* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The dnrM gene in Streptomyces peucetius contains a
Zinc finger nuclease wikipedia , lookup
Transposable element wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Molecular ecology wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Genomic imprinting wikipedia , lookup
Ridge (biology) wikipedia , lookup
Biosynthesis wikipedia , lookup
Expression vector wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Molecular cloning wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Gene therapy wikipedia , lookup
Genetic engineering wikipedia , lookup
Gene expression wikipedia , lookup
Genomic library wikipedia , lookup
Gene desert wikipedia , lookup
Gene nomenclature wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Gene regulatory network wikipedia , lookup
Point mutation wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Gene expression profiling wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Community fingerprinting wikipedia , lookup
MivObiOlOgy (1996), 142,269-275 Printed in Great Britain The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-productiongene cluster Mark A. Gallo,l Joanne Wardlt and C. R. Hutchinson'#* Author for correspondence: C. R. Hutchinson. Tel: e-mail : crhutchia facstaff.wisc.edu School of Pharmacy' and Department of Bacteriologyz, University of Wisconsin, Madison, WI 53706, USA + 1 608 262 7582. Fax: + 1 608 262 3134. A 2.7 kb BamHl fragment of the daunorubicin biosynthetic cluster in Streptomyces peucetius ATCC 29050 was shown to contain two ORFs, dnrL and dnfM#whose deduced products exhibit a high sequence similarity to a number of glucose-l-phosphate thymidylyl transferases and TDP-D-glucose dehydratases, respectively. Although these genes were believed to be necessary for the synthesis of the deoxyaminosugar, daunosamine, a constituent of daunorubicin, the dnrM gene contains a frameshift in the DNA sequence that causes the premature termination of translation. A gene encoding another TDP-glucose 4#6=dehydratase, previously isolated from 5. peucetius, was identified by PCR amplification of genomic DNA. The presence of this gene explains why a dnrM::aphll mutation did not block daunorubicin production. Keywords: daunosamine, deoxyamino sugar, inactive gene, TDP-D-glucose 4,6deh ydratase, glucose-1-phosphate thymidylyl transferase INTRODUCTION The Streptomyes are filamentous, spore-forming soil bacteria that produce an array of products termed secondary metabolites. Many are useful in agriculture and medicine, for example as antiparasitic, antimicrobial, or anticancer agents. Relatively little is known in general about the biochemistry, physiology and genetics of production of secondary metabolites. The anthracyclines are a prevalent class of such compounds isolated from Streptomyes sp. and other actinomycetes. Two anthracyclines, daunorubicin (DNR) and doxorubicin (DXR), have received considerable attention due to their potent antitumour activity (Arcamone, 1981). Our laboratory has been interested in determining the biochemical and genetic processes involved in the synthesis of DNR and DXR in Streptomyes pezlcetins ATCC 29050. t Presetnt address: Sandoz Clinical DevelopmentCentre, The Quadrangle, Frimley Business Park, Frimley, Camberley, Surrey, GU16 SSQ, UK. Abbreviations: DNR, daunorubicin; DXR, doxorubicin; GST, glutathione S-transferase; RHO, c-rhodomycinone; TDP, thymidylyl diphosphate. The GenBanWEMBL accession number for the sequence reported in this paper is L47163. 