beu inoa mica

toa ica n ofw ifiedf e forg -r idB Yee iona asc 03;R al.,2 -b tories

using either nylon membranes (Richmond et al., 1999)or glass slides (Khodursky et al., 2000; Oshima et al.,2002; Polen et al., 2003; Wei et al., 2001). Among thefirst applications ofE. coliDNA microarrays was thecharacterization of the global gene expression responseto tryptophan and the presence or absence of TrpR byKhodursky et al. (2000). Here, we determined the ef-fects of the addition of phenylalanine and shikimic acidto the growth medium on global gene expression ofE.coli and have assessed the role of TyrR in the responsesto phenylalanine.

2. Materials and methods

2.1. Bacterial strains and plasmid

The strains and the plasmid used in this study arelisted in Table 1. For microarray experimentsE. coliwild-type strain LJ110 and itstyrR disruption strainF101 were used. ThelacZ fusion strains were based onF105 and F104. ThefliC′–′lacZfusion strains F108 andF107 were obtained by P1 transduction from IMW356(Miller, 1972). ThefliA′–′lacZ fusion strains were ob-tained by transforming F104 and F105 with plasmidpMW198 (Sambrook et al., 1989). Strains F101 andF104 were derived by transducing thetyrR::cat alleleof strain TK743 into LJ110 and F105 (Miller, 1972).

2.2. Media and culture conditions

i ioni m-i etaaAC31

en-s e-t m).I r0 dt cids

ase I (AroK) and shikimate kinase II (AroL) cansed to produce intermediates of the aromatic amcid pathway such as dehydroshikimic acid or shikicid (Kramer et al., 2003).

In this study, we employed DNA microarraysnalyse the effects ofl-phenylalanine and shikimcid on global gene expression. The generatiohole genome microarrays based on PCR-ampl

ragments of all genes of an organism and their usene expression analysis was pioneered forSacchaomyces cerevisiaeby the labs of Pat Brown and Davotstein (DeRisi et al., 1997; Schena et al., 1995;t al., 2001). Subsequently, genome-wide expressnalysis was established for model bacteria suchE.oli (e.g.Khodursky et al., 2000; Polen et al., 20ichmond et al., 1999; Wei et al., 2001; Yoon et003; Zimmer et al., 2000). ForE. coli, PCR-productased genome arrays were made in several labora

All cultures were grown aerobically at 37◦Cn 1000 ml shake flasks with 180 rpm agitatn sterile filtered mineral medium (pH 7.2) silar to the precultivation medium of Gerigkl., but containing 6 g l−1 glucose, 0.075 g l−1 thi-mine and 1 ml l−1 trace element solution 2.0 g l−1

l2(SO4)3·18H2O, 0.7 g l−1 CoSO4·7H2O, 2.5 g l−1

uSO4·5H2O, 0.5 g l−1 H3BO3, 20 g l−1 MnSO4·H2O,.0 g l−1 Na2MoO4·2H2O, 2.0 g l−1 NiSO4·6H2O and5.0 g l−1 ZnSO4·7H2O (Gerigk et al., 2002).

Cell growth was monitored as the optical dity at 620 nm (OD620, UV–VIS spectral photomer, Lambda 10; Perkin-Elmer, Zaventem, Belgiuf necessary, 0.5 g l−1 or 5 g l−1 phenylalanine o.5 g l−1, 5 g l−1 or 50 g l−1 shikimic acid was adde

o the cultures. The phenylalanine and shikimic atocks were adjusted to pH 7.2 with 5 M KOH.E. coli


Table 1Escherichia colistrains and plasmid used in this study

Relevant genotype Reference/source

StrainsLJ110 W3110 Fnr+, wild-typeE. coli Zeppenfeld et al., 2000TK743 �tyrR::cat Katayama et al., 1999IMW356 �(argF-lac)169zah::Tn10 �(�(fliC′–′lacZ)hyb)bla Lehnen et al., 2002F101 LJ110�tyrR::cat P1(TK743)× LJ110F105 LJ110�(lacZYA)::kan Laboratory stockF104 LJ110�(lacZYA)::kan�tyrR::cat P1(TK743)× F105F107 LJ110�(lacZYA)::kan�tyrR::cat�(�(fliC′–′lacZ)hyb)bla P1(IMW356)× F104F108 LJ110�(lacZYA)::kan�(�(fliC′–′lacZ)hyb)bla P1(IMW356)× F105

PlasmidpMW198 (� (fliA′–′lacZ)hyb)bla Lehnen et al., 2002

strains F104/pMW198 and F105/pMW198 were culti-vated with ampicillin (0.1 g l−1). Precultures were in-oculated into main cultures and contained the same ad-ditives as the main cultures. For DNA microarray anal-ysis, cultures were grown exponentially for about 10generations by repeated dilution to ensure adaptationto the medium before preparation of RNA.

2.3. Assay of�-galactosidase and glutamic aciddecarboxylase enzyme activities

Aliquots from replicate cultures in the exponen-tial growth phase were withdrawn and specific�-galactosidase activities (Miller units, MU) were mea-sured in three independent replicates and calculated asdescribed (Miller, 1992). To assay glutamic acid decar-boxylase activity, cells were collected by centrifuga-tion and resuspended in 1 mM pyridoxal 5′-phosphate,1 mM dithiothreitol buffered in 0.2 M pyridine–HCl,pH 4.6. Cells were disrupted by sonication and enzymeactivity in crude extracts was measured as described(De Biase et al., 1996).

