APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.8.3603–3609.2001

Aug. 2001, p. 3603–3609 Vol. 67, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

The Phosphinomethylmalate Isomerase Gene pmi, Encoding anAconitase-Like Enzyme, Is Involved in the Synthesis of

Phosphinothricin Tripeptide in Streptomyces viridochromogenesE. HEINZELMANN, G. KIENZLEN, S. KASPAR, J. RECKTENWALD, W. WOHLLEBEN,

AND D. SCHWARTZ*

Mikrobiologie/Biotechnologie, Eberhard-Karls-Universitat Tubingen, D-72076 Tubingen, Germany

Received 31 January 2001/Accepted 7 May 2001

Streptomyces viridochromogenes Tu494 produces the antibiotic phosphinothricin tripeptide (PTT). In thepostulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles theaconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out bythe corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222, in L. C. Viningand C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCAcycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene,pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligo-nucleotides which were derived from conserved amino acid sequences of aconitases. The deduced proteinrevealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins fromeucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertionmutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inabilityof AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologouslyproduced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as asubstrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmiand AcnA are highly specific for their respective enzymatic reactions.

The structurally identical antibiotics phosphinothricin tri-peptide (PTT) and bialaphos are produced by Streptomycesviridochromogenes and by Streptomyces hygroscopicus (4, 18),respectively. They consist of two molecules, L-alanine and onemolecule of the unusual amino acid phosphinothricin (PT). Abiosynthetic pathway for bialaphos, consisting of at least 13steps, was postulated following analysis of nonproducing S.hygroscopicus mutants (summarized in reference 35). Severalenzymes were purified, and various genes of the PTT biosyn-thetic gene cluster were mapped in S. hygroscopicus (35), aswell as in S. viridochromogenes (1, 12, 28, 32). It was shown thatthe respective genes and enzymes were highly similar (up to80%) on the DNA and amino acid levels (29, 39, 40). As thegenetic organizations of the two clusters are basically identical,it has been concluded that the biosynthesis in both producingstrains proceeds in the same way.

The biosynthetic steps 6, 7, and 8 were found to be similar tothe citrate synthase, aconitase, and isocitrate dehydrogenasereactions of the tricarboxylic acid (TCA) cycle, respectively(Fig. 1). In contrast to the step 6 reaction, for which a specificPTT biosynthetic gene and protein were identified (15), thesubsequent steps, especially the isomerization of phosphinom-ethylmalate in step 7, were speculated to be catalyzed by theenzymes of the primary metabolism (35). Three facts sup-ported this. First, inhibition of aconitase resulted in a PTT-

negative phenotype; second, no mutants blocked in these stepscould be generated by nonspecific mutagenesis; and third, bio-transformations using crude cell extracts from Streptomyceslividans or Brevibacterium lactofermentum were possible (35).

The isolation and characterization of a PTT biosynthesis-specific aconitase-like gene in S. viridochromogenes, describedin this paper, casts doubt on this hypothesis.

MATERIALS AND METHODS

Bacterial strains, plasmids, phages, and growth conditions. The bacterialstrains, phages, and plasmids used in this work are listed in Table 1. Themorphological and physiological properties of wild-type S. viridochromogenesand of the pmi mutant were examined on yeast malt medium (YM) (28). Culti-vation was carried out at 30°C; liquid cultures were incubated in 100 ml ofmedium in an orbital shaker (180 rpm) in 500-ml Erlenmeyer flasks with steelsprings. The isolation of spores was done as described by Hopwood et al. (16).

Cloning, restriction mapping, and in vitro manipulation of DNA. Methods forisolation and manipulation of DNA were as described by Sambrook et al. (26)and Hopwood et al. (16). Restriction endonucleases were purchased from vari-ous suppliers and used according to their instructions. The oligonucleotides usedfor identification of aconitase genes were Ac1 (59-GGSAACCGSAACTTCGAGGGSCGS-39) and Ac2 (59-GTSACSACSGACCACATCTS-39).