0002-0162 0 1996 SGM Previous reports have identified a 45 kb region that presumably contains all the genes required for the synthesis of and resistance to DNR and DXR (Otten e t al., 1990; Stutzman-Engwall 8c Hutchinson, 1989; Madduri & Hutchinson, 1995a). DNR and DXR consist of two parts, a polyketide-derived aglycone and the trideoxyamino sugar daunosamine. All of the genes required for synthesis of the aglycone carbon skeleton have been characterized (Guilfoile & Hutchinson, 1991; Grimm e t al., 1994; Madduri & Hutchinson, 1995b), and most of them share a high degree of homology with other type I1 polyketide synthases and cyclases at the level of DNA and deduced protein sequence (Hopwood & Sherman, 1990; Katz & Donadio, 1993; Hutchinson & Fujii, 1995). By contrast, little is known about the genes involved in biosynthesis of daunosamine (Krugel etal., 1993;Thorson etal., 1993; Otten etal., 1995b). In this paper we report the sequence of a 2682 bp DNA segment containing two putative ORFs, dnrL and dnrM (Fig. l), located within the known boundaries of the 45 kb dnr gene cluster. We show that dnrL likely encodes a glucose-1-phosphate thymidylyl transferase and dnrM encodes a thymidylyldiphospho(TDP)-D-glucose 4,6-dehydratase7on the basis of sequence comparisons to other known transferases and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 269 M. A. GALLO, J. W A R D a n d C. R. H U T C H I N S O N I B B I 1 dnrR2 deoxysugar B B I I regulatory Bg B // I 1 oxidation dehydratases involved in deoxysugar metabolism. However, the dnrM sequence was unexpectedly found to contain a frameshift that results in the formation of a truncated protein. Thus, inactivation of dnrM in S. pezlcetim 29050 did not prevent DNR o r DXR production, suggesting that another dehydratase-encoding gene existed outside the 45 k b dnr region. Southern analysis of chromosomal DNA using dnrM as a probe showed, in addition to dnrM, several weakly hybridizing bands ; one of these appeared t o be the gene for another dehydratase that, from the results of initial PCR amplification and D N A sequence analysis, encodes a dehydratase previously purified from S. pet/cetit/s (Thompson e t a/., 1992). METHODS Bacterial strains and plasmids. S. peucetius ATCC 29050 was obtained from the American Type Culture Collection. Escber i c h coli strains DH5aMCR (Life Technologies) and JM105 (Yanisch-Perron e t al., 1985) were used for routine subcloning and preparation of ssDNA. The glutathione S-transferase (GST) vector pGEX-4 was obtained from Pharmacia. The high-copy number E. coli-Streptomyces shuttle vector pWHM3 was from Vara e t al. (1989). M13 phage-derived mp18 and mp19 vectors (Yanisch-Perron e t al., 1985) were used for sequencing by previously published methods (Summers e t a/., 1992). The pUC4-KIXX plasmid containing the neomycinlkanamycin resistance gene (aphll) from Tn5 was obtained from Pharmacia. The plasmid pDH5 (Hillemann et al., 1991) was used to generate ssDNA for transformation of Streptomyces sp. and gene disruption ; pWHM3 was used to clone the wild-type dnrLM genes. Media and growth conditions. S. peucetius was grown at 30 OC on R2YE (Hopwood etal., 1985) for propagation of cells, DNA isolation, and protoplast preparation. ISP4 medium (Difco) was used to produce spores. Transformants of S. peucetius were selected on 10 pg thiostrepton ml-', 25 pg apramycin ml-' or 50 pg neomycin ml-'. E. coii transformants were selected with 100 pg ampicillin ml-', 100 pg apramycin ml-' or 50 pg neomycin ml-'. S. peucetius strains were grown in 5 ml seed medium (Guilfoile & Hutchinson, 1991) for 2 d, then transferred to GPS production medium (Dekleva e t al., 1985) and grown in 50 ml broth in a 250 ml baffle-bottom flask for an additional 3 d at 30 "C with shaking at 300 r.p.m. Cultures were acidified with oxalic acid, heated at 60 OC for 45 min, adjusted to pH 8.5, and extracted with chloroform (Otten e t al., 1990). The extracted anthracycline metabolites were analysed by TLC or HPLC (Otten e t al., 1990). DNA isolation and manipulation. DNA isolation, restriction 270 2kb dnrR1 I B 1 resktanc ..................................... ......... .............................. ................................ Fig. I . Partial restriction map of a selected region of the dnr cluster of S. peucetius 29050. dnrN, 0 (Otten et a/., 1995a); dnrF, A, B (Guilfoile & Hutchinson, 1991); dnrl, I (Madduri & Hutchinson, 199513; StutzmanEngwall et a/., 1992). Restriction sites: B, BamHI; Bg, Bglll. endonuclease digestions, and ligations were performed according to standard techniques (Sambrook e t al., 1989). Hybridization analysis was carried out using the Genius nonradioactive kit (Boehringer Mannheim) according to the manufacturer's instructions. DNA was electrophoresed on 0.7 YO agarose gels, and alkali transfer to Hybond-N (Amersham) was performed for Southern analysis. DNA sequencing. DNA fragments were subcloned into M13mp18 and M13mp19, and single-stranded templates were sequenced by the dideoxy chain-termination method using (a35S)dCTP and Sequenase version 2.0 (USB) according to the manufacturer's instructions. 7-Deaza-dGTP was used instead of dGTP to avoid compressions. Disruption of dnrM. A 725 bp BglII fragment internal to dnrM was removed from the 2.7 kb BamHI fragment containing dnrL and dnrM, and the DNA was blunt-ended by treatment with the Klenow fragment and ligated to a 1.1 kb SalI fragment containing apbll. The resulting 3.1 kb fragment was subcloned into pDH5 (Hillemann e t al., 1991) to give pWHM209, and ssDNA was produced and used to transform S. peucetitls 29050 protoplasts. Transformants were selected on R2YE plates containing thiostrepton or neomycin to verify integration into the 5'.peucetius genome by homologous recombination. Several of the transformants were subjected to two rounds of sporulation, and single colonies were picked and scored for the expected neomycin-resistant thiostrepton-sensitive phenotype of a double crossover recombination event. Four mutants were analysed by Southern analysis to verify gene disruption and replacement by digesting total chromosomal DNA with BamHI and probing with either the 725 bp BglII or the 2.7 kb BamHI fragment. All neomycin-resistant, thiostrepton-sensitive mutants lacked the 725 bp BglII fragment and contained a BamHI fragment of 3.1 kb, properties one would expect of gene replacement mutants (data not shown). One dnrM: :aphll clone was chosen and named WMH1590. PCR amplification. PCR was used to amplify an additional putative dehydratase from S. peucetius 29050 chromosomal DNA. The PCR mixture contained 1 x PCR buffer [SO mM KC1, 10mM Tris/HCl, pH 9.0 (at 25 "C), 0.1 YOTriton X-100, and 2-5 mM MgCl,] supplied by Promega. Each primer was present at 0.3 pM, and each dNTP was present at 0.1 mM. The codon bias of Streptomyces genes (Wright & Bibb, 1992) was taken in to account in designing the following two primers: GTCAACGTCACCGTCACCGGCGCCGCCGGCCAGATC- GG(G/C) TACGC(G/C)CT, which corresponds to the Nterminal peptide sequence reported previously (Thompson e t al., 1992); and TCA(G/C)AG(G/C)GGCTCCCACCAG(G/C)(A/T)(G/C)CG, which corresponds to the complementary sequence of a conserved peptide in the C-termini of TDP-Dglucose dehydratases. Taq polymerase (1 U, Promega) and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 Daunorubicin synthesis genes approximately 100 ng target DNA was added in a final reaction volume of 100 pl. Amplification was performed in a thermal cycler (model 480, Perkin-Elmer Cetus) by denaturing the samples at 100 "C for 5 min and then subjecting them to 25 cycles of denaturing (97 "C, 30 s), annealing (55 "C, 45 s) and elongation (70 "C, 90 s). Amplification products were bluntended with Klenow and 0.1 mM dNTPs, separated by agarose gel electrophoresis, and purified with Qiaex resin (Qiagen). The resulting DNA fragment was ligated into M13mp18 and 19 for sequencing. Expression of dnrM in E. coli. The GST gene fusion system (Pharmacia)was used to express dnrM in E. coli. Plasmid pGEX4T-1 was cut with XhoI, and linkers were added to introduce an SstI site. This modified plasmid was cut with EcoRI, bluntended by treatment with