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CNRS UMR8621, Université Paris-Sud, Institut de Génétique et Microbiologie, Bâtiment 400, F-91405 Orsay Cedex, France
Correspondence
Jean-Luc Pernodet
jean-luc.pernodet{at}igmors.u-psud.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Centre de Biotechnologie de Sfax, B.P ‘K’, 3038 Sfax, Tunisia.
Present address: Université d'Avignon, IUT, Site Agroparc, BP 1207, F-84911 Avignon Cedex 9, France.
Present address: Unité Bactéries Lactiques et Pathogènes Opportunistes, Bâtiment 222, INRA, F-78352 Jouy en Josas Cedex, France.
The GenBank/EMBL/DDBJ accession numbers for the sequences of the regions downstream and upstream of the PKS genes are AM709783 and AM709784.
Details of the construction of pWED2 and pOSV238 are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Hansen et al., 2002). They also inhibit ribosome assembly in some bacteria such as Staphylococcus aureus (Champney & Tober, 2000). Macrolides are an old family of antibacterial agents, as erythromycin was introduced in clinical practice in the 1950s, but they still play an important role in the chemotherapy of infectious diseases. This is mostly due to the development of semi-synthetic derivatives, for instance clarithromycin, azithromycin and more recently ketolides, with improvements to features such as stability, pharmacokinetics and activity against resistant bacteria (Schönfeld & Kirst, 2002). The characterization of macrolide biosynthetic gene clusters has opened the way to rational genetic yield improvement of production strains (Stratigopoulos et al., 2004) and to the development of combinatorial strategies for the synthesis of new macrolides (Butler et al., 2002; Hutchinson & McDaniel, 2001; Katz, 1997; Long et al., 2002; Melançon & Liu, 2007).
Streptomyces ambofaciens produces spiramycin, composed of a 16-membered polyketide lactone ring (platenolide) on which two amino sugars (mycaminose and forosamine) and one neutral sugar (mycarose) are attached. Spiramycin is produced as a mixture of three major compounds differing by acyl substitutions at the position of the hydroxyl group at carbon 3 (Fig. 1). Spiramycin is used in human medicine as an antibacterial agent (Smith, 1988) and also for the treatment of Toxoplasma infections (McCabe & Oster, 1989). Several genes that confer spiramycin resistance in heterologous hosts have been cloned from S. ambofaciens (Pernodet et al., 1999; Richardson et al., 1987), and spiramycin biosynthetic genes were shown to be located in the vicinity of one of the resistance genes, srmB (Richardson et al., 1990). The five genes encoding the PKS were characterized (Kuhstoss et al., 1996), and the product of another gene, srmR, was shown to be a transcriptional activator (Geistlich et al., 1992). However, only partial sequence data were available, and the complete cluster has not been characterized.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The construction of some of the vectors used in this work is described in the supplementary material. Standard media and culture conditions were used (Kieser et al., 2000; Sambrook & Russell, 2001). The following antibiotics were added to the medium when required for selection: ampicillin (Amp), apramycin (Apr), hygromycin B (Hyg), puromycin (Pur) and thiostrepton (Tsr). For spiramycin production, S. ambofaciens strains were grown in MP5 liquid medium as described previously (Pernodet et al., 1993). The detection and quantification of spiramycin in culture supernatants was performed by bioassay or HPLC as described previously (Gourmelen et al., 1998).
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; Sambrook & Russell, 2001). Radiolabelled DNA probes were prepared with the Megaprime DNA labelling system (GE Healthcare).
Construction and screening of cosmid libraries.