Gene insertion mutagenesis and transformation. The mutant Mapra1 wasgenerated by polyethylene glycol-mediated transformation of wild-type proto-plasts with plasmid pEH14 as described by Hopwood et al. (16). The Escherichiacoli and Streptomyces plasmids used in the transformation of S. viridochromoge-nes were isolated from the methylase-negative strain E. coli ET 12567 (20) andS. lividans TK23, respectively. Transformation of E. coli was performed using theCaCl2 method described by Sambrook et al. (26). For standard cloning experi-ments, E. coli XL1 Blue was used.

Southern hybridization. Southern hybridization was carried out using thenonradioactive DIG DNA labeling and detection kit from Roche (Basel, Swit-zerland). Hybridizations using the oligonucleotides Ac1 and Ac2 were performedat 57°C with a stringent washing step with 13 SSC (13 SSC is 0.15 M NaCl plus0.015 M sodium citrate)–0.1% sodium dodecyl sulfate (SDS). In oligonucleotidehybridization experiments using chromosomal DNA as a template, the detection

* Corresponding author. Mailing address: Mikrobiologie/Biotech-nologie, Eberhard-Karls-Universitat Tubingen, Auf der Morgenstelle28, D-72076 Tubingen, Germany. Phone: 49 7071 29–74638. Fax: 497071 29–5979. E-mail: schwartz@molbio.biol.biologie.uni-tuebingen.de.

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was carried out by the chemiluminescence method (Roche). Hybridization ex-periments with the 2-kb EcoRI/SacI pmi fragment as a probe were done at 68°Cwith a stringent washing step with 0.13 SSC–0.1% SDS.

DNA sequencing and analysis. pmi genes containing DNA fragments weresubcloned in the sequencing vectors pK19, pUC18, and pBluescript SK(1). TheDNA sequences were determined by standard techniques (27). The DNA frag-ments were examined for open reading frames with the codon usage program ofStaden and McLachlan (5, 31). The programs BLAST (2), CLUSTAL W (36),GeneDoc (22), and TreeView (23) were used for homology searches, multiplealignments and phylogenetic trees. The accession numbers of genes used forconstruction of the phylogenetic tree are deposited in GenBank (J05224,X82841, Z73234, M33131, L22081, U17709, AF002133, U56817, X60293,U46154, D29629, AE000121, AE000590, Z75208, X53090, X84647, M31047,M58510, and U20180) and SwissProt (Q23500, P17279, P55251, and P55811).

Isolation of the pmi gene by PCR. The pmi gene was isolated by PCR. Thefollowing reaction mixture was used: 0.5 mg of pDS201 (a pK19 derivativecontaining a StuI fragment of l-WT8 carrying the pmi gene, which is subclonedinto the HincII site of the plasmid) as a template, 1.0 mM primer 1 (59-AAAGATCTCGCCGATTCCAAGAGGCC-39) and primer 2 (59-TTTAAGCTTTCACGCCGATTCCAAGAG-39), 10 ml of 103 reaction buffer (with 2 mM MgCl2),5% dimethyl sulfoxide, 0.2 mM deoxynucleoside triphosphates, and 0.5 ml of PwoDNA polymerase (Roche). After denaturation (3 min at 94°C), 25 cycles ofamplification (1 min at 94°C, 1.5 min at 60°C, and 2 min at 72°C) were performedin a PTC100 thermocycler from MJ Research, Inc. (Watertown, Mass.). ThePCR products were electrophoretically separated in a 1% agarose gel, isolated bygel elution (Qiaquick; Qiagen, Hilden, Germany), and directly employed forcloning.

