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Identification of DNA amplifications near the center of the Streptomyces coelicolor M145 chromosome
FEMS Microbiology Letters 191 (2000) 123^129
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Identi¢cation of DNA ampli¢cations near the center of the Streptomyces coelicolor M145 chromosome Matthias Redenbach *, Eveline Kleinert, Alexander Stoll Department of Genetics, Genome Research Unit, Kaiserslautern University, 67663 Kaiserslautern, Germany Received 3 May 2000; received in revised form 5 August 2000 ; accepted 12 August 2000
Abstract Linear streptomycete chromosomes frequently undergo spontaneous gross DNA rearrangements at the terminal regions. Large DNA deletions of the chromosome ends are in many cases associated with tandemly reiterated DNA amplifications, found at the border of the deletable areas. In contrast to previous reports, we have discovered amplifications near the center of the Streptomyces coelicolor M145 chromosome. The detected amplified units of DNA are 19.9 kb and 16 kb in length and exist in copy numbers of 30 and 40, respectively. Both amplifications were located in the same region and share at least 3.6 kb. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptomyces ; Genome structure ; DNA ampli¢cation
1. Introduction Analyses of the genomic architecture of various Streptomyces chromosomes showed that all species studied thus far carry an 8-Mb linear chromosome with terminal inverted repeats and protein attached to the 5P ends [1^4]. Replication of the linear chromosome proceeds bidirectionally from an origin of replication (oriC) located in the center of the chromosome [5,6]. The regions close to the ends of the chromosome are devoid of essential genetic markers but were shown to include a signi¢cant number of genes involved in the degradation of carbohydrates [7,8]. The end regions of the Streptomyces chromosome are highly dynamic under laboratory conditions, spontaneously undergoing gross DNA rearrangements with frequencies above 0.1% [9^11]. Large deletions, which a¡ect the chromosome ends, can reach a size of 2 Mb and are frequently associated with tandemly reiterated DNA am-
* Corresponding author. Tel. : +49 (631) 205-3250; Fax: +49 (631) 205-4090; E-mail : [email protected]
pli¢cations. In addition, spontaneous deletion mutants with a circularized chromosome have been reported which indicate that the Streptomyces chromosome can exist in both topological forms [1,2,12,13]. The mechanism of chromosomal instability in Streptomyces is not known. It was suggested that the loss of the terminal protein or transposition of insertion sequence (IS) elements/transposons, which were found at both termini of the S. lividans chromosome [11], induce instability. Collapse of the replication forks moving towards the ends, due to stallation by tightly bound proteins at certain areas [11], was speculated to induce genomic instability in streptomycetes as well. S. coelicolor A3(2) is the most studied Streptomyces species with respect to general genetics and physiology, but reports on the chromosome instability of this particular strain are rare [14]. The closely related species S. lividans 66, which shows a similar genome organization [15], was intensively characterized with respect to genomic instability and ampli¢ed units of DNA (AUDs) of two di¡erent types were assigned to the ends of the chromosome. These areas were shown to be present in S. coelicolor A3(2) also [7,14]. Here we describe the isolation and the ¢rst characterization of two di¡erent S. coelicolor M145 mutants with DNA ampli¢cations that could be located, in contrast to all previously analyzed ampli¢cations, in a central position of the chromosome.
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 8 1 - 5
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color reaction recommended by the kit manufacturer (Roche).
2. Materials and methods 2.1. Strains and culture conditions The parental S. coelicolor strain used for this study was S. coelicolor M145 [16]. Streptomyces strains were grown and handled as described [16]. YEME medium was used for growing cultures, for the preparation of DNA and protoplasts. After transformation with plasmid DNA, thiostreptonresistant Streptomyces clones were selected by plating on R2YE agar and overlaying with 500 Wg ml31 thiostrepton. Thiostrepton-resistant clones were transferred to minimal medium (MM) or R2YE medium containing 50 Wg ml31 thiostrepton. Cosmid clones Q11, D25, D72, D46, D86, D52, 6Fll, D65, D69, D4, D39, D40, D74, D31, 6G4, D63, D77, D1 and 5G8 were from the ordered S. coelicolor library [7]. Escherichia coli cells were grown in either Luria-Bertani (LB) medium or LB with 50 Wg ml31 ampicillin [17] for the isolation of cosmid DNA. 2.2. DNA preparations, standard gel electrophoresis Total DNA from S. coelicolor was isolated using procedure 1 [16]. Plasmid and cosmid DNA isolations were done according to the alkaline lysis method [18]. Restriction of DNA and agarose gel electrophoresis was carried out as described by Maniatis et al. [17]. 2.3. Pulsed-¢eld gel electrophoresis (PFGE) PFGE DNA was prepared as described by Redenbach et al. [7]. Restriction digests were performed in volumes of 200 Wl with 50 U of enzyme for 12 h at 37³C. The digested DNA was subjected to PFGE on 1% agarose gels using a CHEF apparatus from Bio-Rad with 0.5UTBE. Saccharomyces cerevisiae YNN255 chromosomes (Bio-Rad) and V-HindIII were used as molecular mass standards. 2.4. DNA labelling and hybridization DNA was labelled by random priming [19] using a non-radioactive digoxigenin labelling kit (Roche). DNA fragments from agarose gels were transferred to Porablot NY membrane (Macherey-Nagel) by the method of Smith and Summers [20]. Hybridizations with digoxigeninlabelled DNA probes were carried out as recommended by the supplier (Roche). After pre-hybridization of the ¢lters (1 h in 5USSC, 0.1% (w/v) n-lauroylsarcosine, 0.02% (w/v) SDS, 1% (w/v) blocking reagent at 68³C), probes (that had been heat-denatured for 10 min) were added and hybridization was allowed to proceed for 16 h at 68³C. Membranes were washed with 2USSC, 0.2% SDS for 2U5 min at room temperature, followed by high-stringency washes for 2U15 min with 0.2USSC, 0.2% SDS at 68³C. Signals were detected using the
