ORIGINAL PAPER

C. Olano á A. M. Rodriguez á J.-M. Michel á C. Me ndezM.-C. Raynal á J. A. Salas

Analysis of a Streptomyces antibioticus chromosomal region involvedin oleandomycin biosynthesis, which encodes two glycosyltransferasesresponsible for glycosylation of the macrolactone ring

Received: 3 December 1997 /Accepted: 12 May 1998

Abstract A 6-kb region from the chromosome ofStreptomyces antibioticus, an oleandomycin producer,was cloned and sequenced. This region was located be-tween the 3¢ end of the gene encoding the third subunitof the oleandomycin type I polyketide synthase and theoleP and oleB genes, which encode a cytochrome P450monooxygenase and an oleandomycin resistance gene,respectively. Analysis of the nucleotide sequence re-vealed the presence of ®ve genes encoding a cytochromeP450-like protein (oleP1), two glycosyltransferases (oleG1and oleG2) involved in the transfer of the two 6-deoxy-sugars (L-oleandrose and D-desosamine) to the olean-domycin macrolactone ring, a methyltransferase(oleM1), and a gene (oleY) of unknown function. In-sertional inactivation of this region by gene disruptiongenerated an oleandomycin non-producing mutantwhich accumulated a compound that, according to massspectrometry analysis, could correspond to the olean-domycin macrolactone ring (oleandolide), suggestingthat the mutation a�ects oleandrosyl glycosyltransfer-ase.

Key words Macrolides á Glycosyltransferase áDeoxyhexoses á Actinomycetes

Introduction

Macrolides constitute a group of antibiotics that areactive mainly against gram-positive bacteria and have

clinical application in the treatment of bacterial infec-tions (Nakayama 1984). Oleandomycin (Fig. 1) is a14-membered macrolide synthesized by Streptomycesantibioticus that is structurally quite similar to ery-thromycin. It contains a macrolactone ring (oleandolide)which is synthesized by the assembly of one starteracetylCoA and six extender methylmalonylCoA units.Later on, two 6-deoxysugars (L-oleandrose andD-desosamine) are attached to the aglycon. As latesteps, C8 hydroxylation and epoxide formation occurafter the transfer of the two sugar residues onto themacrolactone (Spagnoli et al. 1983). Several genes of theoleandomycin biosynthetic pathway have been charac-terized. A large gene encoding the third subunit of theoleandomycin type I polyketide synthase has beencloned and sequenced, revealing a coding sequence thatis unusual for Streptomyces genes (Swan et al. 1994). Inaddition, two oleandomycin resistance genes (oleB andoleC) have been cloned and characterized; both encodeABC transporters (Rodriguez et al. 1993; Olano et al.1995). In the case of oleB it has been demonstrated thatthis transporter can secrete an inactive glycosylated in-termediate (Olano et al. 1995). This glycosylated olean-domycin is generated by the action of an intracellularglycosyltransferase activity, which transfers a glucosemoiety to the 2¢-hydroxyl group of the desosaminemoiety of oleandomycin (Vilches et al. 1992; Quiro s andSalas 1995). This glycosyltransferase has been puri®ed tonear homogeneity and found to act by a compulsory-order mechanism, having a high speci®city for olean-domycin, and acting with lower a�nity only on a fewother macrolides (Quiro s and Salas 1995).

We have initiated a characterization of the biosyn-thetic pathway for the deoxysugars of oleandomycin, inorder to gain a better understanding of the genetic andbiochemical basis of oleandomycin biosynthesis, and inorder to generate new compounds with modi®ed sugars.Here we report the characterization of a region of thechromosome of the oleandomycin producer Streptomy-ces antibioticus containing ®ve genes, two of which(oleG1 and oleG2) share signi®cant similarities with

