Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK1
Author for correspondence: Eric Cundliffe. Tel: +44 116 252 3451. Fax: +44 116 252 3369. e-mail: ec13{at}le.ac.uk
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ABSTRACT |
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Keywords: tylosin production, Streptomyces fradiae, mycarose biosynthesis, deoxyhexose metabolism
Abbreviations: MGT, macrolide glycosyltransferase
The GenBank accession number for the sequence determined in this work is AF147704.
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INTRODUCTION |
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METHODS |
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DNA sequencing and computer analysis.
The sequence of orf6 was obtained by combining that given under accession numbers U08223 (Merson-Davies & Cundliffe, 1994 ) and AF145042 (Bate et al., 1999
). The St. fradiae tyl DNA sequenced here was obtained from pMOMT4 (Beckmann et al., 1989
). Fragments were subcloned in pIJ2925 (Janssen & Bibb, 1993
) and both strands of the DNA were sequenced independently in overlapping fashion by a combination of nested deletion analysis and primer walking. Sequencing was done on an ABI 377 automated DNA sequencer using Taq FS polymerase with dye terminator chemistry (Perkin Elmer). DNA sequences together with the corresponding chromatograms were imported into Seq Ed version 1.0.3 and aligned using AUTO ASSEMBLER. Sequences were analysed using the University of Wisconsin GCG software programmes. ORFs were identified using CODONPREFERENCE, BLASTX and 6 frame translation with DNA STRIDER. Deduced products were analysed using BLASTP.
Targeted gene disruption through gene transplacement.
The DNA fragment used to disrupt orf7* was generated by PCR using pMOMT4 as template (Beckmann et al., 1989 ). The primers used to create the orf7*-disruption fragment were 5'-ACTTGAATTCATGACGACGGGTCCGGGACG-3' and 5'-GGCGAAGCTTGGCCGGAGGTCAGGCCGCGG-3'. These contained, respectively, EcoRI and HindIII restriction sites (underlined) that were used to ligate the product (1513 bp with a unique central SalI site) into pIJ2925. The hygromycin resistance cassette,
hyg, together with its flanking transcriptional terminators (Blondelet-Rouault et al., 1997
) was then inserted at the SalI site, thereby interrupting the orf7* coding sequence 640 bp downstream of the earliest possible translational start.
A 2272 bp KpnI fragment containing orf6 together with flanking DNA was excised from pSET552 (Beckmann et al., 1989 ) and inserted into pIJ2925. The hygromycin resistance cassette was inserted at the unique SexAI site, 584 bp downstream from the start of orf6 and 784 bp upstream of the translational stop.
Disrupted ORFs plus flanking DNA were ligated into the BamHI site of pOJ260 and introduced into St. fradiae. Transconjugants that had undergone gene transplacement were resistant to hygromycin and sensitive to apramycin.
Complementation of disrupted strains.
Three different DNA fragments were reintroduced into the orf7*-disrupted strain. These were: (i) a 2167 bp fragment containing orf7* plus orf6* selected from a nested deletion library; (ii) an 825 bp fragment containing orf6* together with flanking DNA (227 bp upstream and 4 bp downstream) also selected from a nested deletion library; and (iii) the 1513 bp PCR product containing orf7* plus flanking DNA (118 bp downstream and 9 bp upstream). The orf6-disrupted strain was complemented using a 1630 bp NcoISmaI fragment generated from pSET552. This contained the whole of orf6 together with flanking DNA (150 bp upstream and 112 bp downstream). DNA fragments for complementation analysis were ligated into pLST9828 downstream of ermEp*.
Antibiotic-production fermentation and metabolite analysis.
Fermentation of St. fradiae strains and the analysis of products by HPLC was done as described elsewhere (Butler et al., 1999 ).
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RESULTS AND DISCUSSION |
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orf7* (tylCV).
