Characterization of the Streptomyces violaceoruber SANK95570plasmids pSV1 and pSV2

Kerstin Spatz, Henning Ko«hn, Matthias Redenbach �

Department of Genetics, Genome Research Unit, Kaiserslautern University, 67663 Kaiserslautern, Germany

Received 18 March 2002; received in revised form 27 May 2002; accepted 27 May 2002

First published online 17 June 2002

Abstract

We have analyzed the structure of two extrachromosomal elements of the methylenomycin producing actinomycete Streptomycesviolaceoruber SANK95570. The presence of the circular plasmid pSV1 which was supposed to contain the genes for methylenomycinbiosynthesis could be verified. Physical mapping of pSV1 revealed a size of 175.35 kb for this plasmid. In addition we generated arestriction map for the 100-kb linear plasmid pSV2. Cloning and sequencing of the terminal ends of pSV2 indicated the presence of 426-bpterminal inverted repeats. Both pSV2 termini show significant homology to the chromosome ends of Streptomyces coelicolor A3(2) whichis a closely related strain to S. violaceoruber SANK95570. ; 2002 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Plasmid; Terminal inverted repeat; Streptomyces

1. Introduction

One of the most intriguing features of the Streptomycesgenome organization apart from chromosome linearityand the large chromosome size is the frequent presenceof linear extrachromosomal replicons. These might haveplayed a signi¢cant role in the evolution of the linearStreptomyces chromosomes. Linear plasmids are discussedto have evolved from phages and might be responsible forthe generation of linear chromosomes by integration into achromosome with circular topology [1]. Linear streptomy-cetes plasmids have been demonstrated to interact withStreptomyces chromosomes leading to integration of theelements [2]. In addition, linear actinomycetes elementswere shown to exchange ends with their host chromosomeand thus mobilize large DNA regions of the chromosomeends which frequently carry genes involved in secondarymetabolite synthesis [3].

Streptomyces coelicolor A3(2) contains the 360-kb linearplasmid SCP1 which possesses the genes of the methyle-nomycin biosynthesis cluster (mmy) [4] and is the geneti-cally best characterized linear Streptomyces plasmid. Mostreports about the interaction of linear plasmids with thehost chromosome derived from this system [5]. Recently,the complete genomic sequence of S. coelicolor includingSCP1 was obtained (ftp://ftp.sanger.ac.uk/pub/S_coelicol-or/whole_genome/Sco.dna; AL590464; AL59463). Strep-tomyces violaceoruber SANK95570 belongs to the samegenus as S. coelicolor and synthesizes methylenomycin,too [6]. This strain was supposed to contain two extra-chromosomal elements. The 110-MDa (166.6 kb) largecircular plasmid pSV1 was postulated based on electronmicroscopy analysis [7]. Further experiments provided ad-ditional evidence that pSV1 is a large circular, conjugativeplasmid containing the genes for methylenomycin biosyn-thesis although a physical separation leading to a moredetailed analysis was not possible [8]. In addition, pSV2,an approximately 70 kb linear plasmid of S. violaceoruberSANK95570, was identi¢ed by pulsed-¢eld gel electropho-resis (PFGE) [9].

We have started to compare the genomic structure ofS. coelicolor A3(2) with that of S. violaceoruber SANK-95570 to get insight into the genome evolution of thisspeci¢c genus. In a ¢rst step we therefore characterized

0378-1097 / 02 / $22.00 ; 2002 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 2 ) 0 0 7 8 7 - 5

* Corresponding author. Tel. : +49 (631) 205 3250;Fax: +49 (631) 205 4090.

E-mail address: redenbac@rhrk.uni-kl.de (M. Redenbach).

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www.fems-microbiology.org

the extrachromosomal elements of S. violaceoruberSANK95570.

2. Materials and methods

2.1. Strains and culture conditions

S. coelicolor M138 (SCP1þ) [10], M171 (SLP2þ, SCP1þ)[10] and S. violaceoruber SANK95570 [7] were used forthis study. Streptomyces strains were grown and handledas described in [10]. YEME medium was used for growingcultures and for the preparation of DNA and protoplasts.Cosmid clones from the ordered SCP1 library [11] wereused. Escherichia coli cells were grown in either Luria^Bertani (LB) medium or LB with 50 Wg ml31 ampicillinfor the isolation of cosmid DNA.

2.2. DNA manipulations

Total DNA was isolated using procedure 1 of [12]. Plas-mid and cosmid DNA isolations were done according tothe alkaline lysis method [13]. Isolation of end fragmentsfrom the linear plasmid pSV2 was done according to [14].A cosmid library of S. violaceoruber SANK95570 was es-tablished as described by [11]. A 1284-bp large internalfragment of the methylenomycin resistance gene (mmr)was ampli¢ed with the primers mmr1 (5P-CCC ACAGGA GCA GAA CAG-3P) and mmr2 (5P-GAC GAGTGA CGG CTA GAC-3P) based on sequence M18263using a PTC-100 thermocycler (MJ Research, Waltham,MA, USA).

2.3. Pulsed-¢eld gel electrophoresis (PFGE)

PFGE-DNA was prepared as described by [11]. Restric-tion digests were performed in volumes of 200 Wl with50 U of enzyme for 12 h at 37‡C. The digested DNAwas subjected to PFGE on 1% agarose gels using aCHEF apparatus from Bio-Rad (Munich, Germany)with 0.5UTBE. S. cerevisiae chromosomes (NEB, Frank-furt, Germany), V-concatemers and V-HindIII were used asmolecular mass standards.

2.4. DNA labeling and hybridization

DNA was labeled by random priming and by generatinglabeled T3/T7 transcripts using a non-radioactive digoxi-genin labeling kit (Roche, Mannheim, Germany). DNAfragments from agarose gels were transferred to porablotNY membrane (Macherey-Nagel, Du«ren, Germany) bythe method of Smith and Summers [15]. Hybridizationswith digoxigenin-labeled DNA probes were carried outas recommended by the supplier (Roche). After pre-hy-bridization of the ¢lters (1 h in 5USSC, 0.1% (w/v)n-lauroylsarcosine, 0.02% (w/v) SDS, 1% (w/v) blocking

reagent at 68‡C), probes (that had been heat denaturedfor 10 min) were added and hybridization was allowedto proceed for 16 h at 68‡C. Membranes were washedwith 2USSC, 0.2% SDS for 2U5 min at room temper-ature, followed by high-stringency washes for 2U15 minwith 0.2USSC, 0.2% SDS at 68‡C. Signals were detectedusing the color reaction recommended by the kit manufac-turer (Roche).

2.5. Sequencing

DNA was sequenced on both strands with the thermosequenase £uorescent labeled primer cycle sequencing kit(Amersham/Life Science, Freiburg, Germany) includingdeaza-dGTP and applied on a LI-COR sequencer, Model4000 (Bad Homburg, Germany). In addition, obtainedsequences were checked with a Genetic Analyzer 310 capil-lary sequencer (Applied Biosystems, Weiterstadt, Ger-many) using the bigdye terminator cycle sequencing kit2.0 (Applied Biosystems).

3. Results and discussion

3.1. Analysis of the linear plasmid pSV2

PFGE analysis of undigested S. violaceoruber SANK-95570 PFGE-DNA revealed a very intense band corre-sponding to the linear plasmid