the Klenow fragment, then the DNA was cut with JstI and ligated to a 1 kb BspHIlblunt-JstI fragment containing dnrM to give pWHM204. Expression of GST:DnrM in E. coli(pWHM204) strains was induced with 1 mM IPTG. The formation of a novel, unique protein was demonstrated by SDS-PAGE analysis. RESULTS Sequence analysis of dnrL and dnrM Sequence analysis by CODONPREFERENCE (Devereux e t al. , 1984) of a 2.7 kb BamHI fragment from within the dnr gene cluster (Fig. 1) revealed two ORFs, dnrL and dnrM. The region encompassing nt 1-655 appeared to contain no long ORFs with any recognizable similarity to known proteins by TFASTA analysis (Devereux e t al., 1984). The most likely translational start site for dnrL is the ATG beginning at nt 655 and ending with TGA at nt 1708-1710, producing a polypetide of 351 aa. The translational start site for dnrM is believed to be the ATG beginning at nt 1707. This start site overlaps the TGA stop of dnrL, implying a possible translational coupling of these two proteins. A T G A stop codon beginning at nt 1890 allowed for production of a 61 aa polypeptide (6572 Da). CODONPREFERENCE analysis revealed a switch in reading frames approximately 97 nt beyond the ATG, at nt 1707. If the D N A sequence data are interpreted in a manner that allows introduction of a frameshift, a TGA stop codon beginning at nt 2680 is found in this new frame, implying production of a polypeptide of 324 aa (35 177 Da) for the combined ORFs from nt 1707 to 2679. Codon usage analysis of this frame showed very few rare Streptomyes codons (Wright & Bibb, 1992). Characteristics of the deduced gene products of dnrL and dnrM The deduced product of dnrL has a high sequence similarity to several glucose-1 -phosphate thymidylyl transferases (Table l),enzymes that are involved in transferring a TDP moiety to glucose. This irreversible reaction is an important first step in the activation of sugars for eventual transfer to other molecules (Liu & Thorson, 1994). Consequently, dnrL most likely provides this enzyme for daunosamine biosynthesis. The deduced product of the truncated dnrM gene shares a significant sequence similarity to the N-terminal portion of several TDP-glucose 4,6-dehydratasesmTranslation of the second reading frame identified by CODONPREFERENCE analysis reveals a remarkable sequence similarity to the remaining amino acid residues of this family of dehydratases (Table 1). Therefore, we believe that these two frames should be linked (Fig. 2). A simple sequencing error was not responsible for the shift in translational sequence revealed in Fig. 2 because the region was sequenced several times in both directions from several Table I . Comparisons between the amino acid sequences of DnrL and DnrM with other deoxysugar biosynthesis enzymes having similar functions (Devereux et al., 1984) analysis was used for comparisons. DnrM in this table refers to the combined reading frames as noted in Fig. 2 and the text. BESTFIT Protein Number of amino acids Similarity Identity Reference (%) DnrL StrD TylAl RfbA 355 304 76.3 59.3 293 55.6 59.7 36.9 31-5 Pissowotzki et al. (1991) Merson-Davies & Cundliffe (1994) Stevenson e t al. (1994); Yao & Valvano (1994) 61.6 46.7 58.1 Pissowotzki et al. (1991) Stevenson e t af. (1994) Linton e t al. (1995) DnrM StrE RfbB Gdh 328 361 329 76.2 63.0 71.4 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 271 M. A. GALLO, J. W A R D a n d C. R. H U T C H I N S O N 1 121 241 361 481 601 721 841 961 1081 1201 1321 1441 1561 1681 G A C C A ~ T C T D D S R V E I S S . C C 7 C A l Q A C C A l G M G A X C T W l C k ~ ~ C T A & H - > f M T Y K I L V T G G A G P I G S H Y V R A L L G P R G D G S V G T 1801 GGGTCACKiCPCGACAGCCKiACCTATOCAOaClUTCCOO AC C V T A G Q P D L C R Q S G Q P R S R P R R R A V R L R P R * O C T G A C A ~ G ~ G A ~ * L L D S L T Y A G N P A N L D P V R G D E R F A , F V H G D I C D A P L V D R 1921 m -- C A