Two cosmid libraries of S. ambofaciens DNA were constructed, using the cosmid pWED1 for the strain ATCC 23877 library and pWED2 for the strain OSC2 library. OSC2 is a derivative of ATCC 23877. These two S. ambofaciens strains differ only by the presence of pSAM2, which is absent in OSC2; their growth, differentiation and antibiotic production abilities are indistinguishable (Raynal et al., 2006). Total DNA was partially digested with BamHI and fragments of 35 to 45 kb were ligated with BamHI-digested cosmid vectors. In vitro packaging was performed with the Packagene Lambda DNA packaging system (Promega), according to the manufacturer's protocol. The phage particles were used to infect the E. coli SURE strain. Colony hybridization was performed on about 2000 clones with various radiolabelled probes. Several hybridizing clones were obtained, the cosmids they contained were characterized by their restriction patterns and some were chosen for sequencing and further studies. The cosmids pOS49.1, pSPM5 and pSMP7 came from the first library, constructed with pWED1. The cosmid pSPM36 came from the second library, constructed with pWED2.
DNA sequencing, sequence analysis and annotation.
DNA sequencing of cosmids pSPM5 and pSPM7 was performed by standard shotgun cloning followed by automated DNA sequencing carried out on double-stranded DNA templates to obtain at least sixfold coverage. Sequencing was done by Eurogenetec or Genome Express. Primer walking was used to close the gaps. Fragments from the cosmids pOS49.1 and pSPM36 were sequenced by primer walking. A software program, combining an analysis similar to that performed by Frame plot (Ishikawa & Hotta, 1999) with an analysis similar to that performed by Glimmer (Salzberg et al., 1998) (C. Gerbaud, unpublished), was used to identify protein-coding regions. Sequence analysis and comparisons were performed using BLAST (Altschul et al., 1997) and CD-Search (Marchler-Bauer & Bryant, 2004).
Targeted disruption of spiramycin biosynthetic genes.
The construction of an orf3 in-frame deletion mutant (strain SPM24) was described previously (Raynal et al., 2006).
An in-frame orf2 deletion mutant was also created by PCR targeting (Chaveroche et al., 2000; Gust et al., 2003; Yu et al., 2000) followed by in vivo excision of the interrupting resistance cassette (Raynal et al., 2006). For this purpose, a 4.5 kb EcoRI–BamHI fragment containing orf2 and flanking genes, from srmGI to orf3, was cloned into the plasmid pUC19, yielding pOS49.99. This plasmid was introduced into E. coli strain KS272 already containing pKOBEG (Chaveroche et al., 2000) and expressing the 
-RED recombination system. The Apr resistance cassette att3
aac– (Raynal et al., 2006) was amplified from pOSV211 using the primers ORF2A (5'-CCCGCGCGGCAGCCTCTCCGTGATCGAGTCCGGCGTGACCATCGCGCGCGCTTCGTTCGG-3') and ORF2B (5'-GCTCCGTGCGTCATGCAGGAAGGTGTCGTAGTCGCGGTAGATCTGCCTCTTCGTCCCGAA-3') [sequences identical to the beginning (ORF2A) and end (ORF2B) of the orf2 coding sequence are underlined]. The resulting PCR product was used to transform E. coli strain KS272 containing pKOBEG and pOS49.99. After selection for Apr resistance, clones were obtained in which most of the plasmid-borne orf2 coding sequence has been replaced by the att3aac– cassette, through -RED-mediated recombination. The resulting plasmid was named pSPM17. The insert of pSPM17, obtained as a EcoRI/Klenow–XbaI DNA fragment, was cloned in the plasmid pOSV238 (HygR), previously digested by BamHI/Klenow–XbaI, yielding pSPM21. It was introduced into S. ambofaciens OSC2 via protoplast transformation and Apr selection was applied. AprR transformants were screened for sensitivity to Hyg, indicating a double-crossover allelic exchange. This was confirmed by PCR and Southern blot analysis. One orf2 : : att3aac– S. ambofaciens mutant strain was chosen for further studies and named SPM21. In order to excise the att3aac– cassette, the plasmid pOSV508, expressing the excisionase and the integrase from pSAM2, was introduced into strain SPM21. As a result, the Apr-resistance determinant was excised through site-specific recombination, leading to an in-frame orf2 deletion mutant. After curing of pOSV508, the resulting strain, verified by PCR amplification and sequencing of the PCR products, was named SPM22.
