characterization isocitrate lyase gene corynebacterium ...3476 reinscheid et al. naoh(ph7.3), 5...

Vol. 176, No. 12JOURNAL OF BACTERIOLOGY, June 1994, p. 3474-34830021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Characterization of the Isocitrate Lyase Gene fromCorynebacterium glutamicum and Biochemical

Analysis of the EnzymeDIETER J. REINSCHEID, BERNHARD J. EIKMANNS,* AND HERMANN SAHM

Institut fuir Biotechnologie des Forschungszentrums Jiulich, D-52425 Julich, Germany

Received 25 January 1994/Accepted 4 April 1994

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growthon acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the aminoacid-producing organism Corynebacterium ghltaml'cum. In crude extracts of C. glutamicum, the specific activitiesof isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein aftergrowth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, andcharacterized. The predicted gene product of aceA consists of 432 amino acids (M1, 47,228) and shows up to57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential forthiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of thechromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitratelyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. Thenative enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism ofcatalysis, and to be eflectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate,fructose-1,6-bisphosphate, and succinate.

Aerobic microorganisms growing on acetate as the solecarbon source require the glyoxylate cycle for the biosynthesisof cellular substances (25). The key enzymes of this cycle areisocitrate lyase (ICL) (EC 4.1.3.1) and malate synthase (EC4.1.3.2), with the former cleaving isocitrate to succinate andglyoxylate and the latter condensing glyoxylate with acetylcoenzyme A to give malate. ICL competes with the tricarbox-ylic acid cycle enzyme isocitrate dehydrogenase (ICD) for theircommon substrate isocitrate. Flux control between the twocycles is achieved by changing the activity of either one of thetwo enzymes and by changing their affinities towards isocitrate(27, 28; for a review, see reference 46). Particularly in Esche-richia coli, regulation of carbon flux between the two cycles hasbeen subject to intensive investigation. It was found that in E.coli, ICL is formed only when acetate is the sole carbon sourceof the medium (24). ICL shows low affinity towards isocitrate,and ICD exhibits high affinity for its substrate. To allowsignificant amounts of carbon to enter the glyoxylate cycle,ICD in E. coli is substantially inactivated by reversible phos-phorylation during growth on acetate (27, 28, 49). Bothphosphorylation and dephosphorylation is catalyzed by oneenzyme, ICD kinase/phosphatase (28). When acetate is thesole carbon source, the kinase activity of this enzyme leads tophosphorylation and thus to inactivation of ICD. When glu-cose is added, ICD is dephosphorylated, resulting in therestoration of ICD activity. Besides this regulation of ICD, theE. coli ICL is activated by phosphorylation (37) and is inhibitedby a variety of metabolites, e.g., phosphoenolpyruvate (PEP),3-phosphoglycerate, and succinate (31, 38), thereby allowingfine control of the carbon flux.

Since ICL has a key position in the central metabolism,increased attention has been focused on the genetics of ICL in

* Corresponding author. Mailing address: Institut fur Biotechnolo-gie 1 des Forschungszentrums Julich, D-52425 Julich, Germany.Phone: 49-2461-613967. Fax: 49-2461-612710.

a variety of organisms. The E. coli ICL gene (aceA) and therespective genes from several eucaryotes have recently beenisolated and sequenced (1-3, 15, 16, 33, 36, 52). The E. coliaceA gene was found to form an operon with the genes aceB,which encodes malate synthase, and aceK, which encodes ICDkinase/phosphatase (28, 32). The comparison of the deducedICL protein sequence from E. coli with those from eucaryoticorganisms revealed a high level of similarity between the E. coliand the eucaryotic enzymes. However, the E. coli enzymeturned out to be about 16 kDa smaller than those fromeucaryotes (33). This result is in agreement with the findingsthat by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) analysis, most ICLs from eucaryotesare significantly larger than their procaryotic counterparts (fora review, see reference 46). Since the aceA gene from E. coli isthe only procaryotic gene that has so far been sequenced, it isnot known whether the region lacking in the E. coli ICL occursat the same site within other procaryotic ICLs or whether it isunique to the E. coli enzyme. Therefore, other procaryoticaceA genes have to be characterized in detail to clarify thisquestion.

Corynebactenium glutamicum and several related organismswidely used for large-scale amino acid production are capableof growth on acetate-containing medium (29). The ICL fromC. glutamicum subsp. flavum has been partially purified andfound to be inhibited by several metabolites, e.g., succinate,malate, and 2-oxoglutarate (35). C. glutamicum subsp. flavummutant strains exhibiting increased ICL activity were found toform significantly higher amounts of glutamate than the pa-rental strain did (43), demonstrating the key position of ICL inthe carbon flux. This prompted us to analyze ICL from C.glutamicum both genetically and biochemically. Here we reportthe isolation and characterization of the aceA gene from C.glutamicum, its homologous overexpression, and its inactiva-tion within the chromosome. Furthermore, we present both a

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristicsa Source or

StrainsE. coliK8-5m lacZ4 (or lacY1) tsx-6 supE44 galK2 rspL159 xyl-5 (or xyl-7) met-24 aceA3 iclR13 48DH5 supE44 hsdRJ7 recAl endAl gyrA96 thi-I reWl 17S17-1 thi-l endAR1 hsdRJ7 (r- m+) supE44 pro 44

C. glutamicumATCC 13032 Type strain ATCCbASK1 ATCC 13032 aceA::pSUP301::internal aceA This workJAAl ATCC 13032 thiX::pSUP301::internal thiX This work

Plasmids or cosmidspHC79 Apr Tetr 19pHC79-based gene library Apr, pHC79 containing 40-kb chromosomal C. glutamicum Sau3A inserts 6pUC18 Apr, on of ColEl 47pSUP301 Apr Kmr oniT 44pEKO Kmr, on of pBL1, on of ColEl 13pACB1 pHC79 containing aceB-complementing C. glutamicum Sau3A insert This workpEKAlC pEKO containing 6.2-kb NcoI insert from pACB1 This workpEKA2C pEKO containing 2.4-kb HpaI-NciI insert from pEKA1 This work

a Apr, ampicillin resistant; Tetr, tetracycline resistant; Kmr, kanamycin resistant.b ATCC, American Type Culture Collection.c Fragments were ligated in a blunt-end ligation into the BamHI site of pEKO.

purification scheme for the C. glutamicum ICL and a biochem-ical characterization of this enzyme.

MATERUILS AND METHODSBacterial strains, plasmids, and growth conditions. The

bacterial strains and plasmids used are listed in Table 1. M9medium (39) was used as minimal medium for E. coli. Theminimal medium for C. glutamicum has been described previ-ously (14). Carbon sources were added to the minimal mediumat final concentrations of 4% for glucose, 2% for acetate, and2% each for glucose plus acetate. Luria-Bertani medium (39)was used as the complex medium for both organisms. In orderto grow E. coli K8-5m on minimal medium, L-methionine wasadded at a final concentration of 2 mM. When appropriate,ampicillin (100 ,ug/ml) or kanamycin (50 ,ug/ml) was added tothe medium. Both organisms were grown aerobically. C.glutamicum was grown at 30°C, and E. coli was grown at 37°C.