In database searches, the deduced product of orf7* (461 amino acids; Mr 50100) displayed greater than 30% sequence identity to various macrolide glycosyltransferases (MGTs), such as OleI and OleD from St. antibioticus, producer of oleandomycin (Quirós et al., 1998 ; Hernandez et al., 1993
), GimA from Streptomyces ambofaciens, producer of spiramycin (Gourmelen et al., 1998
) and Mgt from Streptomyces lividans (Jenkins & Cundliffe, 1991
). Such enzymes inactivate their substrates and are involved in antibiotic efflux and/or resistance. Less close matches (2025% sequence identity) were also seen with glycosyltransferases involved in antibiotic biosynthesis such as OleG2 (Olano et al., 1998
), EryCIII and EryBV (Gaisser et al., 1997
; Summers et al., 1997
; Salah-Bey et al., 1998
). Nevertheless, when the role of orf7* was addressed by targeted gene disruption it proved to be a mycarose-biosynthetic gene and not an mgt. Thus, the orf7*-disrupted strain accumulated desmycosin during fermentation (Fig. 4a
) and tylosin production was restored following complementation with cloned DNA, but only when orf6* was added together with orf7* (Fig. 4b
), in this case on a single DNA fragment. Reintroduction of either gene alone did not restore tylosin production (Fig. 4c
, d
) although unidentified material, which was clearly separable from both tylosin and desmycosin, accumulated together with the latter when complementation was attempted with orf7* alone (Fig. 4c
). These data established that orf7*, like orf6*, must be a mycarose-biosynthetic gene and that normally the two genes are probably co-transcribed. Disruption of orf7* using the
hyg cassette (which is flanked by transcriptional terminators) evidently caused a downstream polar effect on the expression of orf6*.
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orf8* (tylCIII).
The deduced product of orf8* (418 amino acids maximum; Mr 46600) displays extremely close end-to-end similarity (with 70% sequence identity) to that of ery orf3 (Haydock et al., 1991 ), now renamed eryBIII (Gaisser et al., 1998
), and is much more closely related to that protein than to any other in the database. Moreover, Orf8* and EryBIII each possess multiple sequence motifs characteristic of S-adenosylmethionine-dependent methyltransferases (Kagan & Clarke, 1994
). Since a strain of Sa. erythraea with a point mutation in eryBIII accumulated 3'-C-desmethyl-erythromycin A during fermentation (Gaisser et al., 1998
), the unequivocal conclusion was that eryBIII encodes a 3-C-methyltransferase activity required during mycarose biosynthesis. An equivalent role is proposed for the orf8* product, designated TylCIII.
orf9* (tylCIV).
The deduced product of orf9* (maximum size, 347 amino acids; Mr 36200) displays 49% sequence identity to that of eryBIV (Gaisser et al., 1997 ; Summers et al., 1997
). The only other significant end-to-end sequence match (46% identity) was to an uncharacterized protein, PCZA 361.4, from vancomycin-producing Amycolatopsis orientalis (van Wageningen et al., 1998
). Mutants of Sa. erythraea, altered in eryBIV, accumulated modified erythromycins in minor amounts and mass spectrometric analysis (Salah-Bey et al., 1998
) revealed that the neutral sugar (mycarose) appeared to lack two hydrogen atoms. From these data, it was concluded that eryBIV encodes 4-ketoreductase activity involved in mycarose metabolism. An equivalent role is proposed here for the orf9* product, TylCIV. Consistent with this proposal, TylCIV and EryBIV are distantly related to UDP-glucose 4-epimerases and to various oxidoreductases that manipulate sugars at the 4-position, including NDP-glucose 4,6 dehydratases such as TylAII (formerly TylA2; Merson-Davies & Cundliffe, 1994
) and StrE (Pissowotzki et al., 1991
). Moreover, TylCIV contains a candidate NADPH-binding sequence (GRGVIAVTAG) with a downstream arginine cluster (RDLLDVR) positioned suitably to form a binding site for the 2'-phosphate of NADPH (N. S. Scrutton, personal communication).
orf10* (tylCII).
The deduced product of orf10* (329 amino acids; Mr 36100) is very similar (74% sequence identity) to EryBII from Sa. erythraea (Summers et al., 1997 ; Salah-Bey et al., 1998
). Less close matches (41% and 30% sequence identity, respectively) were found with MocA from Agrobacterium tumefaciens (Kim & Farrand, 1996
), which is weakly related to glucose-6-phosphate dehydrogenase, and with an authentic aryl alcohol dehydrogenase from the white rot fungus, Phanerochaete chrysosporium (Reiser et al., 1994
). Despite the absence of any obvious NAD(P)H-binding motif in the amino acid sequence, these data suggest that Orf10* might be one of the two reductases involved in mycarose biosynthesis. The function of EryBII was revealed during incisive analysis of Sa. erythraea mutant BII92 (Salah-Bey et al., 1998
), a specifically deleted strain that produced a 2',3'-unsaturated derivative of 3'-C-desmethyl-erythromycin C. Those authors suggested that eryBII encodes 2,3-enoyl reductase activity involved in mycarose biosynthesis and a similar role is proposed here for the orf10* product, designated TylCII.
orf6 (tylCVI).