2.4. Preparation of total RNA and cDNA synthesis

Preparation of RNA and cDNA synthesis were car-ried out as described (Polen et al., 2003; Wendisch etal., 2001). Portions (∼25 ml) of exponentially grow-ing E. coli cultures (OD600 0.4) were added to 15 go di-as -t dis-

rupted by bead-beating (Silamat S5, Vivadent, Ellwan-gen, Germany) using zirconia/silica beads (0.1 mm,ROTH, Karlsruhe, Germany). After centrifugation(1 min, 13,000×g, RT) the supernatant was pro-cessed using the RNeasy system (Qiagen, Hilden, Ger-many) as recommended by the manufacturer. The iso-lated RNA was treated with DNase I (RNase-free,Roche Diagnostics GmbH, Mannheim, Germany) asdescribed (Polen et al., 2003) and purified by phe-nol/chloroform/isoamylalcohol (25:24:1) and by chlo-roform/isoamylalcohol (24:1) extractions followed byethanol precipitation (Sambrook et al., 1989). RNAconcentrations were determined photometrically andRNA quality was checked on formamide agarose gels(Sambrook et al., 1989).

Equal amounts of total RNA (15–25�g) were usedfor random hexamer-primed synthesis of fluorescentlylabeled cDNA with the fluorescent nucleotide ana-logues Cy3-dUTP or Cy5-dUTP (Amersham Pharma-cia) (Khodursky et al., 2003; Wendisch et al., 2001).

2.5. Gene expression analysis by DNA microarrayanalysis

To characterize gene expression changes due to thepresence of phenylalanine or shikimic acid in minimalmedia, mRNA levels for nearly every gene (94%) oftheE. coliMG1655 genome were compared by usingDNA microarrays. These DNA microarrays (Lehnen eta lys osysO am-b s

f ice (−20◦C) and cells were harvested immetely by centrifugation (3 min, 4500×g, 4◦C), re-uspended in 350�l RLT buffer of the RNeasy sysem (Qiagen, Hilden, Germany) and mechanically

l., 2002; Polen et al., 2003) were made by roboticalpotting PCR products generated using the GenRFmer primer set (Genosys Biotechnologies, Cridgeshire, England) onto poly-l-lysine coated glas


microscope slides as described (Khodursky et al., 2003;Khodursky et al., 2000; Polen et al., 2003; Wendischet al., 2001; Zimmer et al., 2000).

DNA microarrays were hybridized for 10 h at 65◦Cto mixtures of Cy3- and Cy5-labeled cDNA probes con-taining 1 g l−1 polyA (Sigma, Germany) as competi-tor, 3× SSC (0.45 M sodium chloride, 0.045 M sodiumcitrate, nuclease-free) and 25 mM HEPES. After hy-bridization arrays were washed in a solution containing1× SSC and 0.03% SDS and finally in 0.05× SSC. Af-ter washing, slides were dried by centrifugation (5 min,50×g). Detailed procedures for microarray hybridiza-tion and stringent washing have been described else-where (Khodursky et al., 2003; Rhodius et al., 2002).After drying, fluorescence at 532 nm (Cy3-dUTP) and635 nm (Cy5-dUTP) was determined at 10-�m res-olution using an Axon GenePix 4000 laser scanner(Axon Instruments, Union City, CA, USA). Acquiredraw fluorescence data were analyzed using GenePixPro 4.1 (Axon Instruments, Union City, CA, USA).For each spot the background-subtracted Cy5/Cy3 ra-tio of the medians was calculated, log-transformed andnormalized based on the fluorescence signals ofE. coliMG1655 genomic DNA spots (Khodursky et al., 2003;Khodursky et al., 2000; Polen et al., 2003; Wendisch etal., 2001; Zimmer et al., 2000). For each DNA microar-ray experiment, these ratios were stored in a mySQLdatabase for further analysis (Polen and Wendisch,2004, in press). For hybridization signals exceedingthe background noise by at least a factor of three thel them un-d rderso sig-n ack-g

sion(w l-i orm oneh f hy-b herh

siond dm s ofE (a)

reliable detection with signal-to-noise ratios exceed-ing a factor of three; (b) in a paired Student’st-testrelative RNA levels of replicate experiments were sig-nificantly different from the genomic DNA controls(p< 0.05;Polen et al., 2003); (c) relative mRNA lev-els changed by a factor of 2 or more in at least oneDNA microarray experiment. The color image showsthe color coding of the log-transformed numerical rel-ative mRNA levels according to the method introducedby Eisen et al. (1998)with grey color indicating thatno reliable signal was measured.

3. Results

3.1. Global gene expression changes due to theaddition of phenylalanine

Global gene expression profiling promises targetgene identification for efficient biotechnological strainimprovement (Wendisch, 2003). As the biotechnolog-ical production of amino acids is accompanied by highproduct titers, e.g. up to 50 g l−1 phenylalanine dur-ing the production of this aromatic amino acid byE.coli (Backman et al., 1990), many genes likely changeexpression not as a prerequisite of amino acid produc-tion, but rather as a direct or indirect consequence ofhigh product titers. It is known that high phenylala-nine titers inhibit growth (Grinter, 1998). To identifyspecific effects of phenylalanine in the growth mediumo wthc hi-b wasu( nceo ntr

in3 ntly( neo it-i em-b -i andt1 e-s hates

og-transformed and normalized Cy5/Cy3 ratio ofedians was taken to reflect relative RNA level abance. The dynamic range achieved up to three of magnitude. When Cy3- and Cy5-fluorescenceals were less than three times greater than the bround, signals were not considered further.

