INTRODUCTION

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Many bioactive drugs contain sugars attached to their aglycones which are usually important or, in some cases, essential for bioactivity. Most of these sugars belong to the family of the 6-deoxyhexoses (6-DOH) (18, 20, 27) and are transferred to the different aglycones as late steps in biosynthesis. Genes involved in the biosynthesis of different 6-DOH have been reported elsewhere and participate in the biosynthesis of erythromycin (9, 12, 31, 38, 39), daunorubicin (13, 26, 36), mithramycin (22), granaticin (2), streptomycin (10, 28), and tylosin (14, 23). However, information about the glycosyltransferases (GTFs) responsible for the transfer of the sugars to the respective aglycones is quite scarce. So far, only two GTFs from antibiotic producers have been biochemically characterized in detail, and they are involved in macrolide inactivation: Mgt, from Streptomyces lividans, a nonmacrolide producer (7, 17); and OleD, from the oleandomycin producer Streptomyces antibioticus (15, 29), which inactivates oleandomycin by addition of glucose to the 2'-OH group of the desosamine attached to the macrolactone ring (40). In the last several years, a few genes have been proposed to encode GTFs involved in the transfer of sugars to various aglycones during biosynthesis: dnrS and dnrH, from Streptomyces peucetius, involved in daunorubicin (26) and baumycin (36) biosynthesis, respectively; gra-orf5, involved in granaticin biosynthesis (2); eryCIII and eryBV, involved in the transfer of desosamine and mycarose, respectively, in erythromycin biosynthesis (12, 32, 38); and tylM2, from Streptomyces fradiae, involved in sugar transfer during tylosin biosynthesis (14).

Mithramycin (Fig. 1) is an aromatic polyketide which shows antibacterial activity against gram-positive bacteria and also antitumor activity (30, 37). Together with the chromomycins and the olivomycins, mithramycin constitutes the so-called aureolic acid group of antitumor drugs. The polyketide moiety of mithramycin is derived from the condensation of 10 acetate building blocks in a series of reactions catalyzed by a type II polyketide synthase (5, 21). The mithramycin aglycone is glycosylated at positions 6 and 2 with disaccharide (D-olivose- D-olivose) and trisaccharide (D-olivose-D-oliose-D-mycarose) moieties, respectively. All of these sugars belong to the 6-DOH family. In the mithramycin pathway, two genes (mtmD and mtmE) encoding two enzymes (glucose-1-phosphate:TTP thymidylyl transferase and dTDP-4,6-dehydratase, respectively) involved in the biosynthesis of the mithramycin 6-DOH have been cloned, and their participation in mithramycin biosynthesis has been demonstrated by insertional inactivation (22). Here we report the characterization of two Streptomyces argillaceus genes (mtmGI and mtmGII) that encode two putative GTFs responsible for the formation and transfer of the disaccharide chain. Inactivation of these genes by gene replacement showed identical accumulated compounds and allowed the isolation of four glycosylated compounds which are likely to be intermediates in mithramycin biosynthesis.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Structures of mithramycin, premithramycinone, and the new premithramycins.

	MATERIALS AND METHODS

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Bacterial strains, culture conditions, and vectors. S. argillaceus ATCC 12956, a mithramycin producer, was used as a source of chromosomal DNA. For sporulation, it was grown for 7 days at 30°C on plates containing medium A, consisting of MOPS (morpholinepropanesulfonic acid) (Sigma), 21 g/liter; glucose, 5 g/liter; yeast extract (Difco), 0.5 g/liter; meat extract (Lab-Lemco Powder; Oxoid), 0.5 g/liter; Casamino Acids (Difco), 1 g/liter; final pH, 7.0, adjusted with KOH. For protoplast regeneration, the organism was grown on R5 solid medium plates (16). Liquid medium for production and isolation of mithramycin intermediates was modified R5 medium containing the following: sucrose, 100 g/liter; K₂SO₄, 0.25 g/liter; MgCl₂ · 6H₂O, 10.12 g/liter; glucose, 10 g/liter; Casamino Acids, 0.1 g/liter; yeast extract (Difco), 5 g/liter; and MOPS, 21 g/liter. Two milliliters of R5 trace elements solution was added per liter, the pH was adjusted to 6.85, and the medium was sterilized by autoclaving. Escherichia coli XL1-Blue (6) was used as the host for subcloning and was grown at 37°C in Trypticase soy broth medium (Oxoid). M13mp18 and M13mp19 phage vectors were used for DNA sequencing. pUC18, pBSKT (a pBSK derivative containing a thiostrepton resistance cassette), and pIAGO (a pWHM3 derivative [39] containing the promoter of the erythromycin resistance gene [ermE] from Saccharopolyspora erythraea [4]) were used for subcloning.