Mol Gen Genet (1998) 259: 299±308 Ó Springer-Verlag 1998

Communicated by A. Kondorosi

C. Olano á A. M. Rodriguez á C. Me ndez á J. A. Salas (&)Departamento de Biologõ a Funcional, Instituto Universitariode Biotecnologia de Asturias (I.U.B.A-C.S.I.C),Universidad de Oviedo, E-33006 Oviedo, SpainFax: +34-85-103652

J.-M. Michel á M.-C. RaynalInfectious Disease Group, Hoechst Marion Roussel,102 Route de Noisy, 93235 Romainville Cedex, France

glycosyltransferase-encoding genes. Insertional inacti-vation in this region abolishes the transfer of the twosugar residues (D-desosamine and L-oleandrose), dem-onstrating that some, if not all, of these genes encodeenzymatic activities involved in the biosynthesis andtransfer of these two sugar residues in the course ofoleandomycin biosynthesis.

Materials and methods

Bacterial strains, culture conditions and vectors

Streptomyces antibioticus ATCC 11891, an oleandomycin produc-er, was used as a source of chromosomal DNA. For sporulation itwas grown for 7 days at 30°C on GAE plates (Hardisson et al.1978). Typically growth in liquid medium was carried out at 30°Cin TSB medium (trypticase soya broth, Oxoid) or in a syntheticminimal medium (Vilches et al. 1990) for analytical purposes. Forfermentation studies liquid cultures were grown in EP1 and EP2media as described (Salah-Bey et al. 1998). Escherichia coli TG1recO1504::Tn5 (Kolodner et al. 1985) was used as a host for sub-cloning. Bluescript SK(+) (Stratagene), pSL1180 (PharmaciaBiotech) and pUC18 were used as vectors for subcloning experi-ments and M13mp18 and M13mp19 phage vectors were used forDNA sequencing. pOJ260 (Bierman et al. 1992) was obtained fromthe NRRL (US Dept. of Agriculture), and used for gene disruptionexperiments.

DNA manipulation and sequencing

Plasmid DNA preparations, restriction endonuclease digestions,alkaline phosphatase treatments, ligations and other DNA ma-nipulations were performed according to standard procedures forEscherichia coli (Sambrook et al. 1989) and for Streptomyces(Hopwood et al. 1985). Southern hybridization was carried out bystandard procedures (Hopwood et al. 1985). Sequencing was per-formed on single-stranded DNA templates derived from variousclones in M13 using the dideoxynucleotide chain-terminationmethod (Sanger et al. 1977), with [a-35S]dCTP (1200 Ci/mmol;Amersham) and modi®ed T7 DNA polymerase (Sequenase version2.0, USB). To overcome band compression artifacts, 7-deaza-dGTP was routinely used instead of dGTP (Mizusawa et al. 1986).Single-stranded DNA was prepared by the polyethylene glycolprecipitation method described by Sambrook et al. (1989). BothDNA strands were sequenced with primers supplied with the Se-quenase kit or with internal oligoprimers (17mers). Computer-as-sisted database searches and sequence analyses were carried outusing the University of Wisconsin Genetics Computer Groupprograms package (UWGCG; Devereux et al. 1984) and theBLASTP program (Altschul et al. 1990).

Biological assay of oleandomycin

The biological activity of oleandomycin was tested by bioassayagainst Bacillus pumilus ATCC 14884. The bacterium was grownovernight at 37°C in TSB medium, then diluted 1:100 in TSBcontaining 50% (w/v) glycerol and stored at ±20°C until needed.For bioassays, 150 ll of a suspension of B. pumilus cells were addedto 100 ml of premelted 1% w/v agar containing TSB medium athalf nutrient concentration at 55°C. The mixture was poured intopetri dishes and, after cooling, Oxford cylinders containing between50 and 200 ll of crude ethyl acetate extracts of S. antibioticuscultures were placed on the top of the agar. After 2 h at 4°C,incubation was carried out at 37°C overnight.