Complementation of SPM22 and SPM24 mutants.
The plasmid pOS49.52, a pIJ903 derivative expressing orf3 under the control of the ermE* promoter, was described previously (Raynal et al., 2006). It was introduced into strain SPM24 by protoplast transformation.
The orf2 coding sequence was amplified with the primers KF8 (5'-AAGCTTCCGGGTCGACTGGAACTGAACCCGAGGG-3', HindIII site underlined) and KF11 (5'-GGATCCGATGGTCATGTGAGGCTCCGTGCGTCAT-3', BamHI site underlined). The resulting PCR product was cloned into pUWL201 digested by BamHI and HindIII, yielding pSPM30, in which orf2 is expressed under the control of the ermE* promoter. The SPM22 mutant was also complemented by orf1a from Streptomyces fradiae, the producer of tylosin. The primers KF12 (5'-AAGCTTGCGAGGAGCAGCCCGATGGCGGCGAGCA-3', HindIII site underlined) and KF13 (5'-GGATCCCCCTGTCACGGGTGGCTCCTGCCGGCCC-3', BamHI site underlined) were used to amplify orf1a from S. fradiae total DNA. The resulting PCR product was cloned into pUWL201 digested by BamHI and HindIII to obtain pSPM33. The plasmids pSPM30 and pSPM33 were introduced into strain SPM22 by protoplast transformation.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) corresponded to tylB, a gene from the tylosin cluster in S. fradiae involved in the biosynthesis of mycaminose (Merson-Davies & Cundliffe, 1994), a sugar also found in the spiramycin molecule. A tylB orthologue is present in S. ambofaciens, as some cosmids from an S. ambofaciens library restored tylosin biosynthesis when introduced into a tylosin non-producing mutant affected in tylB (Richardson et al., 1990). From a colony hybridizing with the probe P1, the cosmid pOS49.1 was extracted. A 3.3 kb SacI DNA fragment, responsible for the hybridization, was identified, subcloned and sequenced. Sequence analysis showed the presence of four coding sequences (two complete, two partial). One of them encoded a protein showing end-to-end similarity to TylB (64 % identity). Inactivation of this coding sequence (see below) abolished spiramycin production, confirming that the gene identified was part of the spiramycin cluster.
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) (Fig. 2b
). In order to select other cosmids, the library was probed again with three probes. Two of them were fragments of pOS49.1, each located at one end of the sequenced region (probes P2 and P3; Fig. 2a). The third one (P4; Fig. 2a) contained the srmD gene, a spiramycin-resistance determinant cloned previously (Pernodet et al., 1999; M.-H. Blondelet-Rouault, unpublished) and part of the upstream gene. The products of srmD and of the upstream gene were similar to those of mdmA and mdmB from Streptomyces mycarofaciens, involved respectively in midecamycin resistance and biosynthesis (Hara & Hutchinson, 1992). Several cosmids hybridized with these probes, although not with all three of them, and from their restriction maps two cosmids that showed minimal overlap with pOS49.1 were selected. These were designated pSPM5 and pSPM7 (Fig. 2a). Sequence analysis showed that the right end of the pSPM5 insert could still be located within the spiramycin cluster. Therefore, another hybridization experiment was performed using probe P5, corresponding to the right end of the pSPM5 insert (Fig. 2a). One cosmid, named pSPM36, overlapping pSPM5 and spanning the remainder of the spiramycin cluster, was partially sequenced (Fig. 2a).