2.5. Mapping of cosmid clones Isolated cosmid DNA was linearized with AseI. A 0.9kb and a 4.2-kb vector region, which do not hybridize to each other, remain left and right of the cosmid clone after the digest. The AseI-restricted cosmid DNA was then partially digested with the particular mapping enzyme (EcoRI, XhoI, BamHI) and separated on a 0.5% and a 0.8% TAE gel. High Molecular Weight Marker (Gibco BRL) and the 1-kb ladder (MBI Fermentas) were used as DNA size standards. The gels were blotted and hybridized with the DIG-labelled 0.9-kb and 4.2-kb vector fragments as probes. Sizes of hybridization bands were calculated and subtracted from each other to reveal the alignment of the fragments. 2.6. Sequencing Plasmid DNA was sequenced on both strands with the Thermo Sequenase £uorescent labelled primer cycle sequencing kit (Amersham/Life Science) including deazadGTP as recommended by the supplier. For sequencing a Li-Cor sequencer (Model 4000) was used. 3. Results 3.1. Isolation of mutants lacking the AseI D fragment The thiostrepton resistance gene (tsr) was inserted into the AseI E region of the S. coelicolor M145 chromosome by homologous recombination (Fig. 1). Therefore a 4.7-kb EcoRI fragment of the AseI E cosmid clone E46 of the ordered cosmid library [7] was cloned into the integration vector pDH5 [21] to reveal pMR10 which was introduced into S. coelicolor M145 protoplasts by transformation and selection for thiostrepton resistance. Ten thiostrepton-resistant clones were picked and further analyzed by PFGE. Cosmid E46 is located in the AseI E fragment approximately 60 kb from the border between AseI E and AseI H (Fig. 1). The AseI restriction pattern of all 10 selected tsr resistant mutants lacked the AseI E fragment and showed in addition two fragments of 60 kb and 760 kb (Fig. 2a). This proved that the recombinant plasmid, which carries two AseI sites, was successfully integrated into the expected position. Three of those mutants (MR11, 12, 13) showed in addition the loss of the AseI D fragment (Fig. 2). New bands could not be identi¢ed using all various PFGE conditions to identify very small or extremely large fragments. Another mutant was isolated by coincidence. A randomly selected, single colony from M145 was used for a spore suspension. PFGE analysis of DNA derived from
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this strain revealed that it also lacked the AseI D band (Fig. 2). The mutant was called AS1. 3.2. Analysis of the AseI macrorestriction pattern In order to investigate if parts of the AseI D fragment remained in the chromosome, the AseI D internal cosmid D31 and the AseI linking clones 5G8 (AseI O and AseI D) and Q11 (AseI H, Q and D) (Fig. 1) of the ordered cosmid library were hybridized against Southern blots of AseI-restricted DNA of MR11^13 and AS1. The internal cosmid D31 hybridized clearly to the AseI D fragment of M145 which was used as a control. In the mutants a broad smear between the AseI C fragment and the AseI A fragment was detected with this probe (Fig. 2b). The linking cosmid 5G8 hybridized to the AseI O fragment in all strains, as expected. In addition, the DNA smear above AseI C was lit up as seen with the internal cosmid 31 (data not shown). Cosmid Q11 also hybridized to the di¡use region above AseI C and as expected to the fragments AseI H and Q in all mutants (data not shown). This demonstrated that the borders of the AseI D fragment are intact and that the fragment increased its size in a non-uniform fashion. 3.3. Location of the rearrangements in the AseI D fragment DNAs of cosmids representing the AseI D fragment were labelled and hybridized to ¢lters with BamHI-digested DNA of AS1 and representatively MR11. The strains MR12 and 13 were not further used because they
Fig. 2. a: PFGE gel with AseI-digested DNA of S. coelicolor M145 and the identi¢ed ampli¢cation mutants, AS1 and MR11^MR13. The missing AseI D fragment in all selected mutants and one of the two new fragments of MR11^13 (AseI E*), which were generated by the integration pDH5 containing tsr, are indicated. Lane 1, S. cerevisiae chromosomes ; lane 2, S. coelicolor M145; lane 3, S. coelicolor AS1; lane 4, S. coelicolor MR11; lane 5, MR12; lane 6, MR13. Running conditions: 120 s, 24 h, 150 V and 240 s, 24 h, 150 V. b: Hybridization of the PFGE gel with the labelled cosmid clone D31.
seemed to be identical with MR11. The gels with BamHIdigested chromosomal DNA of AS1 and MR11^13 revealed intensive bands of 12.5 kb, 5.2 kb, 2.2 kb and 16 kb, respectively (Fig. 3a). These fragments were shown to hybridize strongly with cosmids D40 (16 kb, 12.5 kb) and D74, D31 (16 kb, 12.5 kb, 5.2 kb, 2.2 kb) (Fig. 3c^e). Hybridization of other labelled cosmids representing the AseI D fragment showed identical patterns in M145 and the mutants. These results suggested that an ampli¢cation was responsible for the increased size of the AseI D fragment in the selected strains. It also appeared that the structures of the rearrangement in AS1 and MR11 were not identical and that no large deletion occurred. 3.4. Localization of the ampli¢cations in cosmids D40, D74 and D31
Fig. 1. Location of the identi¢ed AUD region in the physical map of Streptomyces coelicolor M145. The outer circle indicates the alignment of the AseI fragments. The inner circle represents the orientation of the DraI macrorestriction fragments. Numbered black bars indicate the used AseI linking clones Q11 (H,Q,D), 5G8 (D,O) and the internal AseI E clone E46, which was used for the integration of tsr into the M145 chromosome. Arrows show the position of the replication origin (oriC), the location of tsr, the newly identi¢ed AUD4 locus as well as the already identi¢ed AUD regions (AUD1, AUD2).
Cosmids D40, D74 and D31 were mapped with EcoRI, XhoI and BamHI to localize the ampli¢ed unit of DNA and to determine if both ampli¢cations contain a region in common (Fig. 4). Therefore, individual restriction fragments of the cosmid DNA were gel eluted, labelled and hybridized to BamHI-digested chromosomal DNA of the di¡erent mutants (data not shown). The ampli¢cation of AS1 could be shown to carry one end point in the 6.5-kb BamHI^XhoI fragment of cosmid clones D40/74. The right end point was located in the 2.3-kb XhoI band of D74/ D31. The ends of the ampli¢cations in MR11 were assigned to the 5.9-kb BamHI band in D40/D74 and the 5.2-kb EcoRI^BfrI fragment of D74/D31 (Fig. 4). This suggests that both ampli¢cations overlap by at least 3.6 kb but do not have an end in common. The total size of the ampli¢able region is at least 24 kb.