DNA manipulation and sequencing. Plasmid DNA preparations, restriction endonuclease digestions, alkaline phosphatase treatments, ligations, and other DNA manipulations were performed or made according to standard procedures for E. coli (33) and for Streptomyces (16). Southern hybridization was performed according to standard procedures (16). Sequencing was performed on single-stranded templates derived from different clones in M13 phage by the dideoxynucleotide chain-termination method (34) with [-³⁵S]dCTP (1,200 Ci/mmol; Amersham) and modified T7 DNA polymerase (Sequenase version 2.0; U.S. Biochemicals). To overcome band compression artifacts, 7-deaza-dGTP was routinely used instead of dGTP (24). Single-stranded DNA was prepared by the polyethylene glycol precipitation method as described elsewhere (33). Both DNA strands were sequenced with primers supplied in the Sequenase kit or with internal primers (17-mer). Computer-aided database searching and sequence analyses were carried out with the University of Wisconsin Genetics Computer Group program package (8) and the BLAST program (1).

Insertional inactivation. For inactivation of the mtmGI gene, a 1.6-kb SmaI-XhoI fragment (sites 4 to 12 in Fig. 2A) was subcloned between the SmaI-SalI restriction sites of pUC18. The resulting construct was opened by the unique internal SalI site present in the insert (site 6 in Fig. 2A) and made blunt ended with the large fragment of E. coli DNA polymerase and an apramycin resistance cassette inserted as a 1.5-kb SmaI-EcoRV fragment. A thiostrepton resistance cassette was further added to this latter construct as a 1.7-kb SmaI fragment in the ScaI site located within the ampicillin resistance gene of pUC18, thus causing its inactivation. This final construct was designated pEFG1K (Fig. 3A). For inactivation of the mtmGII gene, a 2.1-kb PstI-NotI fragment (sites 1 to 7 in Fig. 2A) was subcloned into the same restriction sites of pBSKT containing the thiostrepton resistance cassette, and the apramycin resistance cassette was then subcloned in the unique SmaI site (site 4 in Fig. 2A) of the insert as a 1.5-kb SmaI-EcoRV fragment, generating pEFG2K (Fig. 3A). The orientation of the apramycin resistance gene in both constructs was in the same direction as that of the inactivated gene. Both constructs were then used to transform S. argillaceus protoplasts, integrants were selected for apramycin resistance (25 µg/ml) on R5 agar plates, and their susceptibility to thiostrepton (50 µg/ml) was tested. Am^r Thio^r integrants obtained in this primary screening were the consequence of a single-crossover event, and the screening for the second crossover essential to replace the wild-type region by the in vitro-disrupted one was carried out as described elsewhere (22).

View larger version (38K): [in this window] [in a new window]   FIG. 2.   (A) Schematic representation of the region sequenced from cosAR3 and location of the mtmGI and mtmGII genes with respect to previously reported genes from the mithramycin gene cluster. Mithramycin sugar biosynthetic genes (22) and mithramycin polyketide synthase genes (21) were designated mtm genes, while mithramycin resistance genes (11) were designated mtr genes. B, BamHI; G, BglII; H, SphI; L, SalI; N, NotI; P, PstI; S, SmaI; T, StuI; X, XhoI. (B) Alignment of the deduced amino acid sequences of different GTFs involved in antibiotic biosynthesis. MtmGI and MtmGII, mithramycin GTFs from S. argillaceus (this work); OleG1 and OleG2, oleandomycin GTFs from S. antibioticus (25); EryCIII and EryBV, desosaminyl and mycarosyl GTFs from Saccharopolyspora erythraea (12, 38); DnrH, baumycin GTF from S. peucetius (36); DnrS, daunorubicin GTF from S. peucetius (26); DauH, daunomycin GTF from Streptomyces sp. strain C5 (GenBank accession no. U43704); Gra-Orf5, granaticin GTF from S. violaceoruber (2); TylM2, tylosin GTF from S. fradiae (14).