For statistical analysis of global gene expresArfin et al., 2000; Hommais et al., 2001), p-valuesere calculated based on Student’st-test using norma

zed log-transformed RNA levels determined in twoore independent replicate experiments, on theand, and the normalized log-transformed ratios oridization signals of genomic DNA spots, on the otand (Polen et al., 2003).

For hierarchical cluster analysis of gene expresata (Eisen et al., 1998), normalized log-transformeRNA levels of 4108 amplified and arrayed gene. coli were selected using the following criteria:

n global gene expression, ideally, gratuitous groonditions have to be used. To minimize growth inition effects, a low phenylalanine concentrationsed. Growth in the presence of 0.5 g l−1 phenylalanineµ = 0.53 h−1) was compared to growth in the absef phenylalanine (µ = 0.71 h−1) in three independeeplicate experiments.

DNA microarray analysis revealed 47 genes3 operons that change mRNA levels significap< 0.05) by a factor of 2 or more in at least of the replicates (Fig. 2). Among the genes exhib

ng the highest RNA level changes were the mers of the TyrR regulonmtr, tyrP andaroP, encod

ng the tryptophan-specific, the tyrosine-specifiche general aromatic amino acid permeases (Brown,970), as well asaroG, encoding the phenylalaninensitive 3-deoxy-arabinoheptulosonate-7-phospynthase (Wallace and Pittard, 1967). Whereasmtrand


tyrP exhibited 18- and 5-fold increased RNA levels,respectively,aroP andaroG exhibited 8- and 4-folddecreased RNA levels, respectively (Fig. 2). This isconsistent with activation ofmtr andtyrP and repres-sion of aroP andaroG, respectively, by TyrR in thepresence of phenylalanine (Pittard, 1996a,b). The tryp-tophan operon genes,trpEDCBA, exhibited about 3-fold increased RNA levels indicating parallels to theresponse ofE. coli to starvation for the amino acidtryptophan (Khodursky et al., 2000).

Genes of the general stress response likeosmYandotsAB (Hengge-Aronis, 2002) showed 2- to 3-foldincreased expression in the presence of phenylalanine(Fig. 2). Although phenylalanine was added at neutralpH, the acid resistance geneshdeABand alsogadAandgadBencoding the two isoenzymes of glutamicacid decarboxylase (De Biase et al., 1996) exhibited 3-to 6-fold increased RNA levels (Fig. 2). Moreover, thegene downstream ofgadA, gadX, which encodes a reg-ulatory protein of the AraC/XylR family and activatestranscription from thegadAandgadBpromoters (Shinet al., 2001), exhibited 3-fold increased RNA levels(Fig. 2). Consequently, the glutamate decarboxylaseactivity was 3- to 4-fold higher in the presence of0.5 g l−1 phenylalanine (0.10 U mg−1 protein) as com-pared to the absence of phenylalanine (0.03 U mg−1

protein). The genesglnK-amtB controlled by thenitrogen regulatory protein C (NtrC) (Zimmer et al.,2000) and the nitrogen assimilation control proteingenenacexhibited about 3-fold decreased RNA levels,w l.,1 ge-nTl ayu ionall e ofheT p-p eta x-p vels( -im e-s andt tem

Fig. 2. Cluster analysis of genes differentially expressed in the ab-sence or presence of 0.5 g l−1 phenylalanine in the culture medium.The hierarchical cluster analysis comprised 47 genes that showedstatistically significant (p< 0.05) RNA level changes with or withoutaddition of 0.5 g l−1 phenylalanine (red sub-cluster). Additionally theexpression data of these genes determined in the presence or absenceof 5 g l−1 phenylalanine, 0.5 g l−1, 5 g l−1 and 50 g l−1 shikimic acidwere included. Not determinable: fluorescent signals too low (seeSection2).

hereas the Nac repressed genegdhA(Camarena et a998), encoding NADP-specific glutamate dehydroase, exhibited a 2-fold increased RNA level (Fig. 2).he high-affinity leucine transport system geneslivKH-ivF (livMG are not arrayed on the DNA microarrsed), which are repressed by the global transcript

eucine-responsive regulator (Lrp) in the presencigh leucine concentrations (Newman et al., 1996),xhibited 2- to 4-fold decreased RNA levels (Fig. 2).he genesylcBCD, suggested to be involved in coer/silver resistance (Franke et al., 2001; Munsonl., 2000; Outten et al., 2001), exhibited decreased eression with the lowest observed relative RNA le1/4- to 1/17-fold) besidesaroP (Fig. 2). Concernng the carbohydrate metabolism, the genesgatABC,anXZ,malPandmalMof the galactitol- and mannos

pecific PTS system as well as the maltose regulonhe ATP-binding protein of the xylose transport sys