Heterologous expression of pmi and purification of the His-tagged protein.YEME medium (200 ml) (16) with 25 mg of thiostrepton/ml and 10 mg ofkanamycin/ml in 1,000-ml Erlenmeyer flasks (with steel springs) was inoculatedwith 4 ml of homogenized cells of a 2-day-old preculture of S. lividansT7(pEH10) and incubated for 24 h at 30°C and 180 rpm. The cells were har-vested by centrifugation at 5,000 3 g and 4°C for 10 min and then resuspendedand incubated in ice-cold lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl,1 mg of lysozyme/ml, 10 mg of RNaseA/ml, 5 mg of DNase I/ml) (4 ml per g [wetweight]) for 30 min on ice. The cells were broken twice, using a French press(10,000 lb/in2). The insoluble protein fraction was harvested by centrifugation at

13,000 3 g for 30 min. The protein was purified from the soluble crude cellextract by metal chelate affinity chromatography using Ni-nitrilotriacetic acidresin according to the standard protocol provided by Qiagen. The collectedfractions were analyzed by standard SDS-polyacrylamide gel electrophoresis(PAGE) in 10% gels (26). The gels were stained with Coomassie brilliant blue,and fractions containing Hispmi* were pooled.

Determination of aconitase activity. The standard aconitase activity was as-sayed spectroscopically at 240 nm after the conversion of citrate to isocitrate.One unit of enzyme activity could convert 1 nmol of substrate (tri-sodium citratedihydrate) per min, and cellular activities are expressed as units per milligram(17).

Nucleotide sequence accession numbers. The nucleotide sequence data re-ported have been assigned accession no. Y17269 and Y17270 in the EMBL datalibrary.

RESULTS AND DISCUSSION

Identification of aconitase-like genes in the chromosome ofS. viridochromogenes. In order to examine the proposed role ofthe TCA cycle aconitase in PTT biosynthesis (35), we intendedto isolate the S. viridochromogenes aconitase gene. The de-duced amino acid sequences of aconitase genes from differentorganisms were aligned. Two oligonucleotides (Ac1 and Ac2)were deduced from conserved motifs (motif 1, GNRNFRGR;motif 2, VTTDHISPAG) and used in Southern hybridizationexperiments. By hybridization against SacI-restricted totalDNA from S. viridochromogenes, two predominant signals (7.5and 1.6 kb) were identified with both oligonucleotides, suggest-ing the occurrence of at least two genes for this group ofenzymes (Fig. 2). In further examinations, one of the signals (a1.6-kb SacI DNA fragment) was assigned to an internal frag-ment of the TCA cycle aconitase gene acnA (30).

FIG. 1. Comparison of selected reactions of PTT biosynthesis and the TCA cycle. The isomerizations of citrate and of phosphinomethylmalicacid are marked by boxes.

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Isolation of an aconitase-like gene in the PTT biosyntheticgene cluster. To check whether the second signal correspondedto a PTT biosynthetic gene, hybridization experiments with thetwo aconitase-related oligonucleotides were carried out. As atemplate, DNA of a l phage clone (l-WT8) carrying approx-imately 20 kb of the PTT biosynthetic gene cluster (29) wasused. A 7.5-kb SacI fragment hybridizing with Ac1 and Ac2was identified and shown to present the second signal (Fig.2A). In further subcloning experiments, it was subsequentlyrestricted to a 2-kb EcoRI/SacI DNA fragment.

Sequence analysis of pmi. The 2-kb EcoRI/SacI subfragmentwas cloned into pK18, resulting in pDS200. Its DNA sequencewas determined, together with those of adjacent fragments.One complete gene and two incomplete open reading frameswere identified on a 3.5-kb DNA fragment. The complete pmigene has a size of 2,667 bp and encodes a protein of 889 aminoacids. A putative Shine-Dalgarno sequence (59-GAGGAG-39)is located 6 bp in front of the GTG start codon. The gene isflanked upstream by the 39 region of the PTT synthetase Cgene (phsC), whose gene product is involved in the nonribo-somal synthesis of the tripeptide PTT (biosynthetic step 11)

(D. Schwartz, unpublished data), and downstream by a gene ofunknown function called orf2 (Fig. 3). pmi and the previouslydescribed TCA cycle aconitase gene acnA (30) show an iden-tity of approximately 68% at the DNA level. Whereas theG1C content in the coding region is approximately 72%, typ-ical for Streptomyces, the G1C content in the intergenic regionbetween phsC and pmi was determined to be 62 mol%. Nosignificant similarities to Streptomyces or E. coli promoters (6,14, 33) were found.