From the analysis of these sequences and their alignment with the sequences of the five PKS genes (Burgett et al., 1999), it was possible to deduce the gene organization of the region. The five srmG genes, encoding the PKS, occupy about 40 kb. Upstream of srmGI, 34 ORFs (orf1 to orf34c) account for about 40 kb, and downstream of srmGV, 11 ORFs (orf1*c to orf11*) occupy about 12 kb. The inferred products of these ORFs were compared to protein databases. From these comparisons, most of the deduced protein products could be assigned putative functions in spiramycin biosynthesis (Fig. 2; Table 2).
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; Kuhstoss et al., 1996). The organization of the modules within the five SrmG proteins is the same as in other known PKS involved in the biosynthesis of 16-membered macrolides, and the domain organization is similar to that of the tylosin or niddamycin PKS (McDaniel et al., 2005). However, the extender units used for platenolide biosynthesis are different from those used for tylactone biosynthesis. The platenolide PKS was predicted to incorporate seven extender units: four malonyl-CoA, one ethylmalonyl-CoA, one methylmalonyl-CoA and one unknown product, incorporated by module 6 (Kuhstoss et al., 1996).
The product of orf4*c, a crotonyl-CoA reductase, presumably contributes to the flux of ethylmalonyl-CoA. Crotonyl-CoA reductase genes are found in other polyketide biosynthetic clusters where the PKS uses ethylmalonyl extender units, such as the tylosin or concanamycin clusters (Cundliffe et al., 2001; Haydock et al., 2005).
Five proteins, the products of orf5*, orf22c, orf23c, orf24c and orf25c, are similar to FkbG, FkbH, FkbI, FkbJ and FkbK, respectively, proteins that are involved in the biosynthesis of methoxymalonyl-acyl carrier protein (ACP) in Streptomyces hygroscopicus var. ascomyceticus, the producer of the macrolide FK520 (ascomycin) (Wu et al., 2000). The greatest similarities are observed with proteins encoded in the geldanamycin and concanamycin clusters (Haydock et al., 2005; Rascher et al., 2003). Recently, sequence comparison of various acyltransferase domains incorporating methoxymalonate allowed identification of a sequence motif predicting the incorporation of this extender unit (Haydock et al., 2005). The conservation of this motif in the AT domain of module 6 in SrmGIV, together with the presence of the five genes involved in methoxymalonyl-ACP biosynthesis, strongly support the incorporation of methoxymalonate by module 6.
Several other gene products are predicted to be involved in lactone ring biosynthesis or modification. A type II thioesterase is encoded by orf6. Such enzymes play an editing role in the biosynthesis of polyketides (Butler et al., 1999; Heathcote et al., 2001). The product of orf1 is a cytochrome P450 hydroxylase, probably involved in ring hydroxylation at position C20 (Fig. 1). The deduced acyltransferase encoded by orf6* is similar to acyltransferases involved in lactone ring acylation. Therefore, Orf6* might be involved in ring acylation at position 3, leading to the biosynthesis of spiramycins II and III.
Another important ring modification during the early steps of spiramycin biosynthesis is that at position 9. The PKS generates platenolide I, with a keto group at position 9 (Omura et al., 1979), which is later converted to platenolide II, with a hydroxyl group at C9, by a post-PKS ketoreductase. Indeed, the presence of such an activity in S. ambofaciens is attested by the fact that the keto group at C9 in tylactone could be modified and that forosamine could then be attached at this position by enzymes from S. ambofaciens (Omura et al., 1983). Such post-PKS reductases have been described previously, for instance AveF from Streptomyces avermitilis (Ikeda et al., 1999), but none of the spiramycin cluster deduced proteins presented similarity to characterized post-PKS ketoreductases. Nonetheless, the product of orf31 shows similarity to oxidoreductases (COG0667, pfam00248) and could be a candidate to perform this reaction.
No gene encoding the phosphopantetheinyl transferase required for the post-translational modification of the PKS is present in the spiramycin cluster. A phosphopantetheinyl transferase is encoded by alpN in the alp cluster, located in the terminal inverted repeats of the S. ambofaciens chromosome (Pang et al., 2004), but it is not known whether this enzyme is the one involved in the spiramycin PKS modification.