Table 2Expression offliA′–′lacZ andfliC′–′lacZ translational fusions and ratios offliA andfliC mRNA levels in the absence and presence of 5 g l−1

phenylalanine

Gene lacZ-fusion�-galactosidase activity (MU)a Ratio with/without 5 g l−1 Phe

Phe (0 g l−1) Phe (5 g l−1) �-Galactosidase level mRNA level

fliA 45 ± 4 13 ± 1 0.29 0.07fliC 6380± 272 1693± 24 0.26 0.08

a MU: Miller units.

xylG showed 2-fold decreased mRNA levels (Fig. 2).The flagellar genesfliE, fliD, fliK, fliC, fliZ andfliR ex-hibited about 2-fold decreased RNA levels in the pres-ence of 0.5 g l−1 phenylalanine (Fig. 2). The decreasedexpression of the flagellar genes was more pronouncedin the presence of 5 g l−1 phenylalanine asfliDST, fliE,fliFGHIJK, fliLMNOPQR, flgBCDEFHIJK, fliCAZ,flgAM, flhD, flhBAE, motB-cheAWand tap-cheRBYexhibited 2- to 21-fold decreased RNA levels. Ex-pression of the translationalfliC′–′lacZandfliA′–′lacZfusions clearly decreased in the presence of 5 g l−1

phenylalanine although to a lesser degree than theobservedfliA andfliC RNA level changes (Table 2). Incontrast, the RNA levels oftrpEDCBA, hdeAB, gadX,gadAandgadBdid not increase further in the presenceof 5 g l−1 phenylalanine. The glutamate decarboxylaseactivity was 0.48 U mg−1 protein in the presence of5 g l−1 phenylalanine compared to 0.03 U mg−1 pro-tein in the absence of phenylalanine. The RNA level oftyrP was 5-fold increased in the presence of 0.5 g l−1

phenylalanine, but unchanged in the presence of 5 g l−1

phenylalanine (Fig. 2). ThesetyrPRNA level changesare consistent with activation oftyrP transcription bythe dimeric form of TyrR at low phenylalanine con-centrations and its repression by the hexameric formof TyrR at high phenylalanine concentrations (Pittard,1996a,b). Overall, in the presence of 5 g l−1 phenylala-nine 427 genes in 295 operons exhibited significantly(p< 0.05) altered RNA levels by a factor of 2 ormore. Among them, genes involved in the centralm dmnp se

w chg(

changed expression of many genes is likely due toindirect effects by growth inhibition. The 2- to 3-fold decreased expression of the ribosomal proteingenesrpsA, rpsH–rplFR–rpsE–rplO–prlA–rpmJ andrpsJ–rplCDWB–rps–rplv–rplP observed in the pres-ence of 5 g l−1 phenylalanine, but not in the pres-ence of 0.5 g l−1, is likely a consequence of reducedgrowth. Furthermore, the reduced expression of genesof the central metabolism, e.g.sucABand sucCD,encoding the tricarboxylic acid cycle enzymes�-ketoglutarate dehydrogenase and succinyl-CoA syn-thetase, the operonnuoBCEFGHIJKL, encoding theNADH dehydrogenase I andatpIBEFAGDC, encodingmembrane-bound ATP synthase, probably reflected re-duced growth. The presence of 30 g l−1 phenylalanineprecluded growth ofE. coli LJ110 in minimal media(data not shown).

3.2. Global gene expression changes due to theaddition of shikimate

In the presence of 0.5 g l−1 and 5 g l−1 shikimatein the growth medium no significant (p< 0.05) geneexpression changes by a factor of two or more wereobserved inE. coli LJ110 (see alsoFig. 2). Only at avery high concentration (50 g l−1) did shikimate inhibitgrowth (µ = 0.43 h−1) and about 104 genes exhibitedsignificantly altered RNA levels by a factor of two ormore. Many of these gene expression changes weresimilar to those caused by 5 g l−1 phenylalanine as ex-p d ex-pow tea ly int ctso imatec e toa

etabolism (e.g.sucAB, sucCD), the amino acietabolism (e.g.thrABC, argA, argF, argCBH), theucleic acid metabolism (e.g.purF, purMN, purH,yrD, pyrF, pyrG, pyrH) and ribosomal proteinxhibited decreased RNA levels.

Hence, as growth inhibition ofE. coli LJ110ild-type due to phenylalanine addition was mureater at a concentration of 5 g l−1 than at 0.5 g l−1

growth rates ofµ = 0.37 h−1 and of µ = 0.53 h−1),

ression of the flagellar genes was decreased anression of the general stress-response genesotsABandsmY, and to a lesser extent ofgadX, gadAandgadB,ere increased (Fig. 2). The concentration of shikimadded to the medium did not decrease significant

hese experiments making it unlikely that the effen gene expression observed at the highest shikoncentration were due to conversion of shikimatromatic amino acids.


Table 3Exponential phase growth rates ofE. coliLJ110 andE. coliF101 (�tyrR::cat) in minimal media in the absence and presence of the supplementsshikimate (Shi), phenylalanine (Phe), tyrosine (Tyr), and/or tryptophan (Trp)

Strain Relevant genotype Growth rateµ (h−1)

–a Phea Phe + Shia Phe + Tyra Phe + Trpa Phe + Tyr + Trpa

LJ110 0.71 0.37 0.53 0.58 0.56 0.65F101 �tyrR::cat 0.59 0.57 0.59 0.58 0.59 0.59

a As additives, we used 5 g l−1 phenylalanine, 0.4 g l−1 shikimic acid, 0.2 g l−1 tyrosine or 0.2 g l−1 tryptophan, respectively.