Chemicals. All chemicals commercially available were pur-chased by Sigma (Deisenhofen, Germany) or Boehringer(Mannheim, Germany). 4-Methyl-5-(,B-hydroxyethyl)thiazolwas a gift kindly provided by Tadhg P. Begley (Ithaca, N.Y.)and by Hoffmann-La Roche (Basel, Switzerland).DNA isolation, manipulation, and transformation. C. glu-

tamicum chromosomal DNA and plasmid DNA from E. coliwere obtained as described previously (12, 39). Restrictionenzymes, T4 DNA ligase, Klenow polymerase, and calf intes-tine alkaline phosphatase were obtained from Boehringer andused as instructed by the manufacturer. Restriction-generatedfragments were separated on 0.8% agarose gels, isolated, andpurified with the Geneclean kit from Dianova (Hamburg,Germany). Transformation of E. coli was performed by theCaCl2 method (39), and transformation of C. glutamicum wasdone via electroporation as described by Liebl et al. (30).DNA sequence analysis. For sequencing, the 2.4-kb HpaI-

NciI fragment carrying aceA was ligated into the XbaI site ofpUC18. The resulting plasmid was digested with either KpnI-BamHI or SphI-SalI, and subsequently deletion clones wereobtained with the Erase-a-base kit from Promega (Madison,Wis.). Sequencing was performed by the dideoxy-chain termi-

nation method (40) with the AutoRead sequencing kit fromPharmacia (Freiburg, Germany). Electrophoretic analysis ofthe sequencing reactions was done with an automated laserfluorescence DNA sequencer from Pharmacia. Sequence datawere compiled and analyzed by the HUSAR program package(EMBL, Heidelberg, Germany).

Southern blot analysis. About 10 p,g of chromosomal C.glutamicum DNA was digested with NcoI, size fractionated ona 0.8% agarose gel, and transferred onto a nylon membraneNytran 13 (Schleicher und Schuell, Dassel, Germany) byvacuum-supported diffusion using the VacuGene system(Pharmacia). A 2.4-kb HpaI-NciI fragment isolated frompEKA1 was labeled with digoxygenin dUTP and used as aprobe. Labeling, hybridization, washing, and detection wereperformed using the Non-radioactive DNA Labeling andDetection Kit (Boehringer).Gene disruption. Gene-directed mutagenesis was performed

by the method described by Schwarzer and Puhler (41). Forinactivation of aceA, a 0.3-kb blunt-ended XmnI-BglI fragmentcarrying an internal aceA fragment was ligated into the ScaIsite of vector pSUP301. For inactivation of thiX, a blunt-ended0.2-kb TaqI fragment was ligated into the Scal site ofpSUP301.

Preparation of crude extracts. Cells were grown in 60 ml ofminimal medium in a 500-ml baffled Erlenmeyer flask toexponential phase, washed twice in 20 ml of 50 mM Tris-HClbuffer, pH 7.6, and resuspended in 1 ml of the same buffer.Cells were disrupted by sonication with a microtip-equippedBranson sonifier for 10 min (2 min for E. coli) at 0°C andmaximum settings. The supernatant was used after centrifuga-tion for 30 min at 13,000 x g.Malate synthase assay. Malate synthase assay was per-

formed by the method of Dixon and Kornberg (11). One unitof activity is defined as 1 ,umol of malate formed per min.ICL assay. In order to determine the cleavage reaction of

ICL, the glyoxylate formed during catalysis was reduced toglycolate by lactate dehydrogenase with concomitant oxidationof NADH (50). In a final volume of 1 ml, each cuvettecontained 50 mM morpholinopropanesulfonic acid (MOPS)-

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3476 REINSCHEID ET AL.

NaOH (pH 7.3), 5 mM dithiothreitol (D1T), 15 mM MgCl2, 1mM EDTA, 5 mM Ds-threo-isocitrate, 0.2 mM NADH, 18 U oflactate dehydrogenase (pig heart isoenzyme I), and ICL en-

zyme. Decrease of NADH was monitored at 340 nm. Amodification of the assay described by Dixon and Kornberg(11) was used to determine the optimum temperature and pH.In a final volume of 1 ml, the test mixture contained 15 mMMgCl2, 3.4 mM phenylhydrazine hydrochloride, 5 mM Ds-threo-isocitrate, enzyme, and buffer as described below. 50 mMpotassium phosphate buffer with pHs of 6.0 to 7.3 and 50 mMMOPS-NaOH with pHs of 6.7 to 8.0 were used to determinethe optimum pH. Determination of the optimum temperaturewas performed between 20 and 60°C in 50 mM MOPS-NaOH,pH 7.3. In the cleavage reaction, 1 U of activity corresponds to1 ,umol of glyoxylate formed per min. To investigate theinhibition pattern of glyoxylate on ICL cleavage reaction, themethod of Ko and McFadden was used (23). The condensationreaction of ICL was measured as described previously byMacKintosh and Nimmo (31), with the following modifica-tions: 15 mM MgCl2 and 1 U of isocitrate dehydrogenase per

ml were used. One unit equals 1 ,umol of isocitrate formed per

min.In order to determine the effects of metal ions on activity,

purified ICL was separated from MgCl2 by ultrafiltration witha solution consisting of 50 mM Tris-HCl (pH 8.0), 5 mM DYT,and 1 mM EDTA, using an Ultrafree filter cup (Millipore,Bedford, Mass.).

Purification of ICL. For purification of the enzyme, cellswere grown in 60 ml of minimal medium in a 500-ml baffledErlenmeyer flask to exponential phase, washed twice in buffercontaining 50 mM 2-morpholinoethanesulfonic acid (MES)-NaOH (pH 6.0), 5 mM DDT, 5 mM MgCl2, and 1 mM EDTA,and resuspended in the same buffer supplemented with 5 U ofDNase per ml, 15 jig of RNase per ml, and 100 ,uM phenyl-methylsulfonyl fluoride (PMSF). Disruption of the cells andremoval of cell debris were performed as described above. Thebuffers used for the chromatographic purification of ICLcontained 5 mM DTT, 5 mM MgCl2, and 1 mM EDTA. Allpurification steps were carried out at 4°C. Crude extracts werediluted with 50 mM MES-NaOH (pH 6.0) to give a finalvolume of 10 ml and subjected for 2 h to ultracentrifugation atabout 183,000 x g. The supernatant was chromatographed on

an HR5/5 MonoQ column with a fast protein liquid chroma-tography apparatus (Pharmacia) (9). During the first chro-matographic procedure, 50 mM MES-NaOH (pH 6.0) with a

0.1 to 0.4 M NaCl gradient was used. For the second chro-matographic procedure, the buffer of the partially purified ICLwas changed to 50 mM Tris-HCl (pH 8.0) by ultrafiltrationusing an Ultrafree filter cup (Millipore). The second chro-matographic purification was performed with 50 mM Tris-HCl(pH 8.0) and a 0.2 to 0.5 M NaCl gradient. During allchromatographic experiments, a flow rate of 1 ml/min was

maintained.Kinetic analysis of ICL. Michaelis constants were deter-

mined by using a Lineweaver-Burk double-reciprocal plot.Inhibition constants were determined from linear replots ofLineweaver-Burk slopes versus inhibitor concentrations (42).SDS-PAGE. SDS-PAGE was performed in 12.5% polyacryl-

amide gels overnight at 8 mA by the method previouslydescribed by Laemmli (26), using a Hoefer Sturdier verticalslab gel instrument (Serva, Heidelberg, Germany). Densito-metric measurements were performed using photographicdocumentation in combination with the Wincam program(Cybertech, Berlin, Germany).