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Analysis of gene replacement in the generation of mutants M3G1 and M3G2. (A) Scheme representing the replacement in the chromosome of the wild-type mtmGI and mtmGII genes by the in vitro-mutated ones. In the case of the mtmGI gene, an apramycin resistance cassette (black inverted triangle) was subcloned into the blunt-ended SalI site of mtmGI (generating mutant M3G1). In the case of mtmGII, the apramycin resistance cassette was subcloned into the unique SmaI site of mtmGII (generating mutant M3G2). B, BamHI; L, SalI; N, NotI; P, PstI; S, SmaI; X, XhoI. The asterisks above the two PstI sites indicate the boundaries of the probe used. (B) Southern hybridization with the 2.3-kb PstI fragment as the 32P-labeled probe. Chromosomal DNA from the wild-type strain and that from mutants M3G1 and M3G2 were digested with SmaI and analyzed by Southern hybridization. Lane 1, SmaI-digested chromosomal DNA from the wild-type strain. Lane 2, SmaI-digested chromosomal DNA from mutant M3G1. Lane 3, SmaI-digested chromosomal DNA from mutant M3G2.

Production and isolation of mithramycin intermediates. A seed culture was prepared in Trypticase soy broth medium inoculated with spores at an optical density at 600 nm of 0.3. After incubation for 24 h at 30°C and 200 rpm, this culture was used to inoculate (at 2.5% [vol/vol]) eight 2-liter Erlenmeyer flasks, each containing 400 ml of modified R5 medium. Production of mithramycin-related compounds was monitored at intervals as follows. Fifteen milliliters of culture was centrifuged, and the supernatant fluid was adjusted to pH 3.5 with formic acid and extracted with 5 ml of ethyl acetate. The ethyl acetate extract was then run on high-performance liquid chromatography (HPLC) with a µBondapak C₁₈ column (Waters) and eluted with a linear gradient from 10 to 100% acetonitrile in 0.1% trifluoroacetic acid (TFA) in water for 30 min, at 1.5 ml/min. Detection and spectral characterization of peaks were performed with a photodiode array detector (Waters). After 72 h of incubation, the whole culture was centrifuged and the supernatant was filtered (Supor membrane; 0.2-µm pore size; Gelman) and applied to a solid-phase extraction cartridge (Supelclean LC-18; 10 g; Supelco). The cartridge was washed with 0.1% TFA in water and eluted with a mixture of acetonitrile and 0.1% TFA in water. A linear gradient from 0 to 100% acetonitrile in 60 min, at 10 ml/min, was used. Fractions were taken every 5 min and subsequently analyzed by HPLC (see above). Products with spectral characteristics resembling those of mithramycin were found in fractions between 15 and 35 min. The products were purified by preparative HPLC in a µBondapak C₁₈ radial compression cartridge (PrepPak Cartridge; 25 by 100 mm; Waters). Short gradients with acetonitrile and 0.1% TFA in water, at a flow rate of 10 ml/min, were optimized for resolution of individual peaks. The material collected in each case was concentrated in vacuo and finally lyophilized.