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Fig. 3. Gel with BamHI-digested chromosomal DNA of M145, AS1, MR11 and hybridization of the Southern blot with cosmids of the AseI D fragment. a: Gel with BamHI-restricted chromosomal DNA. Lane 1, V-HindIII; lane 2; M145; lane 3, AS1; lane 4, MR11. b^f: Hybridization of labelled cosmid DNA to ¢lters with BamHI-restricted DNA. Lane 2, M145; lane 3, AS1; lane 4, MR11. Hybridization with (b) clone D82; (c) D40; (d) D74; (e) D31; (f) 6G4. Sizes of the ampli¢ed bands are indicated. The asterisk shows the additional BamHI fragment in MR11 which is due to the homology of the integrated pDH5 DNA to Supercos-1 which is the vector of the used cosmid clones.
BfrI is a rare cutting enzyme for GC-rich DNA and generates 30^40 fragments of the S. coelicolor chromosome. In addition to the AseI macrorestriction pattern we analyzed BfrI-digested PFGE DNA of M145 and the mutants. Fig. 5 shows the PFGE gel of BfrI-digested
DNA of M145, AS1 and MR11. In AS1 a new and intense 20-kb fragment could be seen. MR11 revealed two intensive bands above 1 Mb. One band had a size of approximately 1200 kb. The larger fragment corresponds to a high molecular smear, similar to the one seen in the
Fig. 4. Restriction map of the AUDs and adjacent regions. The extent of each cosmid (D40, D74, D31) is indicated below the map. Cosmid D40 is not completely shown and extends to the left (broken line). The AUDs are represented by bold lines. Restriction enzymes used: E, EcoRI; X, XhoI; B, BamHI ; A, BfrI.
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AseI digest, which is compressed due to the applied PFGE conditions. Hybridization with labelled DNA of cosmid D74 to a Southern blot of the BfrI gel showed two signals for M145 of 550 kb and 350 kb. AS1 revealed a ladder of fragments starting from the visible 20-kb fragment. In addition, the 550-kb and the 350-kb bands were detected. MR11 showed intensive signals for the 1200-kb band and the compression zone. A faint hybridization signal was detected for the 350-kb band. AS1 possesses a BfrI site in the AUD and therefore carries the intensive 20-kb BfrI fragment. The presence of both original BfrI bands con¢rmed the results obtained by the hybridization of the AseI D cosmids to Southern blots with BamHI-restricted AS1 DNA, that no large deletion occurred in that strain. The ampli¢cation in MR11^13 does not carry the BfrI site and is located within the 550-kb BfrI fragment, which increases to at least 1200 kb. The smear above 1200 kb might be explained by the presence of a mixed population of chromosomes with di¡erent amounts of ampli¢ed DNA as well as the presence of a BfrI fragment containing the integrated pMR10. 3.5. IS1652 is located close to one end of the AUD in S. coelicolor AS1 One end of the AUD in S. coelicolor AS1 could be assigned to the 2.3-kb XhoI fragment of cosmid D74/ D31 as described above. When the labelled DNA of this fragment was hybridized to BamHI-restricted chromosomal DNA of M145 and the di¡erent mutants, multiple bands could be detected (Fig. 6), which indicates that this area carries homology to other regions of the S. coelicolor chromosome. This was con¢rmed by hybridization
Fig. 5. PFGE gel of BfrI-restricted DNA and hybridization of the Southern blot with cosmid D74. a: PFGE gel. Lane 1, S. cerevisiae chromosomes; lane 2, M145; lane 3, AS1; lane 4, MR11; lane 5, V-HindIII. Running conditions : ramping 60^90 s, 36 h, 180 V. b: Hybridization with labelled DNA of clone D74. Lane 1, M145; lane 2, AS1; lane 3, MR11.
Fig. 6. Hybridization of the 2.3-kb XhoI fragment to BamHI-restricted DNA of S. coelicolor M145, AS1 and MR11-13. Lane 1, M145; lane 2, AS1; lane 3, MR11; lane 4, MR12; lane 5, MR13. Asterisks show the additional 17-kb band in MR11^13, the missing 8.5-kb band in all mutants and the missing 4.2-kb BamHI fragment in AS1.