N-terminal sequence analysis. Purified ICL was separatedby SDS-PAGE, transferred to a polyvinylidene difluoride

membrane, and sequenced with an Applied Biosystems 477Asequenator equipped with a Blott cartridge and a model 120on-line high-pressure liquid chromotograph.

Gel filtration. Gel filtration was carried out by fast proteinliquid chromatography on Superose 12 (Pharmacia) in 50 mMTris-HCl buffer (pH 8.0) containing 5 mM DTT, 5 mM MgCl2,and 1 mM EDTA. The molecular weight standards werecatalase (232,000), aldolase (158,000), alcohol dehydrogenase(150,000), lactate dehydrogenase (140,000), albumin (67,000),and ovalbumin (43,000).

Nucleotide sequence accession number. The nucleotide se-quence for the 2.43-kb HpaI-NciI fragment carrying the aceAand thiX genes has been deposited in the EMBL Data Libraryunder accession number X75504.

RESULTS

ICL activity in C. glutamicum. In many organisms, ICLactivity exhibits a drastic increase upon shifting the carbonsource from glucose to acetate. To study whether in C.glutamicum, ICL activity is also regulated by the carbon source,the specific enzymatic activity in crude extracts of C. glutami-cum ATCC 13032 (wild type) was determined after growth inminimal medium containing glucose, glucose plus acetate, oracetate. ICL activity in crude extracts of C. glutamicum grownon glucose was negligible (0.01 U/mg of protein), whereas itwas drastically increased after growth on glucose plus acetate(1.10 U/mg of protein). The highest enzymatic activity wasobtained after growth in medium containing acetate alone(2.80 U/mg of protein). This result shows that ICL activity issubject to tight regulation in C. glutamicum.

Isolation of the aceA gene from C. glutamicum. To isolate theaceA gene from C. glutamicum, an attempt to complement theICL-deficient E. coli mutant K8-5m by heterologous comple-mentation using a pHC79-based cosmid gene library of chro-mosomal C. glutamicum DNA (6) was made. Because of ICLdeficiency, strain K8-5m is not able to grow on acetate as thesole carbon source. After transformation of the cosmid genelibrary into this strain, about 40,000 transformants werescreened for complementation, i.e., for growth on acetate-containing minimal medium. Unfortunately, none of the clonestested was able to grow on acetate, indicating either that theaceA gene from C. glutamicum is not expressed in E. coli, thatits expression is toxic in E. coli, or that the C. glutamicumenzyme bears different biochemical characteristics not allowingcomplementation of strain K8-5m. However, during parallelattempts to isolate the C. glutamicum malate synthase gene(aceB), cosmid pACB1 carrying an approximately 40-kb chro-mosomal C. glutamicum fragment and complementing amalate synthase-deficient E. coli strain was obtained (unpub-lished results). Considering the fact that in E. coli, the aceBgene forms an operon with the aceA gene (32), several largefragments from cosmid pACB1 were ligated into the E. coli-C.glutamicum shuttle vector pEKO, the resulting plasmids weretransformed into wild-type C. glutamicum and the recombinantstrains were tested for their ICL activities. By this approach,plasmid pEKA1 bearing a 6.2-kb NcoI fragment was obtained,resulting in increased ICL activity in C. glutamicum. A restric-tion map of the 6.2-kb NcoI fragment is given in Fig. 1. The6.2-kb NcoI fragment was used for further subcloning experi-ments. By this procedure, plasmid pEKA2 carrying a 2.4-kbHpaI-NciI fragment (Fig. 1) and still capable of conferringincreased ICL activity to C. glutamicum was obtained. Thespecific ICL activities of the wild-type strain C. glutamicum(ATCC 13032), strains ATCC 13032(pEKO), ATCC 13032(pEKA1), and ATCC 13032(pEKA2) after growth in minimal

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NiSt Bg Nr

Il Cl

aceA thiX0 1.0 b

FIG. 1. Restriction map of the chromosomal C. glutamicum 6.2-kbNcoI fragment conferring ICL activity to this organism. The 2.4-kbHpaI-NciI fragment derived from C. glutamicum is indicated by boldletters. Arrows represent ORFs identified after sequencing of the2.4-kb HpaI-NciI fragment. aceA is the gene encoding ICL, and thiX isthe ORF essential for thiamine biosynthesis. Abbreviations: Bg, BglI;Cl, ClaI; Hp, HpaI; Ni, NciI; No, NcoI; Nr, NruI; St, StuI.

medium containing glucose or acetate are given in Table 2. C.glutamicum ATCC 13032 (wild type) and ATCC 13032(pEK0)were indistinguishable in their enzyme activities, showing thatthe pure vector has no influence on ICL activity. After growthin glucose-containing minimal medium, strains ATCC 13032(pEKA1) and ATCC 13032(pEKA2) exhibited 8- and 10-fold-higher ICL activities than that of the wild type, respectively,and after growth in acetate-containing medium, both strainsshowed a 2.5- to 3.0-fold increased ICL activity over that of thehost. In both cases, increase of ICL activity was independent ofthe orientation of the inserts within the vector. This resultindicates that both fragments carried the intact aceA gene fromC. glutamicum, together with the structures necessary forexpression and regulation of aceA in C. glutamicum. It is worthmentioning that neither the 6.2-kb NcoI fragment nor the2.4-kb HpaI-NciI fragment conferred ICL activity to E. coli.This result shows that the C. glutamicum aceA gene is notexpressed in E. coli and explains the failure to obtain this geneby heterologous complementation of the E. coli mutant K8-5m.Since the aceA gene was obtained from cosmid pACB1 thatcomplemented a malate synthase-deficient mutant from E.coli, the two fragments, i.e., plasmids pEKA1 and pEKA2,were also tested for their ability to confer malate synthaseactivity to C. glutamicum or E. coli. However, neither of themcaused increased activity of this enzyme within the two organ-isms.

Southern hybridization was performed to test the structuralintegrity of the cloned fragments. For this purpose, the 2.4-kbHpaI-NciI fragment was labeled with digoxygenin dUTP. Byusing this probe in Southern blots with NcoI-digested chromo-somal C. glutamicum DNA, a signal at about 6.2 kb wasobtained (not shown). This result confirms that the isolatedNcoI fragment is organized in the same way as the chromo-some of C. glutamicum is.

Nucleotide sequence of the aceA gene. The nucleotide se-quence of the HpaI-NciI fragment was determined from both

TABLE 2. Specific activity of ICL in cell extracts of differentC. glutamicum strains after growth in minimal medium

containing different carbon sources

Sp act (U/mg of protein) withC. glutamicum strain carbon source:

Glucose Acetate

ATCC 13032 (wild type) 0.01 2.48ATCC 13032(pEKO) 0.01 2.65ATCC 13032(pEKA1) 0.08 6.20ATCC 13032(pEKA2) 0.10 7.40

strands by the dideoxy-chain termination method. The ob-tained sequence of 2,427 bp is given in Fig. 2. Computeranalysis for potential coding regions identified two openreading frames (ORFs). The first ORF extending from bp 517to 1812 exhibits the codon usage typical of highly expressedcorynebacterial genes (12). It is preceded by a typical ribosomebinding sequence (AAGGAAG) and followed by a structureresembling a rho-independent terminator (Fig. 2). The ORFencodes a polypeptide of 432 amino acids (aa), with a molec-ular weight of 47,228. Data base searches in the SwissProtlibrary revealed high identity to ICLs from other organisms(see below). From this result, it can be concluded that thisORF represents the ICL gene from C. glutamicum and it wastherefore named aceA.The second ORF, designated thiX, is also preceded by a

typical ribosome binding site (GAAAGGA). This ORF startsat bp 2027 and extends to the end of the sequenced fragment,indicating that it represents only the 5' part of a gene. Itscodon usage is in good agreement with that of moderatelyexpressed corynebacterial genes (12). However, data basesearches with the thiX gene and its deduced polypeptiderevealed no similarity to sequences stored in the GenBank orSwissProt data base.Comparison of the ICL from C. glutamicum with those from

other organisms. The primary structure of the C. glutamicumICL, as deduced from the nucleotide sequence of the aceAgene, was compared with the enzyme from other sources (Fig.3). In an alignment of the sequences, the C. glutamicumenzyme shows 57, 37, 38, and 36% identity to the enzymes fromE. coli (36), Candida tropicalis (1), Neurospora crassa (16), andBrassica napus (52), respectively. Considering conservativeexchanges, similarity is as high as 70, 53, 51, and 50%,respectively. The alignment of the two procaryotic enzymeswith those from eucaryotic organisms revealed two gaps of thesame size and location within the enzymes from C. glutamicumand E. coli. Compared with the enzyme from B. napus, the firstgap in the C. glutamicum enzyme starts at aa 16, covering 13 aa,and the second gap starts at aa 253, extending over 102 aa.Further analysis of the aligned ICL sequences showed fourdomains of high identity underlined in Fig. 3. For the E. colienzyme, lysine 193 and cysteine 195, both located within thefirst of these regions, as well as serine 319 and serine 321,situated in the third conserved domain, were identified asessential for catalysis (10, 21, 22, 34). In accordance with thesefindings, the C. glutamicum enzyme possesses identical aminoacids at the respective sites, indicating the importance of theseamino acids in catalysis.

Inactivation of aceA and of thiX in the chromosome of C.glutamicum. To analyze whether aceA is essential for thegrowth of wild-type C. glutamicum, its chromosomal copy wasinactivated by gene disruption, resulting in strain ASK1. Ingrowth experiments, the characteristics of strain ASK1 werecompared with those of wild-type C. glutamicum. Both strainsexhibited an unaltered growth rate on glucose, but mutantstrain ASK1 was no longer able to grow on acetate-containingminimal medium. This result demonstrates the necessity of theaceA-encoded ICL for the growth of C. glutamicum on acetate.When glyoxylate, one of the products of the ICL reaction, wasadded to the acetate-containing medium, growth of strainASK1 was restored. Further testing revealed that both the wildtype strain and mutant strain ASK1 were not able to grow onglyoxylate alone. The inability of strain ASK1 to grow onacetate and the restoration of growth after the addition ofglyoxylate indicate that in this strain glyoxylate is channelledinto the glyoxylate pathway, thereby circumventing its ICLdeficiency. In order to confirm the data obtained by the growth

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GTTAACGGTTGTGAAACTCTTTTAAGAAAGCACTCTGACTACCTCTGGAATCTAGGTGCCACTCTTCTTTCGATTTCAACCCTTATCGTGTTTGGCGA

TGTGATCAGACTAAGTGATCACCGTCACCAGCAAAGGGGTTTGCGAACTTTACTAAGTCATTACCCCCGCCTMACCCCGACTTTTATCTAGGTCACACC

TTCGAAACCTACGGAACGTTGCGGTGCCTGCATTTTCCCATTTCAGAGCATTTGCCCAGTACATCCGTACTAGCMACTCCCCCGCCCACTTTTTCTGCGA

AGCCAGAACTTTGCAAACTTCACAACAGGGGTGACCACCCCCGCACAAACTTMAAMACCCAAACCGATTGACGCACCMATGCCCGATGGAGCAATGTGT

GAACCACGCCACCACGCAAACCGATGCACATCACGTCGAAACAGTGACAGTGCATTAGCTCATACTTTGTGGTCGGCACCGCCCATTGCGAATCAGCACT

TMGGiMGTGACTTTGATGTCAAACGTTGGAAMGCCACGTACCGCACAGGAAATCCAGCAGGATTGGGACACCAACCCTCGTTGGAACGGCATCACCCGCRBS M S N V G K P R T A Q E I Q Q D W D T N P R W N G I T R

601 GACTACACCGCAGACCAGGTAGCTGATCTGCAGGGTTCCGTCATCGAGGAGCACACTCTTGCTCGCCGCGGCTCAGAGATCCTCTGGGACGCAGTCACCCD Y T A D Q V A D L Q G S V I E E H T L A R R G S E I L W D A V T

701 AGGAAGGTGACGGATACATCAACGCGCTTGGCGCACTCACCGGTAACCAGGCTGTTCAGCAGGTTCGTGCAGGCCTGAAGGCTGTCTACCTGTCCGGTTGQ E G D G Y I N A L G A L T G N Q A V Q Q V R A G L K A V Y L S G W

801 GCAGGTCGCAGGTGACGCCMCCTCTCCGGCCACACCTACCCTGACCAGTCCCTCTACCCAGCGAACTCCGTTCCAAGCGTCGTTCGTCGCATCAACAACQ V A G D A N L S G H T Y P D Q S L Y P A N S V P S V V R R I N N

901 GCACTGCTGCGTTCCGATGAAATCGCACGCACCGAAGGCGACACCTCCGTTGACAACTGGGTTGTCCCAATCGTCGCGGACGGCGAAGCTGGCTTCGGTGA L L R S D E I A R T E G D T S V D N W V V P I V A D G E A G F G

1001 GAGCACTCACGTCTACGACTCCAGAGGCATGATCGCAGCTGGCGCTGCAGGCACCCACTGGGAGACCAGCTCGCTTCTGAAMGAGTGTGGCCAG A L N V Y E L Q K A M I A A G A A G T H W E D Q L A S E K K C G H

1101 CCTCGGCGGCAAGGTTCTGATCCCAACCCAGCAGCACATCCGCACCCTGAACTCTGCCCGCCTTGCAGCAGACGTTGCMAACACCCCAACTGTTGTTATCL G G K V L I P T Q Q H I R T L N S A R L A A D V A N T P T V V I

1201 GCACGTACCGACGCTGAGGCAGCAACCCTGATCACCTCTGACGTTGATGAGCGCGACCAACCATTCATCACCGGTGAGCGCACCGCAGAAGGCTACTACCA R T D A E A A T L I T S D V D E R D Q P F I T G E R T A E G Y Y

130 1 ACGTCAAGAATGGTCTCGAGCCATGTATCGCACGTGCAAAGTCCTACGCACCATACGCAGATATGATCTGGATGGAGACCGGCACCCCTGACCTGGAGCTH V K N G L E P C I A R A K S Y A P Y A D M I W M E T G T P D L E L

1401 CGCTAAGAAGTTCGCTGAAGGCGTTCGCTCTGAGTTCCCAGACCAGCTGCTGTCCTACAACTGCTCCCCATCCTTCAMCTGGTCTGCACACCTCGAGGCAA K K F A E G V R S E F P D Q L L S Y N C S P S F N W S A H L E A

1501 GATGAGATCGCTMGTTCCAGMGGAACTCGGCGCAATGGGCTTCAAGTTCCAGTTCATCACCCTCGCAGGCTTCCACTCCCTCAACTACGGCATGTTCGD E I A K F Q K E L G A M G F K F Q F I T L A G F H S L N Y G M F