Physicochemical characterization and structure elucidation of the new premithramycins. R_f values of putative mithramycin intermediates were determined on silica with a CHCl₃/CH₃COOH/CH₃OH/H₂O ratio of 58:34:7:1 as the solvent. Relative retention times (R_rel) in HPLC were determined on a Kontrosorp 10 C₁₈ semipreparative column with a flow rate of 5 ml/min with an H₂O (1% HCOOH)/CH₃CN ratio of 63:37 as the eluent. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Inova 500 instrument in d₆-acetone with instruments at field strengths of 7.05 and 11.94 T. Fast atom bombardment (FAB) mass spectra were recorded with nitrobenzylic alcohol as the matrix. UV and circular dichroism (CD) spectra were recorded in methanol. To obtain an acidic and alkaline UV spectrum, 1 drop of concentrated HCl and NaOH, respectively, was added to the methanol solution. Infrared (IR) spectra were measured as KBr pellets. The new structures were predominantly elucidated through FAB mass spectroscopy (MS) and NMR spectroscopy. Various standard NMR methods including ¹H NMR, broad-band H-decoupled ¹³C NMR, attached proton test (APT), homonuclear correlation spectroscopy, and heteronuclear correlation spectroscopy (hetero-multiple quantum correlation and hetero-multiple bond correlation [HMBC]) have been used to detect all ¹J_C-H and ⁿJ_C-H long-range couplings (n = 2 to 4), which allowed unambiguous assignments of all protons and carbons (if not stated otherwise in Table 1 or 2).

1

5

1

5

1

5

1

5

Nucleotide sequence accession number. The GenBank accession number for the fragment is AF077869.

	RESULTS

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Sequencing of the mtmGI and mtmGII genes. We have previously reported the cloning and characterization of a region of the chromosome of S. argillaceus ATCC 12956 (mithramycin producer) from clone cosAR3 that confers a high level of resistance to mithramycin in a heterologous host (11). We have now cloned (from clone cosAR3) and sequenced a 3-kb PstI-XhoI fragment located approximately 1.7 kb upstream of the mtrX gene and 13.5 kb downstream of the mithramycin polyketide synthase genes, mtmQXPKST1 (21) (Fig. 2A). The nucleotide sequence was analyzed for coding regions with the CODONPREFERENCE program (8), and two divergent open reading frames (ORFs) were observed. The first ORF (designated mtmGII) comprises 1,140 nucleotides and would code for a polypeptide with an estimated M_r of 40,308. The starting codon (ATG) of the second ORF (designated mtmGI) is 220 bp from mtmGII and is divergently transcribed from mtmGII. It comprises 1,182 nucleotides and would code for a polypeptide with an estimated M_r of 41,563. Both genes have a high GC content and the bias in the third codon position characteristic of Streptomyces genes.

Deduced functions of the MtmGI and MtmGII proteins. Computer analysis of mtmGI and mtmGII gene products with the BLAST program (1) identified similarities between the MtmGI and MtmGII proteins and GTFs involved in sugar transfer in biosynthesis of a number of antibiotics and antitumor drugs: DnrS from the daunorubicin pathway in S. peucetius (26), DnrH from the baumycin pathway in S. peucetius (36), DauH from the daunomycin pathway in Streptomyces sp. strain C5 (GenBank accession no. U43704), EryBV and EryCIII from the erythromycin pathway in Saccharopolyspora erythraea (12, 38), Gra-orf5 from the granaticin in Streptomyces violaceoruber (2), TylM2 from the tylosin pathway in S. fradiae (14), and OleG1 and OleG2 from the oleandomycin pathway in S. antibioticus (25). Both MtmGI and MtmGII proteins showed similar percentages of identity and similarity of amino acids with all of these antibiotic GTFs, ranging from 30 to 37% identity and 53 to 60% similarity of amino acids. MtmGI and MtmGII also resemble, although to a lesser extent, two GTFs that have been shown to be involved in macrolide inactivation in S. lividans (Mgt [17]) and in S. antibioticus (OleD [15, 29]). All of these GTFs and also MtmGI and MtmGII contain a characteristic motif present in UDP-glycosyltransferases and UDP-glucuronosyltransferases which is close to the C termini of the proteins (Fig. 2B).