Identification of a promoter in the intergenic region of pmiand phsC. A 228-bp StuI/EcoRI fragment was subcloned intothe promoter probe vectors pIJ486 and pIJ487. S. lividans wastransformed with these plasmids. Using spores of plasmid-carrying strains, the kanamycin resistance was determined af-ter 2 days of incubation. Cloning of the fragment in the direc-tion of pmi transcription in front of aphII in pIJ486 (pDS80)enabled S. lividans to grow on Luria-Bertani medium contain-ing up to 800 mg of kanamycin ml21. When the fragment wascloned in the opposite direction (pDS81), no significant resis-tance (approximately 10 mg ml21) was obtained. pmi and theTCA cycle aconitase gene acnA seem to be differently regu-

TABLE 1. Bacterial strains, plasmids, and phages

Strain, phage, or plasmid Relevant genotype and phenotype Source or reference

S. viridochromogenesTu494 PTT-producing wild type 4Mapral Non-PTT producing; pmi::aprP Aprar This studyACOA TCA cycle aconitase gene mutant; acnA::aphII Kanr 30

S. lividansTK23 spec 16T7 tsr; T7 RNA polymerase gene J. Altenbuchner, personal

communicationE. coli

BL21 (DE3)pLysS F2 ompT hsdFB (rB2 mB

2) gal dcm (DE3) pLysS (Camr) 10, 34ET12567 F2 dam 13::Tn9 dcm-6 hsdM hsdR lacYI 20XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F9 proAB lacIqZD M15Tn10

(Tetr)]8

Phage l-WT8 l phage clone carrying approximately 20 kb of the PTT biosynthetic gene clusterfrom S. viridochromogenes

29

PlasmidspIJ486, pIJ487 pIJ101 derivate; tsr; promoterless aphII gene; promoter probe vector 38pDS77 pUC18 carrying the 7.5-kb SacI fragment of l-WT8 This studypDS80 pIJ486 carrying a 228-bp StuI/EcoRI fragment of the upstream region of pmi This studypDS81 pIJ487 carrying a 228-bp StuI/EcoRI fragment of the upstream region of pmi This studypDS200 pK18 carrying a 2-kb EcoRI/SacI pmi fragment of pDS77 This studypDS201 pK19 carrying native pmi on a 3.3-kb StuI fragment This studypEH5 pRSETB carrying pmi as a PCR-generated BglII/HindIII DNA fragment

(hispmip)This study

pEH7 pEH5 derivate carrying a 3.0-kb KpnI/HindIII fragment of pDS201 (hispmi) This studypEH10 pGM9 derivate carrying pEH7 as a HindIII fragment This studypEH13 pUC21 carrying the 1.8-kb apramycin-PermE resistance cassette This studypEH14 pDS200 derivate; 1.8-kb EcoRV/StuI fragment of pEH13 carrying the apramycin-

PermE resistance cassette (aprP) is inserted in the blunt-ended MluI site of the2-kb EcoRI/SacI pmi fragment

This study

pEH15 pK19 carrying the ermE promoter (PermE) as a 0.3-kb BamHI/KpnI fragment This studypEH16 pEH15 derivate carrying hispmi as a 3.2-kb XbaI/HindIII fragment of pEH7

under the control of PermEThis study

pEH17 pGM8 derivate carrying pEH16 as a HindIII fragment This studypEH20 pEM4 carrying the native pmi gene as a StuI fragment This studypEM4 Streptomyces-E. coli shuttle vector; tsr PermE 24pGM8 tsr aacCI; temperature-sensitive Streptomyces vector 21pGM9 aphII ble tsr; temperature-sensitive Streptomyces vector 21pIJ4026 pUC18 carrying ermE gene from Saccharopolyspora erythraea M. J. Bibb; John Innes Institute

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lated, since the identified promoter region of pmi showed nosimilarity to the corresponding region of acnA. In analogy tothe bialaphos gene cluster in S. hygroscopicus (35), the tran-scription of the pmi promoter is probably affected by the PTTpathway-specific activator PrpA, whose gene is located at theright boundary of the PTT biosynthetic gene cluster (D.Schwartz, unpublished).