1601 ACCTGGCTTACGGATACGCTCGCGAAGGCATGACCTCCTTCGTTGACCTGCAGAACCGTGAGTTCAAGGCAGCTGAAGAGCGTGGCTTCACCGCTGTTAAD L A Y G Y A R E G M T S F V D L Q N R E F K A A E E R G F T A V K

1701 GCACCAGCGTGAGGTTGGCGCAGGCTACTTCGACCAGATCGCMCCACCGTTGACCCGAACTCTTCTACCACCGCTTTGAAGGGTTCCACTGAAGAAGGCH Q R E V G A G Y F D Q I A T T V D P N S S T T A L K G S T E E

1801 CAGTTCCACAACTAGGACCTACAGGTTCTGACAATTTAAATCTCCCTACATCTGTACMCGGATGTAGGGAGTTTTTCCTTATATATGCCCTCCACAAATQ F H N - >1 <

1901

2001

CCCCTATCGTGTGAGATGTGTTTCATAGGTGCCCCCAACGTTGCCTGTTGACTGCAAATTTTCCGAAAGAATCCATAMACTACTTCTTTAAGTCGCCAGA

TTAAAGTCGTCAATGAM6ACATACATGTCTATTTCCCGCACCGTCTTCGGCATCGCAGCCACCGCAGCCCTGTCTGCAGCTCTCGTTGCGTGTTCTCCRBS M S I S R T V F G I A A T A A L S A A L V A C S P

2101 ACCTCACCAGCAGGATTCCCCAGTCCAGCGCACCMTGAGATCTTGACTACTTCTCAGMCCCAACTTCTGCGAGCAGCACCTCMCCTCTTCCGCMCGP H Q Q D S P V Q R T N E I L T T S Q N P T S A S S T S T S S A T

2201 ACTACTTCCTCAGCTCCTGTGGAAGAGGACGTAGAGATCGTTGTTTCACCAGCAGCGTTGGTGGACGGTGAGCAGGTTACCTTCGAAATCTCTGGACTTGT T S S A P V E E D V E I V V S P A A L V D G E Q V T F E I S G L

2301 ATCCAGAGGGCGGCTACTACGCAGCGATCTGCGATTCCGTAGCGMCCCTGGTMCCCAGTTCCTTCTTGCACCGGCGAAATGGCTGATTTCACGTCCCAD P E G G Y Y A A I C D S V A N P G N P V P S C T G E M A D F T S Q

2401 GGCATGGTTGAGCAACTCCCAGCCCGGA W L S N S Q P

FIG. 2. Nucleotide sequence of the 2.43-kb HpaI-NciI fragment carrying the aceA and thiX genes from C. glutamicum. The predicted aminoacid sequences are shown below the sequence. Putative ribosome binding sites (RBS) and the potential terminating structure (inverted arrows) areindicated.

experiments, ICL activity of strain ASK1 was determined aftergrowth in minimal medium containing glucose or glucose plusacetate. In both cases, ICL activity was below the detectionlimit (<0.001 U/mg of protein).To test whether thiX is somehow involved in acetate metab-

olism or its regulation, it was inactivated within the chromo-some of C. glutamicum by the same method used for inactiva-tion of aceA, resulting in strain JAA1. Whereas strain JAA1showed unaltered growth on complex medium compared withwild-type C. glutamicum, it was not able to grow on minimalmedium, irrespective of the carbon source. Further testing of

this strain revealed an auxotrophy for thiamine. Althoughthiamine biosynthesis has not so far been completely eluci-dated, it is known for E. coli that coupling of the twoprecursors 4-methyl-5-(1-hydroxyethyl)thiazol and 4-amino-5-hydroxymethyl-2-methylpyrimidine-phosphate results in thia-mine monophosphate (for a review, see reference 7). There-fore, 4-methyl -5- (,B - hydroxyethyl)thiazol and 4-amino -5-hydroxymethyl-2-methylpyrimidine-phosphate were tested fortheir ability to supplement strain JAA1. Whereas the additionof 4-amino-S -hydroxymethyl-2-methylpyrimidine-phosphatedid not restore growth, supplementation with 4-methyl-5-(P-

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CGICLECICLCTICLNCICLBNICL






MSNVGKPRTAQEIQQD-------------WDTNPRWNGITRDYTADQVADLQGSVIEEHTLARRGSEI LWDAVTQEGD-GYINALGALTGNMKT--RTQQIEELQKE-------------W-TQPRWEGITRPYSAEDVVKLRGSVNPECTLAQLGAAKMWRLLHGESKKGYINSLGALTGGMAYTK----- IDINQEEADFQKEVAEIKKWWSEPRWRKTKRIYSAEDIAKKRGTLKIAYP-SSQQSDKLFKLLEKHDAEKSVSFTFGALDPMAANNMVNPAVDPALEDELFAKEVEEVKKWWSDSRWRQTKRPFTAEQIVSKRGNLKIEYA-SNAQAKKLWKILEDRFAKRDASYTYGCLEPMAASFSVPSMI--MEEEGRFEAEVAEVQTWWSSERFKLTRRPYTARDVVALRGHLKQGYA-.SNEMAKKLWRTLKSHQANGTASRTFGALDP* S * S *S S * SS* SSS S* S S S S S SS S S S S

QAVQQVRAGLKAVYLSGWQVAGDANLSGHTYPDQSLYPANSVPSVVRRINNALLRSDEIARTE-----GDTSVDNWVV----PIVADGEAGQALQQAKAGIEAVYLSGWQVAADANLMSMYPDQSLYPANSVPAVVERINNTFRRADQIQWSA-----GIEPGDPRYVDYFLPIVADAEAGIHVAQMAKYLDSIYVSGWQCSSTASTSNEPSPDLADYPMDTVPNKVEHLWFAQLFHDRKQREERLNMTKEERANTPYIDFLRPI IADADTGTMVTQMAKYLDTVYVSGWQSSSTASSSDEPGPDLADYPYTTCPNKVGHLFMAQLFHDRKQRQERLSVPKDQREKTPYIDFLRPI IADADTGVQVTMMAKHLDTIYVSGWQCSSTHTSTNEPGPDLADYPYDTVPNKVEHLFFAQQYHDRKQREARMSMSREERAKTPFVDYLKPI IADGDTG

S SSSS*S****SSS S SS ** S ** SS *S * SS S * SS S S S S **S**SSS*

FGGALNVYELQKAMIAAGAAGTHWEDQLASEKKCGHLGGKVLIPTQQHIRTLNSARLAADVANTPTVVIARTDAEMTLITSDVDERDQPFFGGVLNAFELMKAMIEAGAAAVHFEDQLASVKKCGHMGGKVLVPTQEAIQKLVARLAADVTGVPTLLVARTDADAMDLITSDCDPYDSEFHGGITAI IKLTKLFIERGMGIHIEDQAPGTKKCGHMAGKVLVPVQEHINRLVAIRASADIFGSNLLAVARTDSEMTLITSTIDHRDHYFHGGLTAVMKLTKLFIEKGMGIHIEDQAPGTKKCGHMAGKVLVPIQEHINRLVAIRAQADIMGSDLLCIARTDAEMTLITTTIDPRDHAFFGGTTATVKLCKLFVERGAAGVHIEDQSSVTKKCGHMAGKVLVAVSEHINRLVMRLQFDVMGTETVLVARTDAVMTLIQSNIDSRDHQF** S S* * SSS ***SS* *** S *****SS****SS S *S * S * *S S S S****S **S** SS * * **