Inactivation of the mtmGI and mtmGII genes. To assay the role of the mtmGI and mtmGII genes in sugar transfer during mithramycin biosynthesis, each gene was insertionally inactivated by insertion of an antibiotic resistance cassette. After the gene replacement experiments, one colony was selected from each experiment (designated M3G1 and M3G2 for inactivation of mtmGI and mtmGII, respectively). To verify that the DNA replacement was actually taking place, DNA from the wild-type strain and from the mutants M3G1 and M3G2 was prepared, SmaI digested, and analyzed by Southern hybridization. With a 2.3-kb PstI fragment (PstI sites are indicated by asterisks in Fig. 3A) as a probe, two SmaI hybridizing bands (1.4 and 1.6 kb) were observed in the wild-type strain, while in the mutants the SmaI bands changed as a consequence of the gene replacement to 1.4 and 3.1 kb (for mutant M3G1) and 1.6 and 2.9 kb (for mutant M3G2) (Fig. 3B). According to the restriction map of the region and that of the inserted apramycin resistance cassette, this Southern analysis indicated that the mtmGI and mtmGII genes had been replaced by the in vitro-mutated copies. To verify that the gene replacement was affecting only the mtmGI or mtmGII gene, both genes were independently subcloned into the shuttle vector pIAGO under the control of the erythromycin resistance promoter (see Materials and Methods) and introduced by transformation into both mutants. Each gene complemented the corresponding mutant, restoring mithramycin biosynthesis. However, cross-complementation was not observed: the mtmGI gene did not complement the mtmGII mutant and vice versa.

Characterization of the products accumulated by the M3G1 and M3G2 mutants. Both mutants (M3G1 and M3G2) were incubated in modified R5 liquid medium for 72 h at 30°C, the culture supernatants were extracted with ethyl acetate, and extracts were analyzed by HPLC (Fig. 4). Interestingly, the HPLC patterns from the extracts of both mutants were nearly identical. In both cases, no mithramycin was detected, but six major peaks showing the characteristic absorption spectra of mithramycin biosynthetic intermediates were observed. The material contained in four of these peaks (A, B, C, and D) from both mutants was purified by preparative HPLC (as described in Materials and Methods), and the structures of the compounds were elucidated and found to be identical in both mutants. Three compounds consist of nearly identical tetracyclic ring frame aglycones and a glycan chain attached at C-12a-O which varies in the amount of its deoxysugar units: premithramycin A1 (peak A) with an aglycone lacking a 9-CH₃ group and one D-olivose moiety and premithramycins A2 (peak C) and A3 (peak D) with aglycones containing a 9-CH₃ group and a disaccharide (D-olivose-D-oliose) and trisaccharide (D-olivose-D-oliose-D-mycarose) chain, respectively. The fourth identified compound, premithramycin A4 (peak B), consists of the tricyclic mithramycinone as the aglycone moiety and contains the same trisaccharide chain as that of premithramycin A3, which is attached at C-2-O (due to the different numbering in this molecular frame [Fig. 1 and below]). Although the four compounds show very similar UV spectra, they exhibit slight differences in their UV maxima: premithramycins A2 and A3 show identical absorption spectra, and premithramycins A1 and A4 show similar patterns but with maxima displaced at higher wavelengths (data not shown). The biosynthesis of these four compounds was monitored during growth, and we found that the patterns of biosynthesis of the different compounds were similar in both mutants (data not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 4.   HPLC analysis of the products accumulated by mutants M3G1 and M3G2. The mobility of mithramycin under the chromatographic conditions used was 17.8 min, a retention time between peaks B and C.

Elucidation of structure of the new premithramycins. The negative ion FAB MS spectra of the four new premithramycins, A1 to A4 (A1, m/z 543; A2, m/z 687; A3, m/z 831; A4, m/z 823), are in agreement with molecular formulae of C₂₇H₂₈O₁₂ (A1), C₃₄H₄₀O₁₅ (A2), C₄₁H₅₂O₁₈ (A3), and C₄₀H₅₆O₁₈ (A4).