3.3. Addition of tyrosine, tryptophan or shikimatealleviated growth inhibition by phenylalanine

Increased expression of the tryptophan operontrpEDCBA (Fig. 2) and of the tyrosine tRNA syn-thetase gene (tyrS, 1.8-fold) suggested an intracellularlimitation for these amino acids caused by the addi-tion of phenylalanine to the growth medium. There-fore, the effect of supplementation with 0.4 g l−1 shiki-mate, 0.2 g l−1 tyrosine or 0.2 g l−1 tryptophan ongrowth inhibition by 5 g l−1 phenylalanine was tested.In the presence of 5 g l−1 phenylalanineE. coli wild-type LJ110 grew faster with shikimate, tryptophanor tyrosine as compared to growth in the absence ofthese supplements (Table 3). When both 0.2 g l−1 ty-rosine and 0.2 g l−1 tryptophan were present,E. coliLJ110 grew nearly as well with 5 g l−1 phenylala-nine as without added phenylalanine (Table 3). Thisis consistent with growth inhibition by high pheny-lalanine concentrations due to internal limitation fortryptophan and tyrosine. Additionally, inE. coli cho-rismate is also a precursor for the biosynthesis of fo-late, ubiquinone, menaquinone and enterobactin. Theperturbed aromatic amino acid biosynthesis by the ad-dition of phenylalanine may led to insufficient choris-mate levels for one of these pathways possibly limitinggrowth. However, we did not test whether e.g. the sup-plementation with the folate precursorp-aminobenzoicacid results in the complete reversal of growthinhibition.

3i

f theT thea wes x-

pression and growth inhibition by phenylalanine. Thegrowth rate of thetyrR deletion strain F101 on glu-cose minimal medium without added phenylalaninewas slightly lower than that of the wild-type (Table 3).Growth ofE. coli F101 was similar in the absence orpresence of 5 g l−1 phenylalanine (Table 3) and thus dif-fered from the wild-type LJ110 which, showed abouthalf-maximal inhibition of growth by 5 g l−1 phenylala-nine (Table 3). Supplementation with 0.4 g l−1 shiki-mate, 0.2 g l−1 tyrosine and/or 0.2 g l−1 tryptophan hadlittle effect on growth ofE. coli F101 in the presenceof 5 g l−1 phenylalanine (Table 3).

3.5. Comparison of global gene expressionchanges by phenylalanine in the absence orpresence of TyrR

To compare the global gene expression changesmodulated by phenylalanine in the absence or pres-ence of TyrR, we used hierarchical cluster analysis ofthe gene expression changes observed in three series ofDNA microarray experiments. We determined genesexhibiting statistically significant (p< 0.05) mRNAlevel changes of a factor of 2 or more in (i) threeDNA microarray experiments comparing the transcrip-tome of F101 (�tyrR::cat) with/without the additionof 5 g l−1 phenylalanine (65 genes in 50 operons);(ii) three DNA microarray experiments comparing thetranscriptomes of LJ110 and F101 in the absence ofp (iii)t thet e of5 Thec 214o from1 d int nineo

.4. Disruption of tyrR alleviated growthnhibition by phenylalanine

As we observed expression changes of genes oyrR regulon as well as growth inhibition due toddition of low concentrations of phenylalanine,tudied the impact of atyrRdeletion on global gene e

henylalanine (eight genes in six operons) andhree DNA microarray experiments comparingranscriptomes of LJ110 and F101 in the presencg l−1 phenylalanine (242 genes in 177 operons).luster analysis of the 293 non-redundant genes inperons also included their expression data taken1 DNA microarray experiments (see above) use

he analysis of the wild-type response to phenylalar shikimate (Fig. 3).


Fig. 3. Cluster analysis of genes differentially expressed inE. coliLJ110 and F101 in response to phenylalanine. See text for details. Sub-clusters1 to 6 are highlighted in different colors. Not determinable: fluorescent signals too low (see Section2).


In E. coli F101, the addition of phenylalanineelicited fewer gene expression changes than in the par-ent strain LJ110. In the presence of 5 g l−1 phenylala-nine 65 genes showed significant mRNA level changesby a factor of two or more (Fig. 3). Expression of theTyrR-regulated genesaroLM, aroP, tyrP, aroF–tyrA,tyrB andaroGdid not change in response to the addi-tion of phenylalanine inE. coli F101 (aroM, tyrP andtyrBnot depicted inFig. 3). Overall, most of the expres-sion changes caused by 5 g l−1 phenylalanine observedin F101 were also found in LJ110 (e.g. sub-cluster 5,Fig. 3). These genes changed expression in responseto 5 g l−1 phenylalanine independently of regulationby TyrR and comprised a number of NtrC-controlledgenes (glnK-amtB, glnA, ddpA of the ddpXABCDEoperon,nacand the Nac-controlled geneyedL; Zimmeret al., 2000). The genes of a putative two-componentregulatory system (ybcZandylcA) and three genes ofthe adjacentylcBCD-ybdEoperon involved in copperand silver resistance (Franke et al., 2001; Munson etal., 2000; Outten et al., 2001) exhibited 3- to 42-folddecreased RNA levels. Genes for uptake systems ofleucine (livKH-livF) and xylose (xylFG) exhibited 3-to 4-fold decreased RNA levels (sub-cluster 5,Fig. 3).Expression of the general stress-reponse genesotsAB,poxB,osmY,gadA,gadBanddpsincreased in responseto phenylalanine in F101 and in LJ110 (Fig. 3). Thetranscriptional regulator genegadX, the catabolic ala-nine racemase genedadXanddadAencoding a sub-unit of ad-amino acid dehydrogenase, the glyoxylate-ie in-c g lp