mCCCAA-lUGGTACGCAM- m L V A E H D Q I V H F A A E S H V D R S V R S G , P R . S S C A P N V M G T Q T L L 2041 G A C O C O O C C C r O C O C C A D A A L R ~ A P R I P V H I S ~ D E V Y O S V O V O A S T E T D P L H P S S A Y ~ m ~ ~ m ~ A P ~ ~ 2161 G C G Q G C A ~ ~ A C C A C C b C G C A C ~ C W X C G G A ~ C A A C T A C G G C M X C A A G T K A S S D L L A L S Y H R T H G L D V R I T R C S N N Y G T H Q H P E K V T G h W W A c G G T G h ~ ~ ~ G G n ~ 2281 A I P R P V T T L L S G G R V P L Y G D G G N L R N W L H V E D H V D A U E L V R ~ C A A C A A ~ ~ ~ 2401 GAGGGA-TCTACAACAE G G S P G E I Y N I G G G T E L S N K E L T A L L L D A V G A G W D S V E L U ~~~ --AGCcG 2521 Ac - - - CAC l A D R K G H D R R Y A V D H G K I T A L L G Y R P R R D F R D S L A E T V A W Y 2641 ~~~~~ R D R R S W W A P L T G S . ..,..........,......,....................................................................,...,,......................................................................,....,.....................,........,........................,.................................................................................................. Fig. 2. Nucleotide sequence of the 2682 bp BamHl fragment containing dnrL and dnrM. Putative RBSs are underlined. The translated amino acid sequences are shown below the DNA sequence. The presumed site of the frameshift in dnrM is indicated by a ' 0 and asterisks indicate stop codons. M13 plaques derived from independent primary clones. Additionally, the region was cloned by PCR from S. pezicetizls 29050 chromosomal DNA and again shown to contain the same frameshift by DNA sequencing. Expression of dnrM in E. co/i A GST: DnrM fusion protein (Methods) appeared as a 32.6 kDa band (Fig. 3), which is 6.6 kDa larger than the band corresponding to GST alone. This indicates that a polypeptide of 61 aa is present at the C-terminal end of GST, in agreement with the predicted size if the truncated 272 DnrM protein is fused to GST. A fusion protein of 60 kDa [equal to GST (26 kDa) plus a full-length DnrM (34 kDa)] was not detected (Fig. 3). This result is consistent with the sequence analysis and corroborates the conclusion that dnrM contains a naturally occurring frameshift mutation. Disruption of dnrM The chromosomal copy of dnrM in S. pezrcetitls 29050 was inactivated by insertion of apb11 to produce the WMH1590 strain (Methods). Wild-type S.pezlcetizls 29050 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 ~ ~ Daunorubicin synthesis genes kDa 94 67 - 43 1 2 3 Isolation of another TDP-D-glucose 4,6-dehydratase gene - - GST: DnrM fusion - 30 GST- 21.5 formation of TDP-D-glucose is not a rate-limiting step in the conversion of RHO to DNR. 4 - Fig. 3. SDS-PAGE of E. coli cell extracts containing the GST: DnrM fusion vector, pWHM204. Lanes: 1, molecular mass markers; 2, E. coli DH5aMCR(pGEX4), induced; 3, DH5aMCR(pWHM204), uninduced; 4, DH5aMCR(pWHM204), induced. Table 2. Effects on secondary metabolite production of the introduction of dnrM into 5. peucetius strains 29050 and WMH1590 ............................................................................ .............................. Values are means of three samples for RHO ( & 417 ) and DNR (& 1.02) and are in pg ml-'. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I Strainlplasmid RHO DNR Ratio 29050/pWHM3 29050/pWHM205 WMH1590/pWHM3 WMH1590/pWHM205 27-7 34.5 35-5 21.2 2.9 3.6 2.4 1.7 9.6 9.6 14.8 12-5 and WMH1590 strains were transformed with the pWHM3 vector DNA (Vara e t al., 1989) or pWHM205, a plasmid containing the wild-type copy of the 2.7 kb BamHI fragment with the dnrLM genes that had been cloned in pWHM3. The levels of erhodomycinone (RHO) or DNR produced by these strains (Table 2) were the same within experimental error. These results indicate the following : first, an uninterrupted copy of dnrM is not required for the synthesis of DNR; second, a gene encoding a