of the ordered cosmid library from S. coelicolor M145 with the labelled 2.3-kb XhoI fragment. Cosmid clones 4G4, H52, D66, D52, D10, D74, D31, 6A9 and 10B8 lit up with the probe. This suggests that the 2.3-kb XhoI fragment has homology to at least seven di¡erent positions (D74/D31 and D52/D10 are overlapping clones) in the S. coelicolor genome. The multiple signals are due to the presence of a complete copy of IS1652 containing a single BamHI site which was found by sequence analysis of the 2.3-kb XhoI fragment (data not shown). In addition, hybridization of the labeled 2.3-kb XhoI fragment revealed signi¢cant di¡erences between the M145 and the mutant hybridization patterns. A 8.5-kb BamHI fragment disappeared in all mutant strains. AS1 revealed the loss of an additional 4.2-kb BamHI fragment. MR11^MR13 showed a new band of 17 kb. 4. Discussion We have discovered two types of S. coelicolor M145 mutants which carry an ampli¢cation in a central position of the chromosome. This is to our knowledge the ¢rst description of an ampli¢cation in streptomycetes outside
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the unstable end regions of the chromosome. The AUDs are 19.9 kb (AS1) and 16 kb (MR11^13) large. The identi¢ed strains seem to contain a mixed population of chromosomes with di¡erent extents of ampli¢cation as indicated by the detected smear in the PFGE analysis. The increase in DNA is approximately 600 kb in all mutants considering that the size of the AseI D fragment (900 kb) shifted to 1500 kb in all strains. Therefore a copy number of 30 (AS1) and 40 (MR11^13) results. Both ampli¢cations, which span a region of more than 24 kb, are located in the same chromosomal area and overlap by at least 3.6 kb. We could not detect any signi¢cant deletions in contrast to the ampli¢cations which were described at the chromosome ends. Streptomycete mutants with rearrangements at the chromosome ends were basically selected based on a visible phenotype such as de¢ciencies of antibiotic biosynthesis/ resistance or morphological variations. The four mutant strains described here did not show any particular phenotype although the a¡ected region belongs to the genetically more densely mapped areas of the genome [7]. For example, the genes involved in translation such as fus, tuf1 and rpsG are located in the overlap between cosmid clones D40 and D74 which would mean that these genes are ampli¢ed most likely. The strA locus which confers resistance to streptomycin and the hyp gene for hypersporulation [22] were assigned to the overlap between cosmids D74 and D31. It cannot be excluded that these genes are located in the ampli¢cation. However, we did not see any characteristic phenotype and the strains grew, sporulated and were as streptomycin-sensitive as the parent strain S. coelicolor M145. Three of four mutants (MR11^13) were discovered by PFGE analyses of strains which carry tsr at a particular region which is approximately 1200 kb away from the ampli¢ed region. The genomic structures in all three mutants seem to be identical although we selected three independent tsr-resistant clones. Protoplastation is known to induce instability of the chromosome ends and leads to ampli¢cations [23,24]. In this respect it could be possible that the protoplastation/transformation induced the ampli¢cation in these strains speci¢cally. On the other hand the ampli¢cation mutant AS1 was isolated from a single colony of a S. coelicolor M145 spore suspension and was not exposed to any selective pressure. We can therefore not exclude at present that the ampli¢cation seen in MR11, 12 and 13 was already present in the M145 spore suspension which we used for the transformation of pMR10. The localization of one copy of IS1652 in the vicinity of the end of the AUD in AS1 may imply that this element is involved in the formation of the ampli¢cation, although we have no direct evidence so far. Accurate analysis of the AUD ends may elucidate the mechanism of the ampli¢cation in that region of the chromosome.
Acknowledgements We are grateful to John Cullum for his support. We thank Alexander Spychaj and Matthias Kotschwar for providing sequence data. References [1] Lin, Y.-S., Kieser, H.M., Hopwood, D.A. and Chen, C.W. (1993) The chromosomal DNA of Streptomyces lividans 66 is linear. Mol. Microbiol. 10, 923^933. [2] Lezhava, A., Mizukami, T., Kajitani, T., Kameoka, D., Redenbach, M., Shinkawa, H., Nimi, O. and Kinashi, H. (1995) Physical map of the linear chromosome of Streptomyces griseus. J. Bacteriol. 177, 6492^6498. [3] Leblond, P., Fischer, G., Francou, F.-X., Berger, M., Guerineau, M. and Decaris, B. (1996) The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats £anking the extremities of the linear chromosomal DNA. Mol. Microbiol. 19, 261^271. [4] Pandza, K., Pfalzer, G., Cullum, J. and Hranueli, D. (1997) Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome. Microbiology 143, 1493^1501. [5] Zakrzewska-Czerwinska, J. and Schrempf, H. (1992) Characterization of an autonomously replicating region from the Streptomyces lividans chromosome. J. Bacteriol. 174, 2688^2693. [6] Musialowski, M.S., Flett, F., Scott, G.B., Hobbs, G., Smith, C.P. and Oliver, S.G. (1994) Functional evidence that the principle DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region. J. Bacteriol. 176, 5123^5125. [7] Redenbach, M., Kieser, H.M., Denapaite, D., Eichner, A., Kinashi, H., Cullum, J. and Hopwood, D.A. (1996) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3 chromosome. Mol. Microbiol. 21, 77^96. [8] Saito, A., Fujii, T., Yoneyama, T., Redenbach, M., Ohno, T., Watanabe, T. and Miyashita, K. (1999) High-multiplicity of chitinase genes in Streptomyces coelicolor A3. Biosci. Biotechnol. Biochem. 63, 710^718. [9] Leblond, P. and Decaris, B. (1994) New insights into genetic instability of Streptomyces. FEMS Microbiol. Lett. 123, 225^232. [10] Dharmalingam, K. and Cullum, J. (1996) Genetic instability in Streptomyces. J. Biosci. 21, 433^444. [11] Vol¡, J.N. and Altenbuchner, J. (1998) Genetic instability of the Streptomyces chromosome. Mol. Microbiol. 27, 239^246. [12] Redenbach, M., Flett, F., Piendl, W., Glocker, I., Rauland, U., Wafzig, O., Kliem, R., Leblond, P. and Cullum, J. (1993) The Streptomyces lividans 66 chromosome contains a 1 Mb deletogenic region £anked by two ampli¢able regions. Mol. Gen. Genet. 241, 255^262. [13] Kameoka, D., Lezhava, A., Zenitani, H., Hiratsu, K., Kawamoto, M., Goshi, K., Inada, K., Shinkawa, H. and Kinashi, H. (1999) Analysis of fusion junctions of circularized chromosomes in Streptomyces griseus. J. Bacteriol. 181, 5711^5717. [14] Flett, F. and Cullum, J. (1987) DNA deletions in spontaneous chloramphenicol-sensitive mutants of Streptomyces coelicolor A3 and Streptomyces lividans 66. Mol. Gen. Genet. 207, 499^502. [15] Leblond, P., Redenbach, M. and Cullum, J. (1993) Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3. J. Bacteriol. 175, 3422^ 3429. [16] 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. and Schrempf, H. (1985) Genetic Manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich.
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[21] Hillemann, D., Pu«hler, A. and Wohlleben, W. (1991) Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation in integration vectors. Nucleic Acids Res. 19, 727^ 731. [22] Ryding, J. (1996) PhD Thesis, University of East Anglia, Norwich. [23] Robinson, M., Lewis, I. and Napier, E. (1981) Occurrence of reiterated DNA sequences in strains of Streptomyces produced by interspeci¢c protoplast fusion. Mol. Gen. Genet. 182, 336^340. [24] Ishikawa, J., Koyama, Y., Mizuno, S. and Hotta, K. (1988) Mechanism of increased kanamycin resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational gene alteration and gene ampli¢cation. J. Antibiot. 41, 104^112.