Genes for deoxysugar biosynthesis
The three deoxysugars present in the structure of spiramycin are also found in other macrolide molecules whose biosyntheses have been thoroughly studied. Mycaminose is found in tylosin, produced by S. fradiae; the genes involved in its biosynthesis have been identified and a biosynthetic pathway has been established (Cundliffe et al., 2001; Melançon et al., 2005). Mycarose is found in erythromycin and tylosin and its biosynthetic genes were first characterized in Saccharopolyspora erythraea (Gaisser et al., 1997, 1998; Summers et al., 1997) and S. fradiae (Bate et al., 2000; Takahashi et al., 2005). Forosamine biosynthetic genes were studied in Saccharopolyspora spinosa, the spinosyn producer (Waldron et al., 2001; Zhao et al., 2005). Sequence comparisons, particularly with proteins from S. fradiae, Saccharopolyspora erythraea and Saccharopolyspora spinosa, whose function in mycaminose, mycarose and forosamine biosynthesis is well characterized, enabled us to identify a potential enzyme for each of the steps of the proposed pathways (Fig. 3).
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Genes for sugar attachment
Four genes, orf3*c, orf17, orf18 and orf26, encode putative glycosyltransferases that could be involved in the attachment of sugars. It was surprising to find four glycosyltransferase genes, as only three sugars are present in the spiramycin molecule. Several hypotheses can be proposed. Firstly, one of these genes might not be expressed or its product could be inactive, but the alignment of the four putative glycosyltransferases with functional glycosyltransferases did not reveal obvious differences suggesting that one of them could be inactive. Secondly, two different glycosyltransferases could perform the same reaction independently, but, to our knowledge, no such case has been described previously. Thirdly, one of the glycosyltransferases could be involved in macrolide inactivation by glycosylation and play a role in resistance towards the produced antibiotic (see below). Sequence comparisons with characterized enzymes that transfer mycaminose, mycarose or forosamine, and analyses performed with SEARCHGTr, a program for analysis of glycosyltransferases involved in glycosylation of secondary metabolites (Kamra et al., 2005), did not allow us to make unambiguous predictions concerning the sugars transferred by these four putative glycosyltransferases. In addition, two genes, orf2*c and orf16, encode products that belong to the recently identified family of glycosyltransferase accessory proteins, required for efficient glycosylation (Borisova et al., 2004; Hong et al., 2007; Melançon et al., 2004). These two genes are located immediately upstream of glycosyltransferase genes, as is generally the case. Their products share 42 % amino acid sequence identity. The presence of two glycosyltransferase/auxiliary protein pairs suggests that each auxiliary protein is preferentially associated with a glycosyltransferase, as shown by Hong et al. (2007).
Resistance genes
srmB (orf11c) was previously isolated from S. ambofaciens as a spiramycin-resistance determinant (Richardson et al., 1987); it encodes the ATP-binding component of an ABC transporter and is thought to be involved in spiramycin efflux (Schoner et al., 1992). orf7* was previously isolated from S. ambofaciens as a spiramycin-resistance determinant called srmD (Pernodet et al., 1999); it encodes a methyltransferase modifying 23S rRNA (M.-H. Blondelet-Rouault; unpublished data).