ITG-----__ -____--_____--_____--ITG-----------------------------------------------------------______________________________I IGATNPES--GDLAALMAEAEAKGIYGDELARIETEWTKKAGLKLFHEAVIDEIKAGNYSNKEAL-- IKKFTDKVNPLSHTSHKEAKKLAI LGCTNPDL--EPLAHLMMKAEAEGKTGAQLQAIEDDWLAKADLKRFDEAVLDVIAKGKFSNAKDL--MKYQMVKG-KQISNREARAIAI LGVTNPSLRGKSLSSLLAEGMAVGNNGPALQAIEDQWLSSARLMTFSDAVVEALKRMNLSENEKSRRVNEWLNHARYENCLSNEQGRELA

---------------ERTAEGYYHVKNGLEPCIARAKSYAPYADMIWMETGTPDLELAKKFAEGVRSEFPDQLLSYNCSPSFNWSAH-LEAD--------------ERTSEGFFRTHAGIEQAISRGLAYAPYADLVWCETSTPDLELARRFAQAIHAKYPGKLLAYNCSPSFNWQKN-WDDKKELTGKDIYFNWDVARAREGYYRYQGGTQCAVMRGRAFAPYADLIWMESALPDYNQAKEFADGVKAVPDQWLAYNLSPSFNWN-KAMPADRQLLGQEI FFDWESPRTREGYYRLKGGCDCSINRAI SYAPYCDAIWMESKLPDYAQAEEFAKGPRVW-PEQKLAYNLSPSFNWK-TAMGRDAKLGVTDLFWDWDLPRTREGFYRFQGSVTMVVRGWAFAQIADLIWMETASPDLNECTQFAEGVKSKTPEVM1AYNLSPSFNWDASGMTDQ

*S **SSS SSS S *S SS*S * S* *S ** S **SS S *S *S** ****** S 5

EIAKFQKELGAMGFKFQFITLAGFHSLNYGMFDLAYGYAR-EGMTSFVDLQNREFKAAEERGFTAVKHQREVGAGYFDQIATTVDPNSST-TIASFQQQLSDMGYKFQFITLAGIHSMWFNMFDLANAYAQGEGMKHYVEKVQQPEFAAAKDGYTFVSHQQEVGTGYFDKVTTI IQGGTSSVEQETYIKRLGQLGYVWQFITLAGLHTTALAVDDFANQYSQ- IGMRAYGQTVQQPEI ---EKGVEVVKHQKWSGANYIDGLLRMVSGGVTSTDQETYIRRLAKLGYCWQFITLAGLHTTALISDQFAKAYSK- IGMRAYGELVQEPEI---DNGVDVVKHQKWSGATYVDELQKMVTGGVSSTQMMEFIPRIARLGYCWOFITLAGFHADALVVDTFAKDYAR-RGMLAYVERIQREER---SNGVDTLAHQKWSGANYYDRYLKTVQGGISST

S SS S*S S*******S*S S* *SS ** SS S S * S S **S *SS* * S SSS

7775859088

159161176181179

250252267272270

253255354358361

329331444447452

418422531534539

CGICL TALKGSTEEGQFHN----------------------- 432ECICL TALTGSTEESQF------------------------- 434CTICL AAMGAGVTEDQFKET------------------KAKV 550NCICL AAMGKGVTEDQFH------------------------ 547BNICL AAMGKGVTEEQFKETWTRPGAAGMGEGTSLVVAKSRM 576

S*S SSS*S**

FIG. 3. Comparison of the predicted ICL sequences from different organisms. Identical (*) and similar amino acids (s) are indicated below thesequences. Four conserved domains are underlined. The protein sequences are from C. glutamicum (CGICL), E. coli (ECICL) (36), Candidatropicalis (CTICL) (1), Neurospora crassa (NCICL) (16), and Brassica napus (BNICL) (52).

hydroxyethyl)thiazol led to normal growth of JAA1 comparedwith the wild-type. From these results, it can be concluded thatthiX is necessary for biosynthesis of the 4-methyl-5-(3-hydroxy-ethyl)thiazol component from which thiamine is formed. How-ever, it remains to be determined whether the thiX geneproduct catalyzes an as yet unknown enzymatic step within4-methyl-5-(3-hydroxyethyl)thiazol biosynthesis or whether itrepresents an already known enzyme with a different primarystructure.

Purification of ICL from C. glutamicum. A purificationscheme for ICL from C. glutamicum ATCC 13032(pEKA2) isgiven in Table 3. The enzyme was purified fourfold to apparent

homogeneity (Fig. 4), with 27% recovery after the last step,indicating that ICL represents about 25% of the solubleprotein fraction within C. glutamicum ATCC 13032(pEKA2).The purified enzyme could be stored at -20°C for 3 weekswithout loss of activity. By SDS-PAGE, the subunit molecularweight of ICL was determined to be about 48,000 (Fig. 4). Gelfiltration experiments revealed a molecular weight of about190,000, indicating that the native ICL from C. glutamicum is atetramer of identical subunits.

N-terminal amino acid sequence. In order to confirm thetranslational initiation site, the N-terminal sequence of puri-fied ICL from C. glutamicum was determined. It was found that

TABLE 3. Purification of the ICL from extracts of C. glutamicum ATCC 13032(pEKA2) after growth in minimal mediumcontaining acetate as the carbon source

Purification step Volume Total amt (mg) Total Sp act Recovery of Purification(ml) of protein activity (U) (U/mg) activity (%) (fold)

Crude extract 2.3 30.4 226 7.4 100 1.0Ultracentrifugation 10.0 12.4 127 10.3 56 1.4MonoQ, pH 6.0 4.0 2.7 76 28.2 34 3.8MonoQ, pH 8.0 2.0 2.0 60 30.0 27 4.1

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kDo M 1 2 3 4

170

85...

56

39

27-

20

14

FIG. 4. SDS-PAGE of crude extracts from wild-type C. glutamicum(ATCC 13032) and strain ATCC 13032(pEKA2) after growth inminimal medium containing glucose or acetate and of purified ICLfrom C. glutamicum. Lane M, molecular mass standards (i.e., a2-macroglobulin [170 kDa], ,B-galactosidase [116 kDa], fructose-6-phos-phate kinase [85 kDa], glutamate dehydrogenase [56 kDa], aldolase[39 kDa], triosephosphate isomerase [27 kDa], trypsin inhibitor [20kDa], and lysozyme [14 kDa]); lane 1, wild-type C. glutamicum grownin glucose; lane 2, wild-type C. glutamicum grown in acetate; lane 3,strain ATCC 13032(pEKA2) grown in acetate; lane 4, purified ICLfrom C. glutamicum.

the enzyme starts with the sequence S-N-V-G-K-P-R-T-A-Q.The obtained N-terminal sequence corresponds to the pre-dicted translational start of aceA at bp 517. The initial methi-onine residue predicted to be present by the DNA sequencewas not found in the purified enzyme, showing that it isremoved by processing (5).

Elfects of pH, metal ions, and temperature on ICL activity.The optimal pH for enzymatic assay was determined to be 7.3by using the MOPS buffer. In the absence of divalent cations,ICL from C. glutamicum showed no activity, whereas theaddition of Mg2+, Co2+, or Mn2+ enhanced enzyme activity.Mg2+ at 15 mM was found to be the most effective cation.Co2+ and Mn2+ were able to replace Mg2+, giving 31 and26%, respectively, of the maximal activity. To determine theoptimal temperature for enzymatic assay and to test enzymaticstability at different temperatures, the assay temperature wasdiscontinuously increased from 20 to 60°C. The enzymeshowed maximal activity at 40°C. At assay temperatures above45°C, the enzyme was denatured within the test, resulting innonlinear plots of activity.

