Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excission nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.
Mithramycin is a member of the family of the aureolic acid drugs . Members of this family show antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria because of a permeability barrier. They also show cytotoxicity against a variety of tumor cell lines and, particularly, mithramycin has clinical application in cancer treatment. All these compounds belong to the important family of polyketides . Structurally, mithramycin is an aromatic polyketide which is synthesized by the condensation of ten acetate units in a series of condensation reactions catalyzed by a type II polyketide synthase [1,3,4]. After the polyketide aglycon has been synthesized, several methylation  and glycosylation [6,7] steps take place originating a tetracyclic intermediate with trisaccharide (D-olivose-D-oliose-D-mycarose) and disaccharide (D-olivose-D-olivose) moieties attached. The final mithramycin molecule is produced after oxidative cleavage of the fourth ring of the glycosylated intermediate by the action of oxygenase . Several genes have been isolated involved in regulation of mithramycin biosynthesis and resistance to mithramycin in Streptomyces argillaceus. At one end of the cluster the mtmR gene codes for a regulatory protein of the SARP (Streptomyces antibiotic regulatory proteins) family . Its inactivation completely abolishes mithramycin biosynthesis and when expressed in the producer organism in a multicopy plasmid it caused a 16-fold increase in mithramycin production . So far, this is the only regulatory gene identified in mithramycin biosynthesis. At the other end of the cluster, a mithramycin resistance determinant has been identified  consisting of a mithramycin secretion system. Here we present further characterization of two additional genes of the mithramycin cluster and experimental evidence suggesting that one of them (mtrX) is possibly involved in self-resistance to mithramycin as a secondary defence mechanism and the other (mtrY) could participate in regulation of mithramycin biosynthesis.
2Materials and methods
2.1Microorganisms, culture conditions and vectors
S. argillaceus ATCC 12956, a mithramycin producer, was used as source of chromosomal DNA. Streptomyces albus G was used as host for transformation. For sporulation they were grown for 7 days at 30°C on plates containing medium A . Protoplast transformation was done according to standard procedures . For protoplast regeneration, the organism was grown on R5 solid medium plates . Liquid medium for production of mithramycin was modified R5 medium (R5A) . Escherichia coli XL1-Blue  was used as host for subcloning and was grown at 37°C in TSB medium (trypticase soy broth, Oxoid). pBSKT  was used for gene replacement experiments; this is a pBSK derivative containing a thiostrepton resistance gene. pIAGO  was used as expression vector for Streptomyces; it is a bifunctional (Streptomyces-E. coli) containing the erythromycin resistance promoter (ermEp) from Saccharopolyspora erythraea and harboring two resistance markers (ampicillin and thiostrepton resistance). pNAE1 is a pSL1108 derived vector (Amersham Pharmacia Biotech) containing an erythromycin resistance gene (ermE) and also containing an ampicillin resistance gene (N. Allende, unpublished results). pKC796  is a bifunctional plasmid with autonomous replication in E. coli and capable of integrating into the Streptomyces chromosome, harboring the apramycin resistance gene. When plasmid containing clones was grown, the medium was supplemented with the appropriate antibiotics: 5 or 50 μg ml−1 thiostrepton for liquid or solid cultures respectively, 25 μg ml−1 for apramycin, 100 μg ml−1 for ampicillin, 20 μg ml−1 erythromycin and 50 μg ml−1 hygromycin.