itedb of ag g lp ster2 sioni gt lowa ster2 thed nlyi , themo itedi onl

in F101 and in the response of the wild-type to 50 g l−1

shikimate (sub-cluster 2,Fig. 3). The genesyghAencoding a putative oxidoreductase and the osmore-sponsive gene b1481 (yddX) of unknown function ex-hibited increased expression only at 50 g l−1 shikimate(sub-cluster 2,Fig. 3). In the presence of 5 g l−1 pheny-lalanine, expression of the phosphoenolpyruvate car-boxykinase genepckAwas reduced 2-fold in LJ110 butincreased 2-fold in F101 (above sub-cluster 5,Fig. 3).

To identify genes differentially expressed in the ab-sence of phenylalanine as a direct or indirect conse-quence of TyrR inactivation, we compared the globalgene expression patterns ofE. coli LJ110 and F101(LJ110 �tyrR::cat) during growth on glucose mini-mal medium without addition of phenylalanine in threeDNA microarray experiments. Compared to the wild-type, six genes exhibited 2- to 4-fold increased RNAlevels in F101 (p< 0.05) and included the TyrR regu-lon membersaroF-tyrAandaroL(sub-cluster 6,Fig. 3).Furthermore, the geneskch (b1250) andyciI (b1251)encoding a potassium channel (Munsey et al., 2002)and a hypothetical protein, as well as the transcriptionalregulator geneycjC (b1299), exhibited 2- to 3-fold in-creased RNA levels in F101 (sub-cluster 6,Fig. 3). Sig-nificantly decreased expression levels (1/2-fold) wereobserved for the NtrC-controlled genesglnK-amtBandnac in F101 compared to the wild-type (sub-cluster 5,Fig. 3).

The vast number of expression changes elicitedlywth,tifye ofs toe ef-nei-byd-

of aand

n theib-

es

nducible glycerate kinase geneybbZ(glxK) andylaCncoding a protein of unknown function exhibitedreased RNA levels in both strains in response to 5−1

henylalanine (Fig. 3).In contrast to gene expression changes elic

y phenylalanine in both strains, the expressionroup of genes was increased in the presence of 5−1

henylalanine in F101 but not in LJ110 (sub-clu, Fig. 3). Among these genes the highest expres

ncrease was exhibited by thetnaLABoperon encodinhe leader peptide, the tryptophanase and theffinity tryptophan permease (up to 8-fold, sub-clu, Fig. 3). Furthermore, expression of all genes ofppABCDFoperon for dipeptide uptake increased o

n F101 in response to phenylalanine. Four genesechanosensitive channel genemscLand theproVWXperon for glycine betaine/proline uptake exhib

ncreased expression in response to phenylalanine
y
by phenylalanine in the wild-type, which are likeindirect, e.g. as a consequence of impaired groprecluded the use of this global approach to idengenes that are regulated by TyrR in the presencphenylalanine. However, the approach allowed udiscriminate between secondary effects and thosfects that were due to phenylalanine addition, butther due to TyrR regulation nor due to perturbationgrowth inhibition. Growth inhibition caused by the adition of 5 g l−1 phenylalanine and 50 g l−1 shikimatein the wild-type correlated to reduced expressioncluster of 50 genes, 36 of which are chemotaxisflagellar genes (sub-cluster 3,Fig. 3). Another groupof 68 genes showed decreased expression wheaddition of phenylalanine, but not shikimate inhited growth ofE. coli LJ110 (sub-cluster 4,Fig. 3).Among these were stress genes (mopAB for GroESand GroEL;sodAfor superoxide dismutase) and genfor the uptake and biosynthesis of amino acids (livJ,


lysP, thrABC, lysC, ilvC,dapB), uptake of peptides (op-pABCD, oppF) and carbohydrates (rbsB,malE,malM,ptsA, ptsHI, gatYZABCD,manXYZ, xylH). A group of40 genes showed increased expression when pheny-lalanine inhibited growth (sub-cluster 1,Fig. 3). Be-sides genes involved in the acid stress response (gadAB,hdeAB, xasA, gadX, yggB, b1836), the genes for thebranched-chain amino acid uptake system (brnQ) andthe tryptophan-specifc uptake system (mtr), as well asgenes for the tRNA synthetases for the amino acidsphenylalanine (pheST), tyrosine (tyrS) and cysteine(cysS) belonged to this group.