functional TDP-Dglucose 4,6-dehydratase must be present in S. pezlcetitls 29050 since DNR was produced in both wild-type and WMHl590 strains. Finally, since additional copies of dnrL do not enhance the conversion of RHO to DNR, the Thompson et al. (1992) have partially purified a TDP-Dglucose 4,6-dehydratase from S.petleetins. The N-terminal sequence of their protein does not agree with that predicted for DnrM, although it does show some similarity to this family of dehydratases (Liu & Thorson, 1994). We synthesized degenerate oligodeoxynucleotides based on highly conserved regions at the N-terminus of the reported amino acid sequence (Thompson e t al., 1992) and the reverse complement of the C-termini of such dehydratases (Liu & Thorson, 1994), then amplified by PCR the targeted region in the DNA from S. pezlcetias 29050. The DNA sequence of a small portion of the 1.1 kb fragment amplified was determined. Its translated sequence is : VNVTVTGAAGQIGYALVTVIGAAGLIG YALQRT; residues in bold are highly conserved in TDP-D-glucose 4,6-dehydratases (Liu & Thorson, 1994). This sequence is not the same as that proposed for DnrM (Fig. 2), but the first 14 aa are identical to those reported by Thompson e t al. (1992). It thus appears that we have located the gene for a functional TDP-D-glucose 4,6dehydratase of S. pezmtizls. The DNA sequence was not identical to any part of the 45 kb region of the dnr gene cluster sequenced, indicating that this other dehydratase is not encoded by a gene inside the defined dnr cluster. We were also unable to amplify this other dehydratase gene from pWHM341 (Otten e t al., 1990), a cosmid containing a 27 kb DNA segment to the left of the defined dnr cluster (Madduri & Hutchinson, 1995a), as in Fig. 1. DISCUSSION The genes for the biosynthesis, regulation, and resistance to secondary metabolites are normally clustered in Streptomzces species, perhaps because this arrangement confers a selective advantage in maintenance and regulation. Coordinate expression of such genes has often been found (Chater & Bibb, 1995). In this regard, we have shown that a transcript containing dnrL and dnrM is present in S. petlcetz'zjs 29050, and is regulated by the dnrI gene encoding a transcription factor (Madduri & Hutchinson, 1995a). On the basis of the above, we were surprised to find an inactive dnrM gene in the dnr cluster. The lack of a functional gene encoding a TDP-D-glucose 4,6dehydratase in this gene cluster implies the presence of another functional gene elsewhere in the organism, as suggested by the results of our exploratory studies and the work of Thompson e t al. (1992). Moreover, the existence of a dnrL homologue at another locus that provides a functional glucose-1-phosphate thymidylyl transferase is not excluded by our data. Since there are many other glycosylated anthracyclines produced by Streptomtees species, it would be interesting to investigate whether other DNR-producing species, or descendants of strain 29050, such as S.peucetitls subsp. caesizls ATCC 27952 (Arcamone e t al., 1969), also contain a mutant dnrM homologue. (It Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 273 M. A. G A L L O , J. W A R D a n d C. R. H U T C H I N S O N would be surprising to find that dnrM was mutated only in the isolate of the 29050 strain in our collection, but we have not tested this possibility.) Although it is unusual to find nonfunctional genes in Streptomyes operons, nonfunctional ketoreductase domains in the products of polyketide synthase genes have been reported in the erythromycin-producing Saccharopobspora eytbraea (Donadio & Katz, 1992) and the avermectin-producing Streptomyes avermitilis (MacNeil e t al., 1994). DNR biosynthesis is unique in this regard since a nonfunctional gene is present, but another gene product from outside the biosynthetic gene cluster complements the missing activity. In erythromycin biosynthesis, the