The missing gene of the mycarose-biosynthetic pathway (tylCVI) is not present in the tylCK region of the St. fradiae genome. Somewhat surprisingly, it is located about 50 kb distant from orf6*10*, on the opposite side of the tylG gene cluster adjacent to the regulatory gene tylR (Bate et al., 1999 ). The maximum length of the orf6 product is 455 amino acid residues, which would give a 50 kDa protein. The involvement of orf6 in mycarose metabolism was first suspected when the upstream gene (orf5) was specifically disrupted and then complemented with intact orf5. The resultant strain produced desmycosin (demycarosyl-tylosin) rather than tylosin, suggesting that disruption of orf5 had caused a downstream polar effect on the expression of a mycarose-biosynthetic gene (Butler et al., 1999
). Validation of that interpretation involved targeted disruption of orf6 via gene transplacement (confirmed by Southern analysis) followed by HPLC screening of fermentation products. This revealed a major peak of UV-absorbing material that co-eluted with desmycosin and was clearly resolved from tylosin (Fig. 5b
). The yield of desmycosin approximated that of tylosin seen in control fermentations with the wild-type strain (Fig. 5a
). More detailed analysis, involving HPLC-MS, revealed that the single peak in Fig. 5
(b) represented a mixture of desmycosin plus 20-dihydrodesmycosin, together with two minor components consistent with N-demethyldesmycosin and N-demethyl- 20-dihydrodesmycosin (D. E. Kiehl & H. A. Kirst, personal communication). Macrolide reductase activity, which converts tylosin to relomycin (20-dihydrotylosin) and also acts on the corresponding aldehyde moieties of other macrolides, was purified from tylosin-producing strains of St. fradiae and was also detected in other Streptomyces spp. (such as St. lividans) that do not produce macrolides (Huang et al., 1993
). Therefore, the presence of 20-dihydrodesmycosin in the orf6-disrupted strain is readily rationalized, although the detection of N-demethyl compounds, albeit at low levels, was unexpected.
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The orf6 product, TylCVI, displays end-to-end similarity (with 51% sequence identity) to the deduced product of eryBVI from Sa. erythraea (Gaisser et al., 1997 ; Summers et al., 1997
) and to the products of dnmT from Streptomyces peucetius (Scotti & Hutchinson, 1996
) and orf3 from Streptomyces sp. C5 (Dickens et al., 1996
), producers of doxorubicin and daunomycin, respectively. Arguing by elimination, it was suggested that EryBVI is the 2,3-dehydratase required for deoxygenation at C-2 during mycarose biosynthesis in the erythromycin producer (Gaisser et al., 1997
; Summers et al., 1997
) and a similar role is invoked for TylCVI during tylosin production in St. fradiae.
Concluding remarks
It will be interesting to study regulation of the tylC genes during tylosin production. The physical organization of orf6*10* would permit possible co-regulation from a pair of divergent (perhaps equivalent) promoters located in the non-coding gap between orf7* and orf8*. Although the minimum size of this gap is 212 bp, we suspect that the TylCV coding sequence is considerably shorter than orf7* and that the intergenic region is accordingly greater than 400 bp. Gaps of this size are rare in the tyl cluster. Since there are no spaces between orf 8*,9* and 10*, and since orf7* and 6* are separated by only 43 bp, this group of genes might constitute two short operons. But what, if anything, is gained by having one mycarose gene displaced so far from the others? And is it significant that the mycaminose-biosynthetic genes are similarly split, with three co-directional tylM genes (orf1*3*) located near the mycarose-gene cluster whereas tylB lies on the other side of tylG, near tylCVI (Merson-Davies & Cundliffe, 1994 ).
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ACKNOWLEDGEMENTS |
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Received 18 June 1999;
revised 29 September 1999;
accepted 11 October 1999.