Metabolite identi®cation

After a preculture period of 48 h in EP1 medium, the S. antibioticusmutant strain A35G1 was cultured for 72 h in EP2 medium at28°C as described (Salah-Bey et al. 1998). Cells were pelleted bycentrifugation, and the supernatant was adjusted to pH 9±10 withsodium hydroxide and extracted with ethyl acetate. The extract wasthen analysed by thin layer chromatography (TLC) and high per-formance liquid chromatography (HPLC) coupled to a massspectrometer (MS). TLC was performed on pre-coated silica gelplates (60 F254, Merck) as described (Salah-Bey et al. 1998).Erythronolide B used as a standard was puri®ed from the eryCImutant strain WH2221 (Dhillon et al. 1989) as described (Salah-Bey et al. 1998). The 6-deoxyerythronolide B aglycon also used as astandard was puri®ed from an eryBIII gene disruption mutant ofSacc. erythraea (K. Salah-Bey, J.-M. Michel and M.-C. Raynal,unpublished results) according to the following procedure. In the®rst step, the ethyl acetate extract was chromatographed on aLichoprep Si60 15±25 l column (Merck) and eluted with a gradientof methylene chloride/methanol (99/1 to 90/10). In the second step,chromatography was performed on a Lichoprep NH2 25±40 llcolumn (Merck) and elution was performed with n-butyl chloridefollowed by an elution gradient of n-butyl chloride/methanol (99/1to 90/10). Lastly, preparative TLC was performed on pre-coatedsilica gel 60 plates using methylene chloride/methanol (90/10) as theeluent. Erythromycin A was obtained from Hoechst MarionRoussel. HPLC analyses were performed at 215 nm on a Gilsonliquid chromatography system equipped with a variable wavelengthdetector, and a Kromasil C18 5 l stainless steel column(250 ´ 4.6 mm i.d.) ®tted with a Kromasil C18 5 l precolumn(50 ´ 4.6 mm i.d.) as described (Salah-Bey et al. 1998). HPLCcoupled to mass spectrometry was carried out on a Waters chro-matography system equipped with a Finnigan TSQ 7000 massspectrometer.

Results

Cloning of the oleG1 and oleG2 genes

The eryCIII gene encoding the desosaminyl transferaseof Sacc. erythraea (Salah-Bey et al. 1998) was used as aprobe for Southern hybridization analysis of a series ofpreviously isolated cosmid clones (Swan et al. 1994;C. Olano, unpublished results) containing the olean-domycin biosynthetic gene cluster. One cosmid clone(cosAB35) included two BamHI fragments of 3.5 and2.7 kb that gave positive signals (Fig. 2). Further sub-cloning and sequencing (see below) showed that thesetwo bands were separated by a small 0.6 kb BamHIfragment that was not detected by Southern hybridiza-tion. The left end of the 3.5-kb BamHI fragment con-tained the 3¢ end of a large gene encoding the third

Fig. 1 Structure of oleandomycin

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subunit of the oleandomycin type I PKS previously se-quenced (Swan et al. 1994). The BamHI fragment ad-jacent to, the hybridizing 2.7-kb BamHI fragment on theright (Fig. 2) corresponded to a 4.5-kb BamHI frag-ment, previously cloned and partially sequenced, thatcontains a gene encoding a cytochrome P450 monooxy-genase (oleP; Rodriguez et al. 1995) and a gene encodingan ATP-binding protein involved in secretion of olean-domycin or intermediates (oleB; Olano et al. 1995). A6-kb SphI-KpnI fragment (sites 4±9 in Fig. 2), locatedbetween the gene encoding the third subunit of the ole-andomycin polyketide synthase and the oleP gene, wassequenced and analysed for coding regions using theCODONPREFERENCE program (Devereux et al.1984). Five open reading frames (orfs) were found, alltranscribed in the same direction. The ®ve orfs showedthe characteristic Streptomyces codon bias (Fig. 3).