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Identi¢cation of DNA ampli¢cations near the center of the Streptomyces coelicolor M145 chromosome Matthias Redenbach *, Eveline Kleinert, Alexander Stoll Department of Genetics, Genome Research Unit, Kaiserslautern University, 67663 Kaiserslautern, Germany Received 3 May 2000; received in revised form 5 August 2000 ; accepted 12 August 2000
Abstract Linear streptomycete chromosomes frequently undergo spontaneous gross DNA rearrangements at the terminal regions. Large DNA deletions of the chromosome ends are in many cases associated with tandemly reiterated DNA amplifications, found at the border of the deletable areas. In contrast to previous reports, we have discovered amplifications near the center of the Streptomyces coelicolor M145 chromosome. The detected amplified units of DNA are 19.9 kb and 16 kb in length and exist in copy numbers of 30 and 40, respectively. Both amplifications were located in the same region and share at least 3.6 kb. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptomyces ; Genome structure ; DNA ampli¢cation
1. Introduction Analyses of the genomic architecture of various Streptomyces chromosomes showed that all species studied thus far carry an 8-Mb linear chromosome with terminal inverted repeats and protein attached to the 5P ends [1^4]. Replication of the linear chromosome proceeds bidirectionally from an origin of replication (oriC) located in the center of the chromosome [5,6]. The regions close to the ends of the chromosome are devoid of essential genetic markers but were shown to include a signi¢cant number of genes involved in the degradation of carbohydrates [7,8]. The end regions of the Streptomyces chromosome are highly dynamic under laboratory conditions, spontaneously undergoing gross DNA rearrangements with frequencies above 0.1% [9^11]. Large deletions, which a¡ect the chromosome ends, can reach a size of 2 Mb and are frequently associated with tandemly reiterated DNA am-
* Corresponding author. Tel. : +49 (631) 205-3250; Fax: +49 (631) 205-4090; E-mail : [email protected]
pli¢cations. In addition, spontaneous deletion mutants with a circularized chromosome have been reported which indicate that the Streptomyces chromosome can exist in both topological forms [1,2,12,13]. The mechanism of chromosomal instability in Streptomyces is not known. It was suggested that the loss of the terminal protein or transposition of insertion sequence (IS) elements/transposons, which were found at both termini of the S. lividans chromosome [11], induce instability. Collapse of the replication forks moving towards the ends, due to stallation by tightly bound proteins at certain areas [11], was speculated to induce genomic instability in streptomycetes as well. S. coelicolor A3(2) is the most studied Streptomyces species with respect to general genetics and physiology, but reports on the chromosome instability of this particular strain are rare [14]. The closely related species S. lividans 66, which shows a similar genome organization [15], was intensively characterized with respect to genomic instability and ampli¢ed units of DNA (AUDs) of two di¡erent types were assigned to the ends of the chromosome. These areas were shown to be present in S. coelicolor A3(2) also [7,14]. Here we describe the isolation and the ¢rst characterization of two di¡erent S. coelicolor M145 mutants with DNA ampli¢cations that could be located, in contrast to all previously analyzed ampli¢cations, in a central position of the chromosome.
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 8 1 - 5
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color reaction recommended by the kit manufacturer (Roche).
2. Materials and methods 2.1. Strains and culture conditions The parental S. coelicolor strain used for this study was S. coelicolor M145 [16]. Streptomyces strains were grown and handled as described [16]. YEME medium was used for growing cultures, for the preparation of DNA and protoplasts. After transformation with plasmid DNA, thiostreptonresistant Streptomyces clones were selected by plating on R2YE agar and overlaying with 500 Wg ml31 thiostrepton. Thiostrepton-resistant clones were transferred to minimal medium (MM) or R2YE medium containing 50 Wg ml31 thiostrepton. Cosmid clones Q11, D25, D72, D46, D86, D52, 6Fll, D65, D69, D4, D39, D40, D74, D31, 6G4, D63, D77, D1 and 5G8 were from the ordered S. coelicolor library [7]. Escherichia coli cells were grown in either Luria-Bertani (LB) medium or LB with 50 Wg ml31 ampicillin [17] for the isolation of cosmid DNA. 2.2. DNA preparations, standard gel electrophoresis Total DNA from S. coelicolor was isolated using procedure 1 [16]. Plasmid and cosmid DNA isolations were done according to the alkaline lysis method [18]. Restriction of DNA and agarose gel electrophoresis was carried out as described by Maniatis et al. [17]. 2.3. Pulsed-¢eld gel electrophoresis (PFGE) PFGE DNA was prepared as described by Redenbach et al. [7]. Restriction digests were performed in volumes of 200 Wl with 50 U of enzyme for 12 h at 37³C. The digested DNA was subjected to PFGE on 1% agarose gels using a CHEF apparatus from Bio-Rad with 0.5UTBE. Saccharomyces cerevisiae YNN255 chromosomes (Bio-Rad) and V-HindIII were used as molecular mass standards. 2.4. DNA labelling and hybridization DNA was labelled by random priming [19] using a non-radioactive digoxigenin labelling kit (Roche). DNA fragments from agarose gels were transferred to Porablot NY membrane (Macherey-Nagel) by the method of Smith and Summers [20]. Hybridizations with digoxigeninlabelled DNA probes were carried out as recommended by the supplier (Roche). After pre-hybridization of the ¢lters (1 h in 5USSC, 0.1% (w/v) n-lauroylsarcosine, 0.02% (w/v) SDS, 1% (w/v) blocking reagent at 68³C), probes (that had been heat-denatured for 10 min) were added and hybridization was allowed to proceed for 16 h at 68³C. Membranes were washed with 2USSC, 0.2% SDS for 2U5 min at room temperature, followed by high-stringency washes for 2U15 min with 0.2USSC, 0.2% SDS at 68³C. Signals were detected using the