Kinetic analysis of ICL. To perform investigations on thekinetic mechanism of ICL, the enzyme was functionally ana-lyzed in both the condensation and cleavage reactions. Instudying the condensation reaction, succinate gave linear dou-ble-reciprocal plots up to 5 mM at fixed glyoxylate concentra-tion. In the analogous experiment, glyoxylate showed linearityup to 1 mM. In both cases, replots of slopes and interceptswere linear with the reciprocal of concentrations of glyoxylateand succinate (data not shown). Inhibition patterns and con-

TABLE 4. Michaelis constants and inhibition characteristics ofpurified ICL from C. glutamicum

Michaelis orReaction and compound Type of inhibition inhibition

constant

CleavageDs-( + )-threo-isocitrate 0.28 mM

Succinate Linear noncompetitive 1.48 mMGlyoxylate Linear competitive 0.54 mMGlycolate Linear competitive 0.69 mMOxalate Linear competitive 7.10 p.M3-Phosphoglycerate Linear competitive 1.15 mM6-Phosphogluconate Linear competitive 1.07 mMItaconate Linear uncompetitive 5.05 ,uMPEP Linear uncompetitive 0.46 mMFructose-1,6-bisphosphate Linear mixed type 1.27 mM

CondensationGlyoxylate 0.34 mMSuccinate 0.61 mMInhibitor versus succinatePEP Linear competitive 0.42 mMGlycolate Linear mixed type 0.23 mM

Inhibitor versus glyoxylatePEP Linear uncompetitive 2.30 mMGlycolate Linear competitive 0.45 mM

stants of the cleavage and condensation reactions were deter-mined by the method of Segel (42) and are given in Table 4. Byanalyzing the cleavage reaction, succinate and glyoxylate, bothproducts of the ICL reaction, turned out to be linear noncom-petitive and linear competitive inhibitors of ICL, respectively.Glycolate and oxalate as structural analogs of glyoxylate werefound to be linear competitive inhibitors with regard toisocitrate. Itaconate and PEP as structural analogs of succinatecaused linear uncompetitive inhibition of ICL. In the conden-sation reaction, PEP was a linear competitive inhibitor withregard to succinate and showed linear uncompetitive inhibitionwith regard to glyoxylate. Glycolate turned out to be a linearmixed-type inhibitor towards succinate and a linear competi-tive inhibitor for glyoxylate. These results support an orderedUni-Bi mechanism for the cleavage reaction (42), in whichsuccinate leaves first and glyoxylate afterwards. This mecha-nism has also been proposed for the ICLs from other organ-isms (for a review, see reference 46).

Regulation of ICL activity. Since in C. glutamicum ICLactivity depended on the carbon source of the medium (seeabove), it was examined whether the responsible regulatorymechanism occurred at the genetic or protein level. SDS-PAGE analysis was performed to determine the portion of ICLin crude extracts of wild-type C. glutamicum and the recombi-nant strain ATCC 13032(pEKA2) after growth in glucose oracetate medium (Fig. 4). This quantitation was done bydensitometric measurements. After growth on glucose, ICLprotein was not detectable in crude extracts from wild-type C.glutamicum, whereas after growth on acetate, nearly 8% of thetotal cell protein consisted of ICL. This result demonstratesthat the activity of ICL is primarily regulated on the geneticlevel. In the aceA-overexpressing strain ATCC 13032(pEKA2),ICL represents approximately 25% of the total cell protein,which is in accordance with the data obtained after purificationof ICL from this strain. The difference in ICL content betweenthe wild-type C. glutamicum and the overexpressing straincorresponds well to the observed difference in enzymaticactivity (see above). The fact that under inducing conditions,

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ICL protein and specific activity in strain ATCC13032(pEKA2) is only threefold higher than in the wild-type C.glutamicum can be explained by the already high level of ICLwithin the wild type.

In order to analyze whether regulation of ICL at the level ofenzyme activity is involved in fine control of carbon fluxbetween the tricarboxylic acid cycle and the glyoxylate cycle,the affinity of ICL towards isocitrate as well as its inhibition bydifferent effectors was studied. The results of this investigationare given in Table 4. ICL from C. glutamicum showed amoderate affinity (Km = 0.28 mM) towards its substrateisocitrate. Whereas the most potent inhibitors oxalate anditaconate are probably of no physiological importance, 3-phos-phoglycerate, 6-phosphogluconate, PEP, fructose-1,6-bisphos-phate, and succinate, all intermediates in central metabolism,were also found to cause significant inhibition of ICL. Wheninhibition of ICL was studied with crude extracts of C.glutamicum instead of with purified enzyme, ICL was found tobe insensitive to PEP at a concentration of 1 mM (data notshown). This finding corresponds to the data obtained withpartially purified C. glutamicum subsp. flavum ICL, which wasshown not to be inhibited by PEP at a concentration of 1 mM(35). However, purified ICL from C. glutamicum was found tobe significantly inhibited by low concentrations of PEP (Table4). It might be speculated that PEP was rapidly metabolized incrude extracts and in the partially purified preparation andthus in both cases no inhibition by PEP was observed.

DISCUSSIONSeveral ICL genes from eucaryotes have been described so

far (1-3, 15, 16, 52), but apart from that from E. coli (33, 36)the aceA gene from C. glutamicum is the only one sequencedfrom a procaryotic organism. It is known from E. coli that theaceA gene forms an operon with the genes aceB and aceK inthe order aceB-aceA-aceK (28, 32). A similar organization hasalso been proposed for the respective genes in Salmonellatyphimurium (51). However, there are several lines of evidenceto suggest that the aceA gene from C. glutamicum has adifferent organization. Since ICL activity in C. glutamicum wasincreased independently from the orientation of the aceA genewithin vector pEKO, the presence of a functional promoterwithin the 0.5-kb region in front of the aceA gene can beproposed. The fact that ICL activity in the aceA-overexpressingstrain was also regulated by the carbon source indicates that inthe 0.5-kb region upstream of aceA, the structures responsiblefor regulation are also present. We therefore suggest that theaceA gene from C. glutamicum is not transcriptionally linked toan upstream gene. About 20 bp behind the aceA gene, apotential rho-independent transcription terminator was iden-tified, and further downstream the thiX gene involved inthiamine biosynthesis was found. Since there is no obviousconnection between acetate metabolism and thiamine biosyn-thesis, it is unlikely that the two genes form a transcriptionalunit. Taking all the data together, we assume that the aceAgene from C. glutamicum has a monocistronic organization.However, it is worth mentioning that the aceA gene wasisolated from cosmid pACB1, which complemented a malatesynthase-deficient E. coli strain. It is therefore likely that in C.glutamicum, the genes aceA and aceB are located in the sameregion within the chromosome. At present, studies to clarifythe genomic and transcriptional organization of these twogenes in C. glutamicum are in progress.