Analysis of the deduced Pmi protein. The deduced Pmiprotein, with 889 amino acids, significantly resembled aconita-ses from plants, bacteria, and fungi and iron regulatory pro-teins (IRP) from eucaryotes. Structurally conserved amino ac-ids and cysteine residues involved in the formation of the[4Fe-4S] cluster typical for this class of enzymes (11) wereidentified. The greatest similarity was found to the TCA cycle

FIG. 2. Detection of aconitase-like genes in the genome of S. viridochromogenes. Chromosomal DNA from S. viridochromogenes and DNA froml phage l-WT8 were digested with the endonuclease SacI and applied in oligonucleotide hybridization experiments using the oligonucleotideprobes Ac1 and Ac2 derived from conserved motifs of aconitases. (A) DNA fragments (7.5 and 1.6 kb) hybridizing with both oligonucleotides wereidentified in the chromosomal DNA of S. viridochromogenes. (B) Lane 1, the hybridizing 7.5-kb SacI fragment was found on phage clone l-WT8carrying a part of the PTT biosynthetic gene cluster. Lane 2, pDS77 carrying the 7.5-kb SacI fragment of l-WT8. M, digoxigenin-labeled DNAmolecular weight marker VII (Boehringer).

FIG. 3. Genetic localization and gene insertion mutagenesis of the pmi gene. (A) Arrangement of genes on a 3.5-kb DNA fragment carryinga part of the PTT biosynthetic gene cluster. pmi, gene encoding Pmi; phsC9, 39 terminus of the PTT synthetase gene C (phsC); orf29, 59 region ofthe orf2 gene from S. viridochromogenes. Restriction sites used in subcloning experiments are marked, and the region with promoter activity isindicated by an arrow. The apramycin-PermE resistance cassette (aprP) and the insertion site used in gene inactivation of pmi are marked.Restriction sites which are destroyed by insertion of the cassette are indicated by brackets. The DNA regions which correspond to theoligonucleotides Ac1 and Ac2 and the EcoRI/SacI DNA fragment used as a probe are marked. (B) The correct gene replacement in mutantMapra1 is shown by Southern hybridization experiments using the 2-kb SacI/EcoRI pmi fragment as a probe. Lane 1, EcoRI-digested chromosomalDNA of S. viridochromogenes wild type; lane 2, EcoRI-digested chromosomal DNA of Mapra1.

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aconitases of Streptomyces coelicolor (GenBank accession no.AF180948) and Mycobacterium avium (GenBank accession no.AF002133) (with identities of 54 and 50%, respectively). Pmiand the previously described S. viridochromogenes TCA cycleaconitase AcnA (30) showed an overall identity of approxi-mately 52%. Comparison of the deduced amino acid sequencesof these two proteins to those of other members of the acon-itase family, such as aconitases, isopropylmalate isomerases,and homoaconitases, showed that both AcnA and Pmi belongto the AcnA-IRP group (Fig. 4). In addition, their domainstructures showed the characteristics of A-type aconitases.Three structurally conserved domains are linked to domain 4at the carboxy-terminal ends of the proteins (13). This impliesthat the specific Pmi and AcnA proteins from S. viridochromo-genes were generated by duplication of an ancestral aconitasegene. In Streptomyces, the duplication of structural genes is nota special feature. The occurrence of secondary-metabolismgenes which have a similar counterpart in the primary metab-olism has also been described for other genes, such as those forthe acyl carrier protein in actinorhodin and fatty acid biosyn-thesis in S. coelicolor (25) or the p-aminobenzoate synthasegene in folic acid synthesis and chloramphenicol biosynthesisin Streptomyces venezuelae (7).