2.5. Mapping of cosmid clones Isolated cosmid DNA was linearized with AseI. A 0.9kb and a 4.2-kb vector region, which do not hybridize to each other, remain left and right of the cosmid clone after the digest. The AseI-restricted cosmid DNA was then partially digested with the particular mapping enzyme (EcoRI, XhoI, BamHI) and separated on a 0.5% and a 0.8% TAE gel. High Molecular Weight Marker (Gibco BRL) and the 1-kb ladder (MBI Fermentas) were used as DNA size standards. The gels were blotted and hybridized with the DIG-labelled 0.9-kb and 4.2-kb vector fragments as probes. Sizes of hybridization bands were calculated and subtracted from each other to reveal the alignment of the fragments. 2.6. Sequencing Plasmid DNA was sequenced on both strands with the Thermo Sequenase £uorescent labelled primer cycle sequencing kit (Amersham/Life Science) including deazadGTP as recommended by the supplier. For sequencing a Li-Cor sequencer (Model 4000) was used. 3. Results 3.1. Isolation of mutants lacking the AseI D fragment The thiostrepton resistance gene (tsr) was inserted into the AseI E region of the S. coelicolor M145 chromosome by homologous recombination (Fig. 1). Therefore a 4.7-kb EcoRI fragment of the AseI E cosmid clone E46 of the ordered cosmid library [7] was cloned into the integration vector pDH5 [21] to reveal pMR10 which was introduced into S. coelicolor M145 protoplasts by transformation and selection for thiostrepton resistance. Ten thiostrepton-resistant clones were picked and further analyzed by PFGE. Cosmid E46 is located in the AseI E fragment approximately 60 kb from the border between AseI E and AseI H (Fig. 1). The AseI restriction pattern of all 10 selected tsr resistant mutants lacked the AseI E fragment and showed in addition two fragments of 60 kb and 760 kb (Fig. 2a). This proved that the recombinant plasmid, which carries two AseI sites, was successfully integrated into the expected position. Three of those mutants (MR11, 12, 13) showed in addition the loss of the AseI D fragment (Fig. 2). New bands could not be identi¢ed using all various PFGE conditions to identify very small or extremely large fragments. Another mutant was isolated by coincidence. A randomly selected, single colony from M145 was used for a spore suspension. PFGE analysis of DNA derived from
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this strain revealed that it also lacked the AseI D band (Fig. 2). The mutant was called AS1. 3.2. Analysis of the AseI macrorestriction pattern In order to investigate if parts of the AseI D fragment remained in the chromosome, the AseI D internal cosmid D31 and the AseI linking clones 5G8 (AseI O and AseI D) and Q11 (AseI H, Q and D) (Fig. 1) of the ordered cosmid library were hybridized against Southern blots of AseI-restricted DNA of MR11^13 and AS1. The internal cosmid D31 hybridized clearly to the AseI D fragment of M145 which was used as a control. In the mutants a broad smear between the AseI C fragment and the AseI A fragment was detected with this probe (Fig. 2b). The linking cosmid 5G8 hybridized to the AseI O fragment in all strains, as expected. In addition, the DNA smear above AseI C was lit up as seen with the internal cosmid 31 (data not shown). Cosmid Q11 also hybridized to the di¡use region above AseI C and as expected to the fragments AseI H and Q in all mutants (data not shown). This demonstrated that the borders of the AseI D fragment are intact and that the fragment increased its size in a non-uniform fashion. 3.3. Location of the rearrangements in the AseI D fragment DNAs of cosmids representing the AseI D fragment were labelled and hybridized to ¢lters with BamHI-digested DNA of AS1 and representatively MR11. The strains MR12 and 13 were not further used because they
Fig. 2. a: PFGE gel with AseI-digested DNA of S. coelicolor M145 and the identi¢ed ampli¢cation mutants, AS1 and MR11^MR13. The missing AseI D fragment in all selected mutants and one of the two new fragments of MR11^13 (AseI E*), which were generated by the integration pDH5 containing tsr, are indicated. Lane 1, S. cerevisiae chromosomes ; lane 2, S. coelicolor M145; lane 3, S. coelicolor AS1; lane 4, S. coelicolor MR11; lane 5, MR12; lane 6, MR13. Running conditions: 120 s, 24 h, 150 V and 240 s, 24 h, 150 V. b: Hybridization of the PFGE gel with the labelled cosmid clone D31.
seemed to be identical with MR11. The gels with BamHIdigested chromosomal DNA of AS1 and MR11^13 revealed intensive bands of 12.5 kb, 5.2 kb, 2.2 kb and 16 kb, respectively (Fig. 3a). These fragments were shown to hybridize strongly with cosmids D40 (16 kb, 12.5 kb) and D74, D31 (16 kb, 12.5 kb, 5.2 kb, 2.2 kb) (Fig. 3c^e). Hybridization of other labelled cosmids representing the AseI D fragment showed identical patterns in M145 and the mutants. These results suggested that an ampli¢cation was responsible for the increased size of the AseI D fragment in the selected strains. It also appeared that the structures of the rearrangement in AS1 and MR11 were not identical and that no large deletion occurred. 3.4. Localization of the ampli¢cations in cosmids D40, D74 and D31
Fig. 1. Location of the identi¢ed AUD region in the physical map of Streptomyces coelicolor M145. The outer circle indicates the alignment of the AseI fragments. The inner circle represents the orientation of the DraI macrorestriction fragments. Numbered black bars indicate the used AseI linking clones Q11 (H,Q,D), 5G8 (D,O) and the internal AseI E clone E46, which was used for the integration of tsr into the M145 chromosome. Arrows show the position of the replication origin (oriC), the location of tsr, the newly identi¢ed AUD4 locus as well as the already identi¢ed AUD regions (AUD1, AUD2).
Cosmids D40, D74 and D31 were mapped with EcoRI, XhoI and BamHI to localize the ampli¢ed unit of DNA and to determine if both ampli¢cations contain a region in common (Fig. 4). Therefore, individual restriction fragments of the cosmid DNA were gel eluted, labelled and hybridized to BamHI-digested chromosomal DNA of the di¡erent mutants (data not shown). The ampli¢cation of AS1 could be shown to carry one end point in the 6.5-kb BamHI^XhoI fragment of cosmid clones D40/74. The right end point was located in the 2.3-kb XhoI band of D74/ D31. The ends of the ampli¢cations in MR11 were assigned to the 5.9-kb BamHI band in D40/D74 and the 5.2-kb EcoRI^BfrI fragment of D74/D31 (Fig. 4). This suggests that both ampli¢cations overlap by at least 3.6 kb but do not have an end in common. The total size of the ampli¢able region is at least 24 kb.