Inactivation of macrolides by glycosylation has been observed in S. ambofaciens extracts. Two distinct glycosyltransferase activities could be identified from their antibiotic substrate profiles. The gene gimA, encoding the glycosyltransferase responsible for one of these activities, has been detected, and is not located within the spiramycin biosynthetic cluster (Gourmelen et al., 1998). However, the gene responsible for the second activity has not yet been identified. One of the four glycosyltransferases might be responsible for this activity. Macrolide inactivation by glycosyltransferases in macrolide-producing strains has been well studied in Streptomyces antibioticus, the oleandomycin producer. It was demonstrated that intracellular glycosylation inactivates oleandomycin, which is subsequently reactivated, after secretion, by an extracellular glycosidase. Two glycosyltransferases, OleI and OleD, show oleandomycin-glycosylating activity but differ in the pattern of substrate specificity, OleI being much more specific for oleandomycin. A glycosidase OleR converts glycosylated oleandomycin into active oleandomycin (Quiros et al., 1998). The genes oleR and oleI are located in the oleandomycin cluster, while oleD is located elsewhere in the genome. As a similar resistance mechanism might operate in S. ambofaciens, we searched for an oleR orthologue, but none was found in the cluster. However, the deduced product of orf29 had a putative glycosylhydrolase domain (pfam02156). Furthermore, analysis of the Orf29 sequence by SignalP (Nielsen et al., 1997) showed that this protein, having a predicted signal sequence of 30 amino acids, was probably excreted. It might therefore be involved in the reactivation of glycosylated spiramycin.
The two genes orf9* and orf8* encode an ABC transporter. To test their possible involvement in spiramycin resistance, these two genes were expressed in the spiramycin-sensitive host Streptomyces lividans OS456 (Pernodet et al., 1996), but no resistant phenotype was observed (data not shown). Therefore, these genes are probably not involved in spiramycin resistance and might belong to the uncharacterized non-ribosomal peptide synthetase (NRPS) cluster flanking the spiramycin cluster.
Regulatory genes
The gene srmR (orf10) has been shown previously to encode a transcriptional activator; its inactivation abolished spiramycin production (Geistlich et al., 1992). Besides srmR, three putative regulatory genes are present: orf13c, orf28c and orf32c. The deduced product of orf13c is a putative GTPase belonging to the HflX family. The cellular functions of proteins from this family remain elusive (Brown, 2005). tylV, an orthologue of orf13c, is present in the tylosin cluster, where it might play a regulatory role in tylosin biosynthesis (Stratigopoulos et al., 2004). The deduced product of orf28c showed high similarity to AcyB2, an activator of carbomycin biosynthesis from Streptomyces thermotolerans (Arisawa et al., 1993), and to TylR, a pathway-specific transcriptional activator of tylosin biosynthesis in S. fradiae (Bate et al., 1999; Stratigopoulos et al., 2004). The gene orf32c encodes a putative transcriptional regulator from the GntR family. It should be noted that neither SrmR nor any of the putative regulators belong to those families of regulatory proteins that are commonly involved in the regulation of secondary metabolism, such as SARP, LAL or 
-butyrolactone-binding proteins (Bibb, 2005).
Limits of the cluster and genes of unknown function
On one side, the resistance gene srmD probably marks the limit of the spiramycin cluster, as the genes orf8* and orf9* are not involved in spiramycin resistance and as the other genes orf10* and orf11* encode an MbtH-like protein and an NRPS, respectively, two proteins for which no role in spiramycin biosynthesis could be proposed. The presence of these last two genes suggests that another secondary metabolite biosynthetic gene cluster, involving an NRPS, is adjacent to the spiramycin cluster. The most likely functions for most of the genes from orf7*c to orf28c are either antibiotic biosynthesis or resistance or regulation of secondary metabolism. After orf28c, the limit is not easily predictable; orf29 might be involved in the reactivation of glycosylated spiramycin, no role could be proposed for orf30c, orf31 could encode a post-PKS reductase and orf32c a transcriptional regulator. The two genes orf33 and orf34c are probably not part of the spiramycin cluster.