Gene insertion mutagenesis of the pmi gene. In order toconfirm the involvement of pmi in PTT biosynthesis, the genewas inactivated by gene insertion (Fig. 3A). To avoid polareffects, the apramycin-PermE resistance cassette (aprP) wasinserted in the direction of transcription of pmi. The geneinsertion mutagenesis was performed using plasmid pEH14(Table 1). Apramycin-resistant, kanamycin-sensitive transfor-mants were analyzed in Southern hybridization experiments,and clones were identified showing a double-crossover eventbetween the chromosomal copy of pmi and the mutated frag-ment located on pEH14 (Fig. 3B). In comparison to the wildtype, the mutant Mapra1 lost the ability to produce PTT, but itshowed normal growth behavior and was able to form aerialmycelium and to sporulate. By genetic complementation usingplasmid pEH20, which carries the native pmi gene, the abilityof the mutant to produce PTT was restored (detected by abiological assay [1]), indicating that the insertion of the resis-tance cassette did not prevent the transcription of the PTTbiosynthetic genes located downstream. Under the conditionsof PTT production (after approximately 70 h of incubation),the crude cell extract of Mapra1 showed a specific aconitaseactivity (0.048 6 0.0037 U/mg of protein) nearly identical tothat of the wild-type extract (0.043 6 0.0013 U/mg of protein).

FIG. 4. Dendrogram showing phylogenetic relationships between Pmi and AcnA from S. viridochromogenes and other members of the aconitasefamily. The dendrogram was generated by the program TreeView (23) using a multiple alignment of aconitases, isopropylmalate isomerases(IPMI), homoaconitases, and IRP deposited in GenBank and SwissProt (according to reference 13).

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Therefore, the inability of the mutant to produce PTT revealedthat AcnA cannot substitute for the function of Pmi, despitethe high similarity of the two proteins.

Heterologous expression of pmi and protein purification. Inorder to characterize the Pmi protein, we intended to heter-ologously express the pmi gene in E. coli using the expressionplasmid pRSETB (Fa. Invitrogen, Groningen, The Nether-lands). A DNA fragment containing the pmi gene was gener-ated by PCR with BglII and HindIII restriction sites at its 39-and 59-terminal ends, respectively. The gene was cloned intothe BglII/HindIII-digested vector, resulting in plasmid pEH5(Table 1). By this cloning strategy, pmi transcription is underthe control of the strong T7 promoter, and a His-tagged codingsequence is fused to the 39-terminal end of the gene (hispmi).To exclude PCR-generated sequence faults, the sequence ofthe 59-terminal end was verified by DNA sequence analysis.Furthermore, a 2.6-kb KpnI/HindIII fragment of pEH5 (with aKpnI site at bp 105 of the pmi gene) was exchanged for a 3-kbKpnI/HindIII fragment of pDS201 carrying the native part ofpmi, resulting in pEH7 (hispmi*). E. coli BL21(DE3)/pLysSwas transformed with pEH7, and the induced cells were exam-ined for hispmi* expression. SDS-PAGE analysis of the crudecell extract showed no overexpression of pmi. The same resultswere achieved after protein purification by affinity chromatog-raphy under native or denaturating conditions and in Westernblotting experiments with anti-His-tag antibodies.

In addition to pRSETB, other expression plasmids (pQE30[Qiagen] and pJOE2775 or pJOE2702 [37]) were used, whichare characterized by other promoters (T5 or rhaP promoter) orby fusion of the His tag at either the 39- or 59-terminal end ofthe gene. In all these experiments, an expression of pmi wasnot detectable. Only by expression as a gst (glutathione S-transferase gene)-pmi fusion could a very small amount ofhybrid protein be obtained (data not shown). Similar problemswere also reported for the expression of other Streptomycesgenes, e.g., the chloroperoxidase gene from S. lividans (3) orthe peptide synthetase gene phsA from S. viridochromogenes(28), which failed or resulted in inactive proteins in E. coli. Itwas speculated (28) that the lack of accessory proteins mayplay a role in this. Since Pmi (like other proteins of the acon-itase family) is likely to possess a [4Fe-4S] cluster in its catalyticsite, specific accessory proteins may be required for the for-mation of this cluster. This has been described for the synthesisof [4Fe-4S] proteins in the nitrogen-fixing bacterium Azoto-bacter vinelandii and in other procaryotes (9, 19, 41).