4. Discussion

Bacteria can degrade and utilize various aromaticcompounds. In the human intestine phenylacetic acid,phenylpropionic acid and benzoic acid are the prin-cipal products of phenylalanine fermentation. Withsome exceptionsE. coli K-12 derivatives are able togrow on phenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid and 3-hydroxycinnamicacid (reviewed inDiaz et al., 2001). Recently, the path-ways involved in phenylacetic acid catabolism inE.coliwere identified by combining functional genomicsand NMR analyses (Ismail et al., 2003). Phenylalanine,however, cannot be used as carbon or nitrogen sourcebyE. coliK-12, whereas it can be incorporated into pro-teins. Consequently, the phenylalanine concentrationi iva-t res-s t re-s , thep wthm theg hus,e heseg icc e re-q

umls enee fectsw wthf enceo ay

analyses. The addition of 0.5 g l−1 phenylalanine led toa moderate growth inhibition, which was much morepronounced in the presence of 5 g l−1 phenylalanine.The response ofE. coli to 0.5 g l−1 phenylalanine com-prised fewer and smaller gene expression changes (47and 427 genes, respectively) as compared to those ob-served with 5 g l−1 phenylalanine (Fig. 2). At 5 g l−1

phenylalanine many of the observed gene expressionchanges resulted from perturbed growth per se ratherthan from specific effects due to phenylalanine. Forexample, in the presence of 0.5 g l−1 phenylalanine ex-pression of six chemotaxis and flagella genes decreasedabout 2-fold, whereas in the presence of 5 g l−1 pheny-lalanine expression of 42 chemotaxis and flagella genesdecreased 2- to 21-fold. Expression oflacZ fusionsto the flagella genesfliA andfliC decreased about 2-fold when 5 g l−1 phenylalanine was present. The ob-served expression patterns of chemotaxis and flagellagenes are consistent with their growth-rate-dependentregulation. Furthermore, phenylalanine causes nega-tive chemotaxis inE. coli and therefore classifies asa repellent (Tso and Adler, 1974).

Expression of the acid stress-response geneshdeAB,gadA, gadBandgadX increased in the wild-type to asimilar extent when either 0.5 g l−1 or 5 g l−1 pheny-lalanine were present irrespective of their differenteffects on the growth rate. The specific activity ofglutamate decarboxylase increased in the presenceof phenylalanine as well. Transcriptional regulationof acid-stress response genes inE. coli involvest dCa oa llu-l an,1 Bp ationo -l theo ue toi eny-l ns-p ea heH Cfp ters( la-

n the growth medium did not decrease during cultion in our experiments (data not shown). Thus, expion changes due to phenylalanine addition did noult from phenylalanine degradation. Furthermoreresence of phenylalanine or shikimate in the groedium did not lead to increased mRNA levels ofenes involved in phenylacetic acid catabolism. Tither glucose repression prevents induction of tenes (Ferrandez et al., 2000) or deaminated aromatompounds rather than amino acids appear to buired.

As the addition of phenylalanine to the medied to a growth inhibition of theE. coli wild-typetrain LJ110, direct and indirect effects on global gxpression had to be distinguished. Transient efere minimized by experimental design, i.e. gro

or at least 10 generations in the absence or presf phenylalanine before performing DNA microarr

he regulators EvgA, YdeO and GadX (Masuda anhurch, 2002; Masuda and Church, 2003). GadAnd GadB catalyse the conversion of glutamate t�-minobutyrate to maintain a near-neutral intrace

ar pH (De Biase et al., 1999; Small and Waterm998; Smith et al., 1992) whereas HdeA and Hderesumably act as chaperones preventing aggregf denatured periplasmic proteins (Gajiwala and Bur

ey, 2000). As phenylalanine was added at pH 7.2,bserved gene expression changes were likely d

ntracellular acidification as a consequence of phalanine uptake, which is coupled to proton traort (Paulsen et al., 2000). Mtr, TyrP and TnaB arromatic amino acid:H+ symport permeases of tAAAP family, AroP and PheP belong to the AP

amily and theshiA-encoded shikimate:H+ symportermease belongs to the MHS family of transporPaulsen et al., 2000). Thus, the uptake of phenyla


nine coupled to proton symport and diffusion of thishydrophobic amino acid out of the cell might resultin intracellular acidification. Due to the low affinityof the shikimate transport system (Brown and Doy,1976), shikimate is likely to elicit similar effects onlyat higher concentrations. Consequently, increased ex-pression of the acid resistance genesgadX, gadAandgadBas well as the general stress-response genesosmYandotsABoccurred only at the very high concentra-tion of 50 g l−1 shikimate in the culture medium. Thefact that expression of the acid-stress-response geneshdeAB, gadA, gadBandgadXdid not increase in F101is commensurate with the observation that AroP trans-port activity is reduced 7-fold in atyrR background(Pittard, 1996a,b).

In both the wild-type and in thetyrR disruptionstrain, phenylalanine led to decreased expression ofthe high-affinity leucine transport system geneslivKH-livF that are repressed by the global transcriptionalregulator Lrp in the presence of high leucine con-centrations (Hung et al., 2002; Newman et al., 1996;Tani et al., 2002). It was shown that thelivK-encodedleucine-specific binding protein can specifically bindl-phenylalanine with aKD of 0.18�M as comparedto a KD of 0.40�M for l-leucine (Luck and Johnson,2000). Recent physiological studies revealed a role forthe branched-chain amino acid transporter with its twoperiplasmic binding proteins LivJ and LivK as the thirdphenylalanine transport system inE. coli (Koyanagi etal., 2004). BesideslivKH-livF , we observed differentialeg( thee duet idb ninea ,1 llym notb st

be-l ek nceo -cl -c

1996a,b). Additionally, at low phenylalanine concen-trations tyrP RNA levels increased presumably dueto activation by the dimeric TyrR and they decreasedat high phenylalanine concentrations due to repres-sion by the hexameric form of TyrR (Pittard, 1996a,b).The addition of 0.5 g l−1 or 5 g l−1 shikimate to thegrowth medium had no effect on gene expression. Thisis consistent with the fact that shikimate, which istaken up intoE. coli by the shiA encoded transportsystem (Whipp et al., 1998), did not perturb growth(Table 3).