The ®rst orf (designated as oleP1) starts at an ATGcodon located 279 bp downstream of the olePKS-orf3and extends to a TGA stop codon, encoding a poly-peptide of 401 amino acids with an estimated Mr of42 141. The second orf (designated as oleG1) starts be-tween 48 and 54 bp downstream of the oleP1 stop co-don; there are three in-frame potential starting codonswithin a very short stretch (GTGATGATG). No clearpotential ribosomal binding site was detected upstreamof these putative starting codons. The oleG1 gene wouldcode for a polypeptide of 426 amino acids and an esti-mated Mr of 46 976. The third orf (designated as oleG2)starts 5 bp downstream of oleG1 with an ATG codonand ends in a TGA codon; it would code for a poly-peptide of 426 amino acids and an estimated Mr of46 006. There are two potential starting codons for thefourth orf (designated as oleM1): an ATG codon thatoverlaps the 3¢-end of the preceeding oleG2 gene by 8 bpand a GTG codon located 55 bp downstream of theoleG2 stop codon. Although at this stage a de®nitivestarting codon cannot be assigned to oleM1, we favourthe ATG codon based on the following assumptions:

(i) 19 of the 20 codons between the two potential startingcodons have G or C in the third codon position, which ischaracteristic of Streptomyces coding regions (Bibb et al.1984); (ii) the ATG codon (but not the GTG codon) ispreceded by a sequence (GGAGG) that possibly repre-sents a ribosomal binding site, since it shows a degree ofcomplementarity to a region close to the 3¢ end of the16S rRNA of S. lividans (Bibb and Cohen 1982) and(iii) overlapping genes that are involved in the samemetabolic process are quite common in bacteria(Normark et al. 1983). The last orf (designated as oleY)starts at an ATG codon 78 bp downstream of the oleM1stop codon, ending in a TAG codon and encoding apolypeptide of 386 amino acids with an estimated Mr of42 484.

Deduced functions of the di�erent gene products

Computer analysis of the deduced products of the dif-ferent genes using the BLAST program (Altschul et al.1990) identi®ed similarities with other proteins in thedatabases. The deduced product of the ®rst gene, OleP1,shows similarity to various cytochrome P450 oxygenases.The highest similarity scores were with the products offour genes from di�erent antibiotic biosynthetic path-ways: EryCII in the erythromycin pathway of Sacc.erythraea (Summers et al. 1997; Salah-Bey et al. 1998),DnrQ in the daunorubicin pathway of S. peucetius(Otten et al. 1995), Tyl-orf1 on the tylosin pathway ofS. fradiae (Gandecha et al. 1997) and the partial se-quence of a protein involved in daunomycin biosynthesisin Streptomyces sp. C5 (Dickens et al. 1995). All theseproteins show similarities with many cytochrome P450monooxygenases, but they do not contain the charac-teristic P450 motif (Bairoch 1992) which contains thehighly conserved heme binding site and includes thecysteine residue that serves as the heme iron ligand(Nelson et al. 1993). In the case of the EryCII protein,experimental evidence demonstrates that it is involved inthe biosynthesis of the deoxysugar D-desosamine duringerythromycin biosynthesis (Weber et al. 1990; Summerset al. 1997; Salah-Bey et al. 1998).

The deduced products of the second and third orfs(oleG1 and oleG2) showed similarities with glycosyl-transferases and glucuronosyltransferases from di�erentsources. In particular, they resemble several glycosyl-transferases that are possibly involved in the transfer ofvarious deoxyhexoses to di�erent aglycons during thebiosynthesis of daunorubicin (DnmS, Otten et al. 1995and DauH, GenBank accesion number U43704) andbaumycin (DnrH, Scotti and Hutchinson 1996) byS. peucetius, erythromycin by Sacc. erythraea (EryCIII,Summers et al. 1997; Salah-Bey et al. 1998; and EryBV,Gaisser et al. 1997; Summers et al. 1997), tylosin byS. fradiae (TylM2, Gandecha et al. 1997) and granaticinby S. violaceoruber (Bechthold et al. 1995) (Table 1).The OleG1 and OleG2 proteins also resemble, althoughto a lesser extent, two glycosyltransferases that have