Because E. coli was unsuitable for pmi expression, we pro-ceeded to express the gene in S. lividans T7 (Table 1). Thisstrain possesses a thiostrepton-inducible T7 RNA polymerasegene (J. Altenbuchner, personal communication). The E. colipmi expression plasmid pEH7, which carries hispmi* under thecontrol of the T7 promoter, was cloned as a HindIII fragmentinto the vector pGM9, resulting in the Streptomyces-E. colishuttle plasmid pEH10 (Table 1). S. lividans T7 was trans-formed with this plasmid, and after induction with thiostrep-ton, an overexpression of hispmi* was detected in the crudecell extract in SDS-PAGE (Fig. 5). The soluble protein waspurified by metal chelate affinity chromatography using Ni-nitrilotriacetic acid resin under native conditions (Fig. 5).

Test of standard aconitase activity. In order to test whetherPmi is able to catalyze the aconitase reaction of the TCA cycle,

the aconitase assay described by Kennedy et al. (17) was per-formed. No aconitase activity could be measured with either 50or 500 mM trisodium citrate. The absence of aconitase activityseems not to be a result of the protein purification, as it waspossible to purify the TCA cycle aconitase protein from S.viridochromogenes in an active form, using the same method(Schwartz, unpublished). In order to exclude the possibilitythat the chelating His tag has a negative effect on the [4Fe-4S]cluster of Pmi, the mutant Mapra1 was complemented withhispmi*. hispmi* was cloned as a 3.2-kb XbaI/HindIII fragmentinto the vector pEH15 downstream of the ermE promoter. Theresulting plasmid, pEH16, was inserted as a HindIII fragmentinto the vector pGM8 (Table 1), and the mutant Mapra1 wastransformed with the resulting plasmid, pEH17. By a biologicaltest described by Alijah et al. (1), it was shown that the com-plemented mutant produced the antibiotic PTT at the samelevel as the wild type, indicating that the His-tagged proteinpossesses sufficient activity to support this phenotype under theconditions used.

Genetic complementation of the TCA cycle aconitase mu-tant ACOA with hispmi*. In order to verify that the Pmi pro-tein is not able to catalyze the TCA cycle reaction, the TCAcycle gene (acnA) mutant ACOA was complemented usingplasmid pEH17. ACOA is characterized by a growth delay andby the inability to develop aerial mycelium and to sporulate(bald phenotype) (30). Furthermore, ACOA has a defect inphysiological differentiation shown by the loss of production ofthe secondary metabolite PTT, probably due to the absence oftranscription of the PTT biosynthetic genes, including pmi.Introducing the plasmid pEH17 had no effect on the pheno-type of ACOA, indicating that Pmi cannot carry out theisomerization of citrate and thus is specialized in the postu-lated secondary-metabolism reaction.

In the complete PTT biosynthetic gene cluster (Schwartz,unpublished), pmi is the only aconitase-like gene. Biochemicalstudies of PTT biosynthesis demonstrated that only the bio-synthetic step 7 (Fig. 1) shares chemical and structural char-acteristics with the aconitase reaction of the TCA cycle (35).Therefore, Pmi probably catalyzes the isomerization of phos-phinomethyl malic acid in this step. As the secondary metab-olite phosphinomethylmalic acid is unfortunately not commer-

FIG. 5. Heterologous expression of hispmi* in S. lividans T7 andpurification of the protein by metal chelate chromatography. Protein-containing fractions were analyzed by SDS-PAGE. Lane 1, crude cellextract; lane 2, flowthrough of unbound protein; lane 3, size marker;lanes 4 to 9, elution of the protein. The size of the overexpressedHis-tagged protein is marked by an arrow.

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cially available, its purification will be the essential step todemonstrate the postulated Pmi activity in further experi-ments.

ACKNOWLEDGMENTS

This research was supported by the DFG (Graduiertenkolleg Mik-robiologie) and by the BMBF (ZSP Bioverfahrenstechnik, D 3.2 E). G.Kienzlen and E. Heinzelmann were supported by grants from theKonrad-Adenauer-Stiftung and the Landesgraduiertenkolleg Baden-Wurttemberg, respectively.

We are very grateful to J. Altenbuchner for providing the S. lividansT7 strain. The antibiotic thiostrepton was kindly provided by S. J.Lucania, Squibb, New York, N.Y.

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