For inactivation of mtrX two adjacent BamHI fragments of the chromosome of S. argillaceus (2.1 kb and 6.5 kb; sites 3–5 and 5–12 in Fig. 1B) were positionated at both sides of a hygromycin resistance cassette in pHYG1 (kindly provided by A. Jiménez). This is a pBSK derivative that contains a hygromycin (hyg) resistance gene without its transcriptional terminator. The 2.1 kb BamHI fragment was first subcloned in the same site of pHYG1 upstream of the hyg gene (pJG103). For cloning of the 6.5 kb BamHI fragment, it was first subcloned in pKC796  and then rescued as a EcoRV-HindIII (blunt-ended) fragment and finally subcloned in the same sites of pJG103, downstream of the hyg gene. This final construct (pJG122) contains the mtrX gene interrupted by the hyg gene orientated in the direction of transcription of mtrX. For inactivation of mtrY, a 7.5 kb BglII fragment (sites 2–11 in Fig. 1B) was subcloned into pBSKT. Then, an erythromycin resistance cassette (ermE; ) was subcloned as a PstI-NsiI fragment from pNAE1 into the NsiI site (site 8 in Fig. 1B) located within the mtrY gene. In this construct (pJG118) the resistance cassette is orientated in the same direction as mtrY. The two final plasmids (pJG122 and pJG118) constructed in suicide vectors (not able to replicate in Streptomyces) were used to transform protoplasts of S. argillaceus. Integrant clones were selected for resistance to the antibiotic marker cloned within the gene to be inactivated (hygromycin for mtrX and erythromycin for mtrY). To facilitate the occurrence of the second crossover, a resistant colony from both transformations was cultivated for 72 h in the absence of antibiotic and finally plated on antibiotic-containing agar plates. The occurrence of the double crossover replacing the wild-type region in the chromosome by the in vitro mutated copy was verified by Southern hybridization. In the case of the mtrY mutant the loss of the plasmid was initially detected by susceptibility to the antibiotic (thiostrepton) marker in the vector (for mtrY). This was not possible for mtrX mutant since only one antibiotic marker was present in the plasmid construct.
Both mtrX and mtrY were independently expressed in a bifunctional (Streptomyces-E. coli) plasmid (pIAGO) under the control of a strong promoter, the erythromycin resistance promoter (ermE*p) from S. erythraea. The mtrX gene was subcloned as a 3.4 kb PstI-NsiI fragment (sites 4–8 in Fig. 1B) in the PstI site of pIAGO and the mtrY as a 1 kb blunt-ended SalI fragment (sites 7–9 in Fig. 1B) into the BamHI site (blunt-ended) of pIAGO. The final constructs were named pJG145 and pJG144, respectively.
Spores of the different strains or clones were initially grown in TSB medium during 24 h at 30°C and 250 rpm. This seed culture was used to inoculate (at 2.5%, v/v) Erlenmeyer flasks containing 50 ml of R5A medium. At different time intervals, samples were removed, centrifuged, filtered, extracted and analyzed by HPLC as described .
3Results and discussion
From a cosmid library of chromosomal DNA of a mithramycin producer, S. argillaceus, two cosmid clones were isolated (cosAR3 and cosAR4) that conferred high level of resistance to mithramycin when transformed into S. albus. The resistant determinant was localized within a PstI-PvuII fragment (sites 6–10 in Fig. 1B) and characterized as an ABC (ATP-binding cassette) transporter coded by the mtrA (hydrophilic ATP-binding protein) and mtrB (hydrophobic membrane protein) genes . Two additional genes were identified immediately upstream of mtrA: mtrX and mtrY. These two genes were not essential for conferring resistance to mithramycin in a heterologous host since subcloning only mtrA and mtrB conferred high level of resistance to mithramycin in S. albus and in frame mutations introduced in the mtrX and mtrY genes did not affect mithramycin resistance in S. albus providing mtrA and mtrB were normally expressed . In order to clarify the role of mtrX and mtrY in mithramycin biosynthesis and resistance both genes were now independently inactivated by gene replacement and the effect on growth, mithramycin biosynthesis and resistance determined.