Fig. 2 Schematic representation of the region sequenced fromcosAB35, the di�erent orfs and their locations with respect to thegene for the third subunit of the oleandomycin polyketide synthase(Swan et al. 1994), oleB (Olano et al. 1995) and oleP (Rodriguez et al.1995). The hatched bars indicate regions that hybridise with theeryCIII gene from the erythromycin biosynthetic pathway. B, BamHI;G, BglII; H, SphI; K, KpnI. Only relevant restriction sites are shown

301

Fig. 3(i)

Fig. 3(ii)

been shown to be involved in macrolide inactivationin S. lividans (Mgt, Jenkins and Cundli�e 1993) andin S. antibioticus (OleD, Vilches et al. 1992; Herna ndezet al. 1993) (Table 1). All these glycosyltransferases re-tain a characteristic motif present in UDP-glycosyl andUDP-glucuronosyl transferases, which is localised closeto the C-terminus of these enzymes (Fig. 4).

Comparison of the deduced product of the fourthgene (oleM1) with proteins in databases revealed clearsimilarities with the SnoX protein in the nogalamycinpathway of S. nogalater (GenBank accession numberS52403), the RdmD protein from S. purpurascens, whichparticipates in the anthracycline pathway (Niemi andMaÈ ntsaÈ laÈ 1995), the SrmX protein in the spiramycinpathway of S. ambofaciens (Geistlich et al. 1992), theTylM1 protein in the tylosin pathway of S. fradiae(Gandecha et al. 1997) and the EryCVI protein in theerythromycin pathway of Sacc. erythraea (Gaisser et al.1997; Summers et al. 1997). The OleM1 protein alsoshows similarity, although to a lesser extent, to severalmethyltransferases: the glmT product from Rattus nor-vegicus (Ogawa et al. 1987), the bchM product fromRhodobacter sphaeroides (Gibson and Hunter 1994) andglycine methyltransferases from human and pig liver(Ogawa et al. 1993). An alignment of speci®c regions ofall these proteins revealed three conserved regions(Fig. 5) that have been described in various S-adeno-sylmethionine-dependent methyltransferases (Kaganand Clarke 1994). TylM1 and EryCVI have been shownto be involved in the synthesis of mycaminose and

desosamine, repectively (Gandecha et al. 1997; Gaisseret al. 1997). Accordingly, and taking into account allabove considerations, OleM1 may well code for amethyltransferase involved in the dimethylation eventthat must occur during desosamine biosynthesis inS. antibioticus.

The deduced product of the last orf (oleY) onlyshowed signi®cant similarity with the SnoY protein(54.3% identity and 32.2% similarity) in the no-galamycin pathway of S. nogalater (Ylihonko et al.1996). On the basis of comparison with proteins in da-tabases no function can be proposed for either SnoY orOleY.

Insertional inactivation of the oleG1 gene

To demonstrate the involvement of the sequenced regionin oleandomycin biosynthesis, a gene disruption experi-ment was carried out. A 0.6-kb BamHI fragment inter-nal to the oleG1 gene (sites 5±6 in Fig. 2) was subclonedinto the BamHI site of pOJ260, and the resultant con-struct (pCO3) was introduced into S. antibioticus byprotoplast transformation, selecting transformants for

Fig. 3 Nucleotide sequence of the 6-kb SphI-KpnI fragment. Thecomplete nucleotide sequence of the fragment is shown, with thededuced amino acid sequence of the di�erent gene products in asingle-letter code. The sequence has been deposited in GenBank underthe accession number AJ002638