The altered expression of genes regulated by TyrRand increased expression of the tryptophan operongenestrpEDCBA, the tyrosine tRNA synthetase genetyrS as well as the growth inhibition due to the ad-dition of phenylalanine indicated starvation for tryp-tophan and tyrosine. In the presence of phenylala-nine, RNA levels ofaroG, encoding the predominantlyphenylalanine-sensitive isoenzyme of DAHP synthase(Ray et al., 1988), decreased 4-fold. DecreasedaroGexpression and inhibition of AroG activity by pheny-lalanine likely led to a shortage of chorismate and thusof tryptophan and tyrosine. Consistent with starvationfor tryptophan and tyrosine, expression of thetrpED-CBAoperon and oftyrSincreased when phenylalaninewas added to the growth medium and the growth inhibi-tion by phenylalanine could be relieved by supplemen-tation with tryptophan and tyrosine (Table 3). Alterna-tively, supplementation with shikimate, the precursorof all three aromatic amino acids, relieved growth in-h

r of5 -mr nd,tc theg ex-p ster2 edb tran-s 2;M ,2 ofTh to ah thus

xpression of other Lrp-regulated genes, e.g.thrLABC,dhA, dppABCDF, oppABCDandoppF, rmfandyggBFig. 3). The effect of phenylalanine addition onxpression of Lrp-regulated genes might either beo the role of Lrp in the coordination of amino aciosynthesis and catabolism with leucine and alas indicators of amino acid sufficiency (Mathew et al.996). Alternatively, phenylalanine might specificaodulate regulation by Lrp, although to date it haseen shown whetherl-phenylalanine specifically bind

o Lrp.Phenylalanine modulated expression of genes

onging to the TyrR regulon (Fig. 2) according to thnown mechanism of TyrR regulation. In the presef phenylalanine, RNA levels ofmtr increased in acordance with activation ofmtr by TyrR while mRNAevels ofaroP, aroG, aroF-tyrA and its own gene dereased in accordance with TyrR repression (Pittard,

ibition due to phenylalanine.In contrast to the wild-type, growth of thetyrRdis-

uption strain F101 was not affected by additiong l−1 phenylalanine (Table 3). The absence of TyrRediated repression ofaroG, aroF andaroLM did not

esult in starvation for tryptophan and tyrosine ahus, expression oftrpEDCBA, pheSTandtyrSdid nothange when 5 g l−1 phenylalanine was present inrowth. ThetnaABoperon showed 8-fold increasedression in F101 but not in the wild-type (sub-clu, Fig. 3). Transcription of this operon is regulaty catabolite repression and tryptophan-inducedcription antitermination (Deeley and Yanofsky, 198cFall and Newman, 1996) and indole (Wang et al.001). In the tyrR disruption strain F101, the lackyrR-mediated repression ofaroF-tyrA and aroLMatigh phenylalanine concentrations might have ledigher chorismate and tryptophan availability and


to tryptophan-induced transcription antitermination oftnaAB.

From the biotechnological point of view, micro-bial phenylalanine production has been remarkably im-proved in the past by industrial process developmentand development of strains with increased precursorsupply and alleviated negative regulation by pheny-lalanine (reviewed inBongaerts et al., 2001). How-ever, growth inhibition by phenylalanine appears tohave a negative effect on process performance (Grinter,1998; Konstantinov et al., 1991). The characterizationof global gene expression changes due to phenylalanineand the absence of TyrR regulation suggested targets toimprove phenylalanine production. Disruption oftyrRresulted in a strain no longer inhibited by phenylala-nine. Moreover, increased expression oftktBandpckA(Fig. 3) might suggest an improved supply of the pre-cursors for phenylalanine biosynthesis, E4P and PEP,in the tyrRdisruption strain. If in this strain increasedtktBmRNA levels in response to phenylalanine resultedin increased pentose phosphate pathway flux, then im-proved supply of E4P would be a further advantageof tyrR disruption. Similarly, 2-fold increased expres-sion ofpckA, the gene of the PEP generating phospho-enolpyruvate carboxykinase, in the TyrR mutant F101in response to 5 g l−1 phenylalanine might result in im-proved supply of PEP. It should be noted that in re-sponse to phenylalanine additionpckAexpression wasdecreased 2-fold in the wild-type (Fig. 3). Thus, com-bining disruption oftyrRwith overexpression oftktBa iono

A

forh on-t uc-t r forh calr wl-e

R

A le,g in

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D ryp-,

D di--acid

D olic. Sci-

D gra-

D A.,lytic

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E d.),81–

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cknowledgements

The authors would like to thank Doris Rittmannelp with DNA microarrays, Hermann Sahm for c

inuous support, Sonja Orf for help in strain constrion and�-galactosidase assays, Susanne Kremeelp with cultivations and Roel Bovenberg for critieading of the manuscript. T.P. and V.F.W. acknodge the support from DSM Biotech.

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the global gene expression response of escherichia coli to l-phenylalanine

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