3.1The mtrX gene could act as a secondary resistance mechanism
It has been previously shown that the mtrX gene product resembles one of the subunits (UvrA) of different ABC excission nuclease systems responsible for DNA repair in bacteria . These proteins have ATPase activity and DNA-binding domains. MtrX protein also resembled DrrC from the daunorubicin gene cluster in Streptomyces peucetius and SnorO from the nogalamycin gene cluster in Streptomyces nogalater (accession number AJ224512) (Fig. 2). The mtrX gene was inactivated by gene replacement by interrupting its expression by the presence of a hygromycin resistance cassette. This was confirmed by Southern hybridization analysis: using as probe a 2 kb PstI fragment (Fig. 1B) the wild-type strain showed a single 2 kb PstI hybridization band while the mutant showed two bands (1 and 1.6 kb) due to the interruption of mtrX by the insertion of the hyg cassette. The mutant generated (M3X1) still produced mithramycin but showed a very slow growth rate on both solid and liquid media and in liquid medium tended to form aggregates which are not so frequent for S. argillaceus under the culture conditions used. When inoculated on solid agar plates it gave rise to isolated colonies with a number of colonies per plate not corresponding with the inoculum size. Consequently, inactivation of mtrX affected in some way the normal growth of this mutant. The normal growth was recovered when the mutant was retransformed with the mtrX gene. To verify if inactivation of mtrX affected mithramycin resistance, the minimal inhibitory concentration of M3X1 mutant was determined in comparison with the wild-type strain. No significative difference in resistance to mithramycin was observed between the mutant and the wild-type strain. The mtrX gene was expressed in S. albus under the control of the erythromycin resistance promoter (ermE*p) in a multicopy plasmid vector (pIAGO) and it was found that it conferred a low, but significant, increase in resistance to mithramycin: growth of S. albus only containing the vector (pIAGO) was totally inhibited in the presence of 2 μg ml−1 mithramycin while clones containing pJG145 (expressing mtrX) grew until 5–6 μg ml−1. The conclusions from these experiments are that mtrX is not essential for biosynthesis of mithramycin (since mithramycin was still produced by M3X1 mutant) but normal growth of the strain is affected if the gene is inactivated. Furthermore, expression of the gene confers to S. albus the capability of surviving in the presence of low mithramycin concentrations. Since the ABC transporter coded by mtrA and mtrB confers higher level of resistance to mithramycin, we propose that the mtrX gene product could act as a secondary self-resistance mechanism responsible for repairing minor DNA damages caused by mithramycin molecules that escape the efficiency of the ABC transporter to remove the drug from the cytoplasm. Consequently, in the absence of this alternative defense mechanism (i.e. in the M3X1 mutant), normal growth during mithramycin biosynthesis is impaired.
3.2The mtrY gene codes for a positive regulatory element involved in mithramycin biosynthesis
Comparison of the mtrY gene product with proteins in databases did not reveal any significant similarity with other proteins that could suggest a possible function for this protein. To assay the importance of a functional MtrY protein in mithramycin biosynthesis the mtrY gene was inactivated by gene replacement. Verification that the replacement took place was carried out by Southern hybridization analysis: using as probe a 1.9 kb SmaI fragment (Fig. 1B) the wild-type strain showed a 5 kb PstI-BglII hybridizing band while in the mutant this band changed to 6.5 kb due to the insertion of the ermE gene within mtrY. Mithramycin was still produced by the corresponding mutant (M3Y1) but its biosynthesis was reduced in approximately 50% (Fig. 3A). Moreover, the mutant was highly resistant to mithramycin indicating that no polar effect on transcription of downstream genes (i.e. mtrA and mtrB) had taken place. Furthermore, the mutant was complemented by expressing the mtrY gene in a plasmid vector. All these experiments suggest that mtrY does not code for a mithramycin structural gene but rather it could code for a regulatory element. Expression of this regulatory gene would be required for production of normal levels of the drug. If this was the case, its expression in the wild-type strain in a multicopy plasmid could cause an increase in the level of mithramycin biosynthesis. This was assayed by cloning mtrY under the control of a strong promoter, the erythromycin resistance gene promoter ermE*p, and transforming the wild-type strain with this construct (pJG144) (Fig. 3B). During the first 2 days of incubation the levels of mithramycin production were similar in the control strain (only containing the vector pIAGO) and in the recombinant strain harboring pJG144; however, in the latter strain, production of mithramycin extended for 3 additional days once the control strain reached a plateau in the production levels. Furthermore, the levels of mithramycin production were higher in the recombinant strain expressing mtrY with approximately 47% increase. These experiments strongly suggest that mtrY gene product probably codes for a positive regulatory element, expression of which is important for normal levels of production of mithramycin. MtrY has no similarity to any known regulatory protein in databases. However, analysis of the MtrY amino acid sequence with the PEPTIDESTRUCTURE program of the GCG package  reveals an amino acid sequence close to its N-terminus (LLLEHAMHPYEMATLREN) that could represents a putative helix-turn-helix motif (HTH) characteristic of proteins that interact with DNA. It must be here emphasized that another regulatory gene (mtmR) has been identified in this pathway . In this case, inactivation of mtmR completely abolished mithramycin biosynthesis. Further experiments will be required to elucidate how these two regulatory proteins (and perhaps other additional ones) control expression of the mithramycin gene cluster.
This work was supported by grants to J.A.S. of the Plan Nacional en Biotecnología (BIO97-0771) and from the European Union (BIO4-CT96-0068).