For Yersinia pestis, it has been reported that inhibition ofICL by itaconate reduced the growth rate of this organism onxylose (18). Recently, Yarrowia lipolytica mutants carrying an

inactivated ICL gene within their chromosome and showing animpaired growth rate on glucose were described (2). It wasspeculated that in these organisms, ICL is needed duringgrowth on carbohydrates to provide glyoxylate for use inglycine biosynthesis via serine (2, 18). However, the data mayalso indicate that in these organisms, ICL supports PEPcarboxylase in meeting the anaplerotic requirements duringgrowth on glucose. Compared with the wild-type C. glutami-cum, mutant strain ASK1, carrying an inactivated copy of aceAwithin its chromosome, exhibited an unaltered growth ratewith glucose as the sole carbon source. This result indicatesthat C. glutamicum requires its ICL during growth on glucoseneither to synthesize glycine via glyoxylate nor to fulfill anaple-rotic functions.

For the majority of ICLs from eucaryotic organisms, asubunit molecular weight of about 65,000 has been reported(for a review, see reference 46). In procaryotes, the ICLsubunit size seems to be more variable. For the ICLs from E.coli and a thermophilic Bacillus species, a weight of about48,000 has been determined (8, 38), whereas the ICLs fromAcinetobacter calcoaceticus and a Rhodopseudomonas speciesexhibited a subunit weight of around 63,000 (20, 45). Acomparison of the predicted ICL sequence from E. coli withthose from eucaryotic organisms revealed a gap of about 100aa in the middle of the E. coli enzyme, which is mainlyresponsible for its smaller size (33). By SDS-PAGE and bysequence analysis, the subunit weight of ICL from C. glutami-cum was determined to be 48,000, which corresponds well tothe size of the E. coli ICL. The alignment of the deduced ICLsequence from C. glutamicum with those from E. coli andeucaryotic organisms showed that for the enzyme from C.glutamicum, there was a gap of the same size and location asobserved within the E. coli enzyme. Therefore, it can besuggested that the location and size of this gap is common forthe small procaryotic ICLs. Since in eucaryotes ICL is synthe-sized in the cytoplasm and has to be transported into glyoxy-somes, it was speculated that the additional sequence withinthe eucaryotic enzymes contains one or more signals forglyoxysomal targeting (16; for a review, see reference 46).However, recently it was shown in an in vitro system that theN-terminal 168 aa of ICL from Ricinus plants were sufficientfor transport of the truncated enzyme into sunflower glyoxy-somes (4). This portion of the enzyme lacked the stretch,unique to the eucaryotic enzymes, that had been proposed tobear signals for sequestration into glyoxysomes. It was there-fore speculated that a signal within the N-terminal part of theeucaryotic enzymes is responsible for translocation of theenzyme into the glyoxysomes (4). It is worth mentioning thatboth the procaryotic enzymes show at their N-terminal part anadditional gap of 13 aa and that the eucaryotic enzymes exhibitwithin this respective region an accumulation of chargedamino acids. Furthermore, by inspection of all ICL sequencesso far known, at the N-terminal region of all eucaryotic ICLs(between aa 90 and 100), a common motif (M-A-K) could beidentified that is not present in the two procaryotic enzymes. Itwould be interesting to investigate whether this additionalstretch of charged amino acids or the 3-amino-acid motif is infact involved in sequestration of ICL into glyoxysomes.

During the analysis of metabolites that may have a physio-logical effect on ICL activity, fructose-1,6-bisphosphate,6-phosphogluconate, 3-phosphoglycerate, PEP, and succinatewere found to inhibit ICL activity at relatively low concentra-tions. In E. coli, the cellular concentrations of PEP and3-phosphoglycerate are assumed to be 0.22 and 2.5 mM,respectively (27, 31). If there were similar concentrations in C.glutamicum, 3-phosphoglycerate would increase the Michaelis

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constant of ICL from 0.28 to 0.89 mM, thereby being ofphysiological significance. In order to attribute a physiologicalrole to the inhibition of ICL by 3-phosphoglycerate, it is alsonecessary to analyze biochemically the ICD from C. glutami-cum. At present, these studies are in progress to obtain moreinformation about the regulation of carbon flux at the branch-ing point between ICL and ICD in C. glutamicum.

ACKNOWLEDGMENTS

This work was supported by grant BIOT-CT91-0264(RZJE) fromthe EC-BRIDGE program.We gratefully thank S. Peters for preparing the photos and J.

Carter-Sigglow for critically reading the manuscript.

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2. Barth, G., and T. Scheuber. 1993. Cloning of the isocitrate lyasegene (ICL1) from Yarrowia lipolytica and characterization of thededuced protein. Mol. Gen. Genet. 241:422-430.

3. Beeching, J. R., and D. H. Northcote. 1987. Nucleic acid (cDNA)and amino acid sequences of isocitrate lyase from castor bean.Plant Mol. Biol. 8:471-475.

4. Behari, R., and A. Baker. 1993. The carboxy terminus of isocitratelyase is not essential for import into glyoxysomes in an in vitrosystem. J. Biol. Chem. 268:7315-7322.

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6. Bormann, E. R., B. J. Eikmanns, and H. Sahm. 1992. Molecularanalysis of the Corynebactenium glutamicum gdh gene encodingglutamate dehydrogenase. Mol. Microbiol. 6:317-326.

7. Brown, G. M., and J. M. Williamson. 1987. Biosynthesis of folicacid, riboflavin, thiamine, and pantothenic acid, p. 521-538. InF. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M.Schaechter, and H. E. Umbarger (ed.), Escherichia coli andSalmonella typhimurium: cellular and molecular biology, vol. 1.American Society for Microbiology, Washington, D.C.

8. Chell, R. M., T. K. Sundaram, and A. E. Wilkinson. 1978. Isolationand characterization of isocitrate lyase from a thermophilic Bacil-lus sp. Biochem. J. 173:165-177.

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10. Diehl, P., and B. A. McFadden. 1993. Site-directed mutagenesis oflysine 193 in Escherichia coli isocitrate lyase by use of uniquerestriction enzyme site elimination. J. Bacteriol. 175:2263-2270.

11. Dixon, G. H., and H. L. Kornberg. 1959. Assay methods for keyenzymes of the glyoxylate cycle. Biochem. J. 72:3P.

12. Eikmanns, B. J. 1992. Identification, sequence analysis, and ex-pression of a Corynebacterium glutamicum gene cluster encodingthe three glycolytic enzymes glyceraldehyde-3-phosphate dehydro-genase, 3-phosphoglycerate kinase, and triosephosphate isomer-ase. J. Bacteriol. 174:6076-6086.

13. Eikmanns, B. J., E. Kleinertz, W. Liebe, and H. Sahm. 1991. Afamily of Corynebacterium glutamicumlEscherichia coli shuttlevectors for cloning, controlled gene expression and promoterprobing. Gene 102:93-98.

14. Eikmanns, B. J., M. Metzger, D. Reinscheid, M. Kircher, and H.Sahm. 1991. Amplification of three threonine biosynthesis genesin Corynebacterium glutamicum and its influence on carbon flux indifferent strains. Appl. Microbiol. Biotechnol. 34:617-622.

15. Fernandez, E., F. Moreno, and R. Rodicio. 1992. The ICLI genefrom Saccharomyces cerevisiae. Eur. J. Biochem. 204:983-990.

16. Gainey, L. D. S., I. F. Connerton, E. H. Lewis, G. Turner, and D. J.Ballance. 1992. Characterization of the glyoxysomal isocitratelyase genes of Aspergillus nidulans (acuD) and Neurospora crassa(acu3). Curr. Genet. 21:43-47.

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