cluster of production genes, as currently defined, lacks genes encoding glucose-1-phosphate thymidylyl transferase and TDP-D-glucose 4,6-dehydratase, but genes for these enzymes have been found elsewhere in S. eytbraea (Linton e t al., 1995; S. Zotchev & C. R. Hutchinson, unpublished results). The unglycosylated intermediate RHO is approximately 10-fold more prevalent than DNR when S. pegcetizls is grown in GPS medium (Dekleva et al., 1985). Therefore, it appears quite likely that one of the steps of daunosamine biosynthesis or attachment is rate-limiting. It is also possible that the TDP-D-glucose 4,6-dehydratase that actually catalyses the second step of daunosamine biosynthesis in S.pezlcetitls is not well-adapted for this function and hence is forming a metabolic bottleneck. Consequently, it would be interesting to determine the effect on DNR production in S. petlcetitls 29050 of repairing the frameshift in dnrM or placing the other TDP-D-glucose 4,6-dehydratase gene under control of dnrl (Madduri & Hutchinson, 1995a), or introducing additional copies of a known TDP-D-glucose 4,6-dehydratase. ACKNOWLEDGEMENTS We are grateful to K. Linton and B. Jarvis for sharing sequence data prior to publication. The authors would also like to thank B. Jarvis and S. L. Otten for helpful discussions. This work was supported by grants from the National Institutes of Health (GM 25799 and 31925) and Pharmacia Spa, Milan, Italy. REFERENCES Arcamone, F. (1981). Doxorubicin. Med Chem Ser Monogr 17,25-31. Arcamone, F., Cassinelli, G., Fantini, G., Grein, A,, Orezzi, P., Pol, C. & Spalla, C. (1969). 14-hydroxydaunomycin, a new antitumor antibiotic from Streptomycespeucetiusvar. caesius. Biotechnol Bioeng 11, 1101-1 110. Chater, K. F. & Bibb, M. J. (1995). Regulation of bacterial antibiotic production. In Products of Secondav Metabolism. Biotechnology, vol. 7. Weinheim : VCH. Dekleva, M. L., Titus, J. A. & Strohl, W. R. (1985). Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium. Can J Microbiol 31, 287-294. Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395. Donadio, 5. & Katz, L. (1992). Organization of the enzymatic domains in the multifunctional polyketide synthase involved in 274 erythromycin formation in SaccbaropoEyspora erythraea. Gene 110, 5140. Grimm, A., Madduri, K., Ali, A. & Hutchinson, C. R. (1994). Characterization of the Streptomyces peucetius ATCC 29050 genes encoding doxorubicin polyketide synthase. Gene 151, 1-10. Guilfoile, P. G. & Hutchinson, C. R. (1991). A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci U S A 88, 8553-8557. Hillemann, D., Puhler, A. & Wohlleben, W. (1991). Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors. Nucleic Acids Res 19, 727-73 1. Hopwood, D. A. & Sherman, D. H. (1990). Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet 24, 37-66. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. & Schrempf, H. (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich : The John Innes Foundation. Hutchinson, C. R. & Fujii, 1. (1995). Polyketide synthase gene manipulation : a structure-function approach in engineering novel antibiotics. Annu Rev Microbiol49, 201-38. Katz, L. & Donadio, 5. (1993). Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol47, 875-912. Krugel, H., Schumann, G., Hanel, F. & Fielder, G. (1993). Nucleotide sequence analysis of five putative Streptomyces griseus genes, one of which complements an early function in daunorubicin biosynthesis that is linked to a putative gene cluster involved in TDPdaunosamine formation. Mol b Gen Genet 241, 326-333. Linton, K. J., Jarvis, B. W. & Hutchinson, C. R. (1995). Cloning of the genes encoding thymidine diphosphoglucose 4,6-dehydratase and thymidine diphospho-4-keto-6-deoxyglucose3,5-epimerase from the erythromycin-producing SaccharopoEyspora evtbraea. Gene 153, 33-40. Liu, H. W. &Thorson, J. 5. (1994). Pathways and mechanisms in the biogenesis of