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Fig. 3. Gel with BamHI-digested chromosomal DNA of M145, AS1, MR11 and hybridization of the Southern blot with cosmids of the AseI D fragment. a: Gel with BamHI-restricted chromosomal DNA. Lane 1, V-HindIII; lane 2; M145; lane 3, AS1; lane 4, MR11. b^f: Hybridization of labelled cosmid DNA to ¢lters with BamHI-restricted DNA. Lane 2, M145; lane 3, AS1; lane 4, MR11. Hybridization with (b) clone D82; (c) D40; (d) D74; (e) D31; (f) 6G4. Sizes of the ampli¢ed bands are indicated. The asterisk shows the additional BamHI fragment in MR11 which is due to the homology of the integrated pDH5 DNA to Supercos-1 which is the vector of the used cosmid clones.
BfrI is a rare cutting enzyme for GC-rich DNA and generates 30^40 fragments of the S. coelicolor chromosome. In addition to the AseI macrorestriction pattern we analyzed BfrI-digested PFGE DNA of M145 and the mutants. Fig. 5 shows the PFGE gel of BfrI-digested
DNA of M145, AS1 and MR11. In AS1 a new and intense 20-kb fragment could be seen. MR11 revealed two intensive bands above 1 Mb. One band had a size of approximately 1200 kb. The larger fragment corresponds to a high molecular smear, similar to the one seen in the
Fig. 4. Restriction map of the AUDs and adjacent regions. The extent of each cosmid (D40, D74, D31) is indicated below the map. Cosmid D40 is not completely shown and extends to the left (broken line). The AUDs are represented by bold lines. Restriction enzymes used: E, EcoRI; X, XhoI; B, BamHI ; A, BfrI.
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AseI digest, which is compressed due to the applied PFGE conditions. Hybridization with labelled DNA of cosmid D74 to a Southern blot of the BfrI gel showed two signals for M145 of 550 kb and 350 kb. AS1 revealed a ladder of fragments starting from the visible 20-kb fragment. In addition, the 550-kb and the 350-kb bands were detected. MR11 showed intensive signals for the 1200-kb band and the compression zone. A faint hybridization signal was detected for the 350-kb band. AS1 possesses a BfrI site in the AUD and therefore carries the intensive 20-kb BfrI fragment. The presence of both original BfrI bands con¢rmed the results obtained by the hybridization of the AseI D cosmids to Southern blots with BamHI-restricted AS1 DNA, that no large deletion occurred in that strain. The ampli¢cation in MR11^13 does not carry the BfrI site and is located within the 550-kb BfrI fragment, which increases to at least 1200 kb. The smear above 1200 kb might be explained by the presence of a mixed population of chromosomes with di¡erent amounts of ampli¢ed DNA as well as the presence of a BfrI fragment containing the integrated pMR10. 3.5. IS1652 is located close to one end of the AUD in S. coelicolor AS1 One end of the AUD in S. coelicolor AS1 could be assigned to the 2.3-kb XhoI fragment of cosmid D74/ D31 as described above. When the labelled DNA of this fragment was hybridized to BamHI-restricted chromosomal DNA of M145 and the di¡erent mutants, multiple bands could be detected (Fig. 6), which indicates that this area carries homology to other regions of the S. coelicolor chromosome. This was con¢rmed by hybridization
Fig. 5. PFGE gel of BfrI-restricted DNA and hybridization of the Southern blot with cosmid D74. a: PFGE gel. Lane 1, S. cerevisiae chromosomes; lane 2, M145; lane 3, AS1; lane 4, MR11; lane 5, V-HindIII. Running conditions : ramping 60^90 s, 36 h, 180 V. b: Hybridization with labelled DNA of clone D74. Lane 1, M145; lane 2, AS1; lane 3, MR11.
Fig. 6. Hybridization of the 2.3-kb XhoI fragment to BamHI-restricted DNA of S. coelicolor M145, AS1 and MR11-13. Lane 1, M145; lane 2, AS1; lane 3, MR11; lane 4, MR12; lane 5, MR13. Asterisks show the additional 17-kb band in MR11^13, the missing 8.5-kb band in all mutants and the missing 4.2-kb BamHI fragment in AS1.