Inactivation of orf3 and orf2
The gene orf3 encodes an NDP-hexose aminotransferase probably involved in mycaminose biosynthesis. The inactivation of orf3 was performed early in the course of this study, to verify that the first genes isolated were indeed part of the spiramycin cluster. Inactivation of this gene by in-frame deletion using an excisable cassette resulted in a spiramycin non-producing mutant (strain SPM24); spiramycin production could be restored by expression of the cloned orf3 gene in this mutant, as reported previously (Raynal et al., 2006). Spiramycin biosynthesis could also be restored by expressing the tylB gene from S. fradiae in strain SPM24 (data not shown). As TylB is the NDP-hexose aminotransferase involved in mycaminose biosynthesis, this complementation experiment thus supports the involvement of Orf3 in mycaminose rather than in forosamine biosynthesis. Moreover, strain SPM24 was shown to produce platenolide aglycone, as expected if Orf3 is involved in mycaminose biosynthesis (data not shown).
The gene orf2 encodes an NDP-hexose isomerase and is highly similar to orf1a from S. fradiae, whose role in mycaminose biosynthesis was demonstrated recently (Melançon et al., 2005, 2007). This gene was inactivated by in-frame deletion using an excisable cassette and the resulting mutant (orf2 : : att3) was named SPM22. This mutant was unable to synthesize spiramycin, but production was restored by expression of orf2 (under the control of the ermE* promoter; plasmid pSPM30) or by expression of orf1a from S. fradiae (under the control of the ermE* promoter; plasmid pSPM33) (data not shown). It is interesting to note that the product of orf2 is also similar (46 % identity, 59 % similarity) to that of SAMR0473, a gene clustered with other deoxyhexose biosynthetic genes, and with large type I PKS genes, in the right arm of the S. ambofaciens chromosome (Choulet et al., 2006). Nothing is known about the expression of this other PKS I cluster and the product synthesized, but SAMR0473 obviously could not complement orf2 deletion.
Concluding remarks
The analysis of the spiramycin biosynthetic gene cluster from S. ambofaciens ATCC 23877 provides information on the origin of the precursors used by the PKS, the biosynthetic route for the deoxysugars and putative regulatory genes. For many of the biosynthetic genes, the closest orthologues are found in the midecamycin and tylosin clusters, in agreement with the chemical similarities of the macrolide molecules synthesized. Despite this high similarity at the protein level, the gene organization in the spiramycin cluster is strictly identical to that of the tylosin cluster only for the PKS genes and the seven genes upstream (orf1 to orf7 in the spiramycin cluster). From the partial data available on the midecamycin cluster (Cong & Piepersberg, 2007), the gene organization seems to be conserved between the spiramycin and midecamycin clusters for the seven genes downstream of the PKS and for the seven genes upstream (with the exception of an inversion that affects two genes). The complex regulation of tylosin biosynthesis has been studied thoroughly in S. fradiae (Bignell et al., 2007; and references therein), but the similarity with S. ambofaciens is probably limited, as only tylR, one of the multiple genes controlling tylosin biosynthesis, has an orthologue (orf10) in the spiramycin cluster. In addition to the question of regulation, this work also raised questions about the genes whose products are involved in the attachment of deoxysugars and about the existence of a mechanism for inactivation/reactivation of the produced antibiotic. The present determination of the sequence and organization of the spiramycin gene cluster will allow such questions to be addressed via targeted mutations.
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ACKNOWLEDGEMENTS |
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Edited by: P. R. Herron
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Received 18 May 2007;
revised 21 August 2007;
accepted 22 August 2007.
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90 kb locus from the S. ambofaciens chromosome. The positions of the fragments hybridizing with the various probes used are indicated (P1 to P5). Probe P1, 1 kb NaeI fragment containing most of the tylB gene from S. fradiae; probe P2, 3.7 kb BamHI–PstI fragment containing part of srmGI, orf1, orf2 and part of orf3; probe P3, 2 kb PstI–BamHI fragment containing orf7 and orf8; probe P4, 1.8 kb PstI–BamHI fragment containing srmD (orf7*c); probe P5, PCR product containing part of orf23c, orf24c and part of orf25c. Lines below correspond to the positions of the inserts of some of the cosmids, thick lines indicating regions of the insert that have been sequenced. Hatched lines indicate the two regions sequenced previously: the PKS genes (Burgett et al., 1999