304

apramycin resistance (25 lg/ml ®nal concentration).S. antibioticus is a streptomycete strain that is very re-fractile to transformation (Swan et al. 1994). After sev-eral attempts a single transformant was obtained (themutant designated A35G1). By Southern hybridizationit was veri®ed that the corresponding region in theS. antibioticus chromosome was altered: the 4.7-kb PstIfragment characteristic of the wild-type chromosomewas replaced in A35G1 by a 2.4-kb and a 6.5 kb PstIfragment (Fig. 6). The products accumulated by thismutant were analysed. After cultivation of the mutant,the culture supernant was extracted as described inMaterials and methods, and the ®nal extract was testedfor antibiotic activity against B. pumilus by bioassay. Noinhibitory e�ect on the growth of B. pumilus was ob-served. In addition, TLC analysis of the extract did notreveal the presence of oleandomycin, and a spot (purplecolour) with higher mobility than erythronolide B andclose to 6-deoxyerythronolide B was clearly visible, aswould be expected for the 8,8a-deoxyoleandolide agly-con (data not shown). Production of oleandomycin andprecursor metabolites was also monitored by HPLCcoupled to MS (in the atmospheric pressure chemicalionization mode). No peak corresponding to olean-domycin (m/z 673) could be detected in the extract, whiletwo major metabolites, M1 and M2, eluting at 6:12 and

Fig. 4 Alignment of the deduced amino acid sequences of variousglycosyltransferases around the conserved motif characteristic ofUDP-glycosyltransferases and UDP-glucuronosyltransferases. Thefollowing sequences were included: DauH, glycosyltransferase fromStreptomyces sp. C5 (GenBank Accession No. U43704); DnmS,daunosamine glycosyltransferase from S. peucetius (Otten et al.1995); DnrH, baumycin glycosyltransferase from S. peucetius (Scottiand Hutchinson 1996); EryCIII and EryBV, the desosaminyl andmycarosyl glycosyltransferases, respectively from Sacc. erythraea(Salah-Bey et al., in press; Summers et al. 1997); Mgt, macrolideglycosyltransferase from S. lividans (Jenkins and Cundli�e 1991);OleD, oleandomycin glycosyltransferase from S. antibioticus (Her-na ndez et al. 1991) Gra-orf5, granaticin glycosyltransferase fromS. granaticolor (Bechthold et al. 1995); TylM2, mycaminose glycosyl-transferase from S. fradiae (Gandecha et al. 1997); OleG1 andOleG2, this study

Fig. 5 Alignment of the deduced amino acid sequences of putativeN-methyltransferases. The following sequences were included: SnoXfrom the nogalamycin pathway of S. nogalater (Ylihonko et al. 1996);RdmD from the rhodomycin pathway in S. purpurascens (Niemiand MaÈ ntsaÈ laÈ 1995); SrmX from the spiramycin pathway ofS. ambofaciens (Geistlich et al. 1992); TylM1 from the tylosinpathway in S. fradiae (Gandecha et al. 1997); EryCVI from theerythromycin pathway in Sacc. erythraea (Gaisser et al. 1997); GlmT,a methyltransferase from Rattus norvegicus (Ogawa et al. 1987);BchM, a methyltransferase from Rhodobacter sphaeroides (Gibsonand Hunter 1994); Hslgmetf and Pigglyn are glycine methyltransf-erases from human and pig liver, respectively (Ogawa et al. 1993)

Table 1 Similarities betweenOleG1 and OleG2 and variousglycosyltransferases from anti-biotic producers