of the ordered cosmid library from S. coelicolor M145 with the labelled 2.3-kb XhoI fragment. Cosmid clones 4G4, H52, D66, D52, D10, D74, D31, 6A9 and 10B8 lit up with the probe. This suggests that the 2.3-kb XhoI fragment has homology to at least seven di¡erent positions (D74/D31 and D52/D10 are overlapping clones) in the S. coelicolor genome. The multiple signals are due to the presence of a complete copy of IS1652 containing a single BamHI site which was found by sequence analysis of the 2.3-kb XhoI fragment (data not shown). In addition, hybridization of the labeled 2.3-kb XhoI fragment revealed signi¢cant di¡erences between the M145 and the mutant hybridization patterns. A 8.5-kb BamHI fragment disappeared in all mutant strains. AS1 revealed the loss of an additional 4.2-kb BamHI fragment. MR11^MR13 showed a new band of 17 kb. 4. Discussion We have discovered two types of S. coelicolor M145 mutants which carry an ampli¢cation in a central position of the chromosome. This is to our knowledge the ¢rst description of an ampli¢cation in streptomycetes outside
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the unstable end regions of the chromosome. The AUDs are 19.9 kb (AS1) and 16 kb (MR11^13) large. The identi¢ed strains seem to contain a mixed population of chromosomes with di¡erent extents of ampli¢cation as indicated by the detected smear in the PFGE analysis. The increase in DNA is approximately 600 kb in all mutants considering that the size of the AseI D fragment (900 kb) shifted to 1500 kb in all strains. Therefore a copy number of 30 (AS1) and 40 (MR11^13) results. Both ampli¢cations, which span a region of more than 24 kb, are located in the same chromosomal area and overlap by at least 3.6 kb. We could not detect any signi¢cant deletions in contrast to the ampli¢cations which were described at the chromosome ends. Streptomycete mutants with rearrangements at the chromosome ends were basically selected based on a visible phenotype such as de¢ciencies of antibiotic biosynthesis/ resistance or morphological variations. The four mutant strains described here did not show any particular phenotype although the a¡ected region belongs to the genetically more densely mapped areas of the genome [7]. For example, the genes involved in translation such as fus, tuf1 and rpsG are located in the overlap between cosmid clones D40 and D74 which would mean that these genes are ampli¢ed most likely. The strA locus which confers resistance to streptomycin and the hyp gene for hypersporulation [22] were assigned to the overlap between cosmids D74 and D31. It cannot be excluded that these genes are located in the ampli¢cation. However, we did not see any characteristic phenotype and the strains grew, sporulated and were as streptomycin-sensitive as the parent strain S. coelicolor M145. Three of four mutants (MR11^13) were discovered by PFGE analyses of strains which carry tsr at a particular region which is approximately 1200 kb away from the ampli¢ed region. The genomic structures in all three mutants seem to be identical although we selected three independent tsr-resistant clones. Protoplastation is known to induce instability of the chromosome ends and leads to ampli¢cations [23,24]. In this respect it could be possible that the protoplastation/transformation induced the ampli¢cation in these strains speci¢cally. On the other hand the ampli¢cation mutant AS1 was isolated from a single colony of a S. coelicolor M145 spore suspension and was not exposed to any selective pressure. We can therefore not exclude at present that the ampli¢cation seen in MR11, 12 and 13 was already present in the M145 spore suspension which we used for the transformation of pMR10. The localization of one copy of IS1652 in the vicinity of the end of the AUD in AS1 may imply that this element is involved in the formation of the ampli¢cation, although we have no direct evidence so far. Accurate analysis of the AUD ends may elucidate the mechanism of the ampli¢cation in that region of the chromosome.
Acknowledgements We are grateful to John Cullum for his support. We thank Alexander Spychaj and Matthias Kotschwar for providing sequence data. References [1] Lin, Y.-S., Kieser, H.M., Hopwood, D.A. and Chen, C.W. (1993) The chromosomal DNA of Streptomyces lividans 66 is linear. Mol. Microbiol. 10, 923^933. [2] Lezhava, A., Mizukami, T., Kajitani, T., Kameoka, D., Redenbach, M., Shinkawa, H., Nimi, O. and Kinashi, H. (1995) Physical map of the linear chromosome of Streptomyces griseus. J. Bacteriol. 177, 6492^6498. [3] Leblond, P., Fischer, G., Francou, F.-X., Berger, M., Guerineau, M. and Decaris, B. (1996) The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats £anking the extremities of the linear chromosomal DNA. Mol. Microbiol. 19, 261^271. [4] Pandza, K., Pfalzer, G., Cullum, J. and Hranueli, D. (1997) Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome. Microbiology 143, 1493^1501. [5] Zakrzewska-Czerwinska, J. and Schrempf, H. (1992) Characterization of an autonomously replicating region from the Streptomyces lividans chromosome. J. Bacteriol. 174, 2688^2693. [6] Musialowski, M.S., Flett, F., Scott, G.B., Hobbs, G., Smith, C.P. and Oliver, S.G. (1994) Functional evidence that the principle DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region. J. Bacteriol. 176, 5123^5125. [7] Redenbach, M., Kieser, H.M., Denapaite, D., Eichner, A., Kinashi, H., Cullum, J. and Hopwood, D.A. (1996) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3 chromosome. Mol. Microbiol. 21, 77^96. [8] Saito, A., Fujii, T., Yoneyama, T., Redenbach, M., Ohno, T., Watanabe, T. and Miyashita, K. (1999) High-multiplicity of chitinase genes in Streptomyces coelicolor A3. Biosci. Biotechnol. Biochem. 63, 710^718. [9] Leblond, P. and Decaris, B. (1994) New insights into genetic instability of Streptomyces. FEMS Microbiol. Lett. 123, 225^232. [10] Dharmalingam, K. and Cullum, J. (1996) Genetic instability in Streptomyces. J. Biosci. 21, 433^444. [11] Vol¡, J.N. and Altenbuchner, J. (1998) Genetic instability of the Streptomyces chromosome. Mol. Microbiol. 27, 239^246. [12] Redenbach, M., Flett, F., Piendl, W., Glocker, I., Rauland, U., Wafzig, O., Kliem, R., Leblond, P. and Cullum, J. (1993) The Streptomyces lividans 66 chromosome contains a 1 Mb deletogenic region £anked by two ampli¢able regions. Mol. Gen. Genet. 241, 255^262. [13] Kameoka, D., Lezhava, A., Zenitani, H., Hiratsu, K., Kawamoto, M., Goshi, K., Inada, K., Shinkawa, H. and Kinashi, H. (1999) Analysis of fusion junctions of circularized chromosomes in Streptomyces griseus. J. Bacteriol. 181, 5711^5717. [14] Flett, F. and Cullum, J. (1987) DNA deletions in spontaneous chloramphenicol-sensitive mutants of Streptomyces coelicolor A3 and Streptomyces lividans 66. Mol. Gen. Genet. 207, 499^502. [15] Leblond, P., Redenbach, M. and Cullum, J. (1993) Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3. J. Bacteriol. 175, 3422^ 3429. [16] 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. and Schrempf, H. (1985) Genetic Manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich.
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[21] Hillemann, D., Pu«hler, A. and Wohlleben, W. (1991) Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation in integration vectors. Nucleic Acids Res. 19, 727^ 731. [22] Ryding, J. (1996) PhD Thesis, University of East Anglia, Norwich. [23] Robinson, M., Lewis, I. and Napier, E. (1981) Occurrence of reiterated DNA sequences in strains of Streptomyces produced by interspeci¢c protoplast fusion. Mol. Gen. Genet. 182, 336^340. [24] Ishikawa, J., Koyama, Y., Mizuno, S. and Hotta, K. (1988) Mechanism of increased kanamycin resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational gene alteration and gene ampli¢cation. J. Antibiot. 41, 104^112.
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