novel deoxy sugars by bacteria. Annu Rev Micrubiol48, 223-256. MacNeil, D. J., Occi, J. L., Gewain, K. M. & MacNeil, T. (1994). Correlation of the avermectin polyketide synthase genes to the avermectin structure : implications for designing novel avermectins. Ann NY Acad Sci 721, 123-132. Madduri, K. & Hutchinson, C. R. (1995a). Functional characterization and transcriptional analysis of the dnrR 1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius. J Bacterioll77, 1208-1215. Madduri, K. & Hutchinson, C. R. (1995b). Functional characterization and transcriptional analysis of a gene cluster governing early and late steps in daunorubicin biosynthesis in Streptomyces peucetius. J Bacterioll77, 3879-3884. Merson-Davies, L. A. & Cundliffe, E. (1994). Analysis of five tylosin biosynthetic genes from the t$lBA region of the Streptomycesfradiae genome. Mol Microbioll3, 349-355. Otten, 5. L., Stutzman-Engwall, K. J. & Hutchinson, C. R. (1990). Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius. J Bacteriof 172, 3427-3434. Otten, 5. L., Ferguson, J. & Hutchinson, C. R. (1995a). Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus. J Bacterioll77, 12161224. Otten, 5. L., Liu, X., Ferguson, J. & Hutchinson, C. R. (1995b). Cloning and characterization of the Streptomyces peucetius dnrQS Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 Daunorubicin synthesis genes Thompson, M. W., Strohl, W. R. & Floss, H. G. (1992). Purification genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis. J Bacterioll77, 6688-6692. Pissowotzki, K., Mansouri, K. & Piepersberg, W. (1991). Genetics of streptomycin production in Streptomyces griseus : molecular structure and putative function of genes strELMBZN. M o l 6 Gen Genet 231, 113-123. Sambrook,J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laborato? Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Thorson, J. S., Lo, S. F., Liu, H.-W. & Hutchinson, C. R. (1993). Stevenson, G., Neal, B., Liu, D., Hobbs, M., Packer, N. H., Batley, M., Redmond, J. W., Lindquist, L. & Reeves, P. (1994). Structure of the 0 antigen of Escbericbia coli K-12 and the sequence of its r- gene oxysugar portion of the erythromycin biosynthesis pathway in Saccbaropo&ora elytbraea (Streptomyces etytbreus). J Bacteriol 171, 5872-5881. cluster. J Bacterioll76, 4144-4156. Stutzman-Engwall, K. J. & Hutchinson, C. R. (1989). Multigene families for anthracycline antibiotic production in Streptomyces peucetius. Proc Natl Acad Sci U S A 86, 3135-3139. Stutzman-Engwall, K. J., Otten, 5. L. & Hutchinson, C. R. (1992). Regulation of secondary metabolism in Streptomyces: its use for the overproduction of daunorubicin in Streptomycespeucetius.J Bacteriol 174, 144-154. Summers, R. G., Wendt-Pienkowski, E., Motamedi, H. & Hutchinson, C. R. (1992). Nucleotide sequence of the tcmll-tcmlV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glawescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase. J Bacterioll74, 1810-1820. and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes. J Gen Microbiol 138, 779-786. Biosynthesis of 3,6-dideoxy-hexoses : new mechanistic reflections upon 2,6-dideoxy, 4,6-dideoxy and amino sugar construction. J A m Cbem Soc 115, 6993-6994. Vara, J., Lewandowska-Skarbek, M., Wang, Y.-G., Donadio, 5.81 Hutchinson, C. R. (1989). Cloning of genes governing the de- Wright, F. & Bibb, M. J. (1992). Codon usage in the G+C-rich Streptomyces genome. Gene 113, 55-65. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119. Yao, 2.81Valvano, M. A. (1994). Genetic analysis of the O-specific lipopolysaccharide biosynthesis region ( r - )of Escbericbia coli K-12 W31100: identification of genes that confer group 6 specificity to Sbigella jexneri serotypes Y and 4a. J Bacterioll76, 41334143. Received 27 June 1995; revised 14 September 1995; accepted 28 September 1995. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:16:23 275