	DISCUSSION

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Many bioactive compounds contain 6-DOH in their structures which, in many cases, are very important features for their biological activities. In mithramycin, DNA binding and antitumor activity are dependent on the presence of the sugars attached to the aglycone (35). The transfer of D-olivose (three molecules), D-oliose (one molecule), and D-mycarose (one molecule) in their activated forms (dTDP-sugars) to the mithramycin aglycone requires the participation of GTFs. We report here the identification of two genes from the mithramycin producer S. argillaceus encoding two GTFs presumably responsible for the transfer of the D-olivose disaccharide to the aglycone. These two genes (mtmGI and mtmGII) were identified in a cosmid clone, cosAR3, in which a mithramycin resistance determinant was previously found (11) and were located approximately 13.5 kb downstream of the mithramycin polyketide synthase (21). Independent insertional inactivation of both genes generated two nonproducing mutants which accumulated the same compounds as determined by HPLC analysis of the culture supernatants of both mutants followed by elucidation of the structures of the four major compounds. All four compounds lack the D-olivose disaccharide normally connected at C-6-O. Thus, the MtmGI and MtmGII GTFs should be involved in the transfer of this disaccharide. There are two alternative ways for the transfer of the disaccharide: (i) sequential and successive addition of both D-olivose molecules by independent GTFs or (ii) formation of the diolivosyl and transfer of the disaccharide to the aglycone. According to the former hypothesis, inactivation of one of the GTFs (that responsible for adding the second sugar) would result in a monosaccharide at C-6-O, while inactivation of the other GTF (responsible for transferring the first sugar unit) would produce compounds with no sugars in this position. In contrast, production of compounds not glycosylated at C-6-O would be obtained as a consequence of the inactivation of either GTF gene on the basis of the latter hypothesis. Identical compounds were isolated from culture supernatants of mutants M3G1 and M3G2 (premithramycins A1, A2, A3, and A4), thus supporting the disaccharide transfer hypothesis.

It has been proposed that the biosynthesis of mithramycin proceeds in its early stage through several tetracyclic intermediates, two of which (premithramycinone and 4-demethylpremithramycinone) have been isolated elsewhere (5, 22, 31). As a late step in biosynthesis, an enzyme (possibly an oxygenase) would be responsible for a C---C bond scission, leading to the typical tricyclic aureolic acid chromophore (31). Experimental evidence in support of this view comes from the isolation of premithramycins A3 and A4, glycosylated tetracyclic and tricyclic compounds differing only in the breakage of the fourth ring. However, it is still not yet clear when the corresponding oxygenase causes this C---C bond scission in the fourth ring, i.e., before, during, or after sugar addition. The isolation of premithramycin A4 (a glycosylated compound containing the trisaccharide at C-2-O but lacking the disaccharide at C-6-O) suggests that fourth ring breakage takes place at least after addition of the trisaccharide chain, but it is still unclear whether this happens before or after the disaccharide has been attached. Chromocyclomycin, a tetracyclic glycosylated compound containing a monosaccharide (D-mycarose) and a trisaccharide (D-olivose-D-oliose and D-mycarose), has been isolated from a mithramycin-producing streptomycete (3). This compound possibly represents a mithramycin-related compound in which the fission of the fourth ring has not yet occurred but which has been monoglycosylated at C-8-O (here with D-mycarose instead of with D-olivose) and suggests that breakage of the fourth ring might be a late step in biosynthesis, probably occurring after all sugars have been transferred into the aglycone (Fig. 5). The isolation of premithramycin A4 from the cultures of mutants M3G1 and M3G2 possibly indicates the existence of some kind of relaxed substrate specificity of the responsible oxygenase acting also on those tetracyclic intermediates in which some sugar moieties are lacking. Four oxygenase genes (mtmOI, mtmOII, mtmOIII, and mtmOIV) have been identified in the mithramycin gene cluster (28a), and recent experimental evidence strongly suggests that the mtmOIV product could be a candidate for breakage of the fourth ring (10a).

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Proposed pathways for mithramycin biosynthesis. CoA, coenzyme A.

Two methyl groups in the mithramycin aglycone must be introduced by methyltransferases: the O-methyl group at carbon 4 and the C-methyl group at carbon 9. Premithramycinone (31) is accumulated by the M7D1 mutant of S. argillaceus in which the mtmD gene encoding a glucose-1-phosphate:TTP thymidylyl transferase has been inactivated (22). Premithramycinone contains the O-methyl group but lacks the C-methyl group, thus indicating that O-methylation occurs before sugar transfer. Recent experimental evidence demonstrates that the mtmMI gene product encodes this O-methyltransferase (11a). Interestingly, premithramycins A2 and A3 contain the C-methyl group at carbon 9 while premithramycin A1 does not. This suggests that C methylation (possibly catalyzed by the MtmMII protein) must take place before the second D-olivose is transferred to the trisaccharide chain to convert premithramycin A1 into premithramycin A2.