Gene product Percentage similarity Percentage identity References

OleG1 OleG2 OleG1 OleG2

DnmS 68.8 70.3 50.0 48.8 Otten et al. (1995)DnrH 68.4 66.0 47.9 47.1 Scotti and Hutchinson (1996)EryCIII 71.9 72.1 53.2 53.1 Salah-Bey et al. (1998)EryBV 64.1 69.6 44.8 50.7 Gaisser et al. (1997)OleD 46.6 48.5 25.6 25.6 Hema ndez et al. (1993)Mgt 45.7 45.4 24.0 20.1 Jenkins and Cundli�e (1991)TylM2 68.8 73.3 47.7 54.6 Gandecha et al. (1997)Gra-orf5 47.2 53.9 20.1 26.5 Bechthold et al. (1995)DauH 66.0 63.1 45.6 44.6 GenBank accession

number U43704

305

17:23 respectively, were characterized by their massspectra (full scan range 0±750 average mass unit) andsubsequent MS-MS analysis (scan range 0±400 a.m.u)(data not shown). In both cases, a parent peak wasdetected at m/z 373, which was consistent with [8,8a-deoxyoleandolide]H+ and gave a fragmentation patternclose to that of the previously described erythronolide Bmacrolactone ring (Mitscher et al. 1972). However, onlycompound M2 has a retention time close to that of6-deoxyerythronolide B (K. Salah-Bey, J.-M. Micheland M.-C. Raynal, unpublished results) and is there-fore fully consistent with 8,8a-deoxyoleandolide. TheM1 compound could possibly correspond to theopen lactone ring or, alternatively, to an isomericstructure.

Discussion

Macrolide antibiotics are dependent for activity on thepresence of sugars attached to the macrolactone ringeither as monosacharides (as in erythromycin, olean-domycin or picromycin) or as disaccharides (as in car-bomycin, spiramycin and tylosin). The biosynthesis ofthe 6-deoxyhexoses attached to the respective lactones isnot yet well understood, and genes involved in thisprocess in the erythromycin (Dhillon et al. 1989; Hay-dock et al. 1991; Gaisser et al. 1997; Salah-Bey et al.1998; Summers et al. 1997) and the tylosin (Merson-Davies and Cundli�e 1994; Gandecha et al. 1997)pathways have been cloned and sequenced. In this paperwe describe the cloning and sequencing of ®ve genesinvolved in the biosynthesis and transfer of 6-deoxy-hexoses during oleandomycin biosynthesis. The OleP1protein resembles cytochrome P450 oxygenases and hashomologous counterparts in the daunorubicin (DnrQ;Otten et al. 1995), erythromycin (EryCII; Salah-Bey et al.1998; Summers et al. 1997) and tylosin (TylM1; Ga-ndecha et al. 1997) pathways. Many cytochromes P450are monoxygenases responsible for introduction of mo-

lecular oxygen into di�erent macrolide aglycones (We-ber et al. 1991; Stassi et al. 1993; Rodriguez et al. 1995).All these proteins, however, lack the critical cysteineinvolved in the binding of haem, suggesting that theyrepresent a new family of proteins with unknown enzy-matic function. In the case of EryCII it has been dem-onstrated that this gene participates in the biosynthesisof dTDP-desosamine (Summers et al. 1997; Salah-Beyet al. 1998) and Salah-Bey et al. (1998) have suggestedthat the encoded enzyme is responsible for the 3±4 iso-merization step during the biosynthesis of the dTDP-desosamine precursor of erythromycin, while Otten et al.(1997) suggest that it is likely to be involved in the de-oxygenation at C-4. Based on the fact that D-desos-amine is also attached to the oleandomycin aglycon,OleP1 could be also involved in D-desosamine biosyn-thesis in the oleandomycin producer.

The deduced product of the oleM1 gene resemblesother proteins involved in the synthesis of spiramycin,erythromycin, tylosin and anthracycline. A featurecommon to all these drugs is the presence of a di-methylamino sugar moiety attached to the aglycon(desosamine in erythromycin and oleandomycin, rho-dosamine in rhodomycin, nogalamine in nogalamycinand mycaminose in tylosin and spiramycin). Based onthis observation, and in light of the fact that all of theseproteins show motifs characteristic of S-adenosylme-thionine-dependent methyltransferases, it is proposedthat OleM1 encodes a N-methyltransferase responsiblefor dimethylation of the amino group at C-3 inD-desosamine.

Fig. 6A, B Insertional inactivation of the oleG1 gene. A Schemerepresenting the integration of pC03 into the homologous region ofthe S. antibioticus chromosomal DNA and indicating the fragmentsgenerated after PstI digestion. Abbreviations: B, BamHI; E, EcoRI;K, KpnI; P, PstI; G, BglII; H, SphI. BAnalysis of chromosomal DNAby Southern hybridization. PstI-digested chromosomal DNA of thewild-type S. antibioticus strain (lane 1) and of the mutant strainA35G1 (lane 2) were analysed by Southern blotting, using as a probethe 0.6-kb BamHI fragment (sites 5±6 in Fig. 2)

306

The transfer of D-desosamine and L-oleandrose intheir activated forms (i.e. dTDP-sugars) to the ole-andolide requires the action of glycosyltransferases thatcould be encoded by the oleG1 and oleG2 genes. In-volvement of the DNA region reported in this paper inoleandomycin biosynthesis was unequivocally demon-strated by insertional inactivation and the subsequentgeneration of an oleandomycin non-producing mutant(A35G1). Desosamine is present in erythromycin andoleandomycin, but the two antibiotics di�er in the other6-deoxyhexoses attached to the lactone ring: mycarose inthe case of erythromycin and oleandrose in olean-domycin. Comparison of the OleG1 and OleG2 aminoacid sequences with those of other glycosyltransferases(particularly with EryCIII, the desosaminyl glycosyl-transferase of Sacc. erythraea) did not reveal any moresigni®cant similarity between OleG1 or OleG2 and Er-yCIII that could help to assign a role to either of theseglycosyltransferases in oleandrose or desosamine trans-fer. During erythromycin biosynthesis, L-mycarose isthe ®rst sugar incorporated into the erythronolide B andno antibacterial activity is associated to the mycarosyl-erythronolide B. Activity is fully acquired when thesecond sugar, D-desosamine, is attached. Assuming thata parallelism exists between the order of sugar additionin erythromycin and oleandomycin biosynthesis, it canbe expected that L-oleandrose is ®rst transferred to theoleandolide and then D-desosamine is added. TheA35G1 mutant accumulated a compound tentativelyidenti®ed as oleandolide, and this suggests that the geneencoding the oleandrosyl glycosyltransferase has beeninactivated in this mutant. However, since the mutantwas generated by gene disruption via the integration ofthe plasmid vector into the chromosome, the existence ofa polar e�ect on the expression of other genes down-stream of oleG1 that prevents transcription of oleG2,oleM1 and oleY cannot be excluded. If this is the case,both glycosyltransferases could be a�ected and no sugarwould be transferred to the oleandolide.

Antibiotic biosynthetic genes tend to be clustered inthe chromosomes of the producer strains, as has beenshown in the various pathways that have been charac-terized. However, this cannot be extended to the genesinvolved in 6-deoxyhexose biosynthesis in these path-ways. In the erythromycin pathway, 6-deoxyhexosebiosynthetic genes are distributed on both sides ofthe polyketide synthase genes, with no clustering ofD-desosamine and L-mycarose genes. In the case oftylosin, genes involved in sugar biosynthesis are alsofound on both sides of the polyketide synthase. A similarsituation occurs in the oleandomycin pathway. The genesdescribed in this paper are located immediately down-stream of the polyketide synthase genes. Other potentialsugar genes located on the other side of the PKS geneswill be reported elsewhere (C. Olano, I. Aguirrezaba-laga, C.Me ndez and J.A.Salas, unpublished).

Acknowledgements We wish to thank R. Legrand and M. Meili forassistance in mass spectrometry analysis and P. Jevardat de Fom-

belle for expert technical assistance. We are also grateful toP. Leadlay for communicating results prior to publication. Thiswork was supported by a grant from the European Community toM.C.R. and J.A.S. (Biotech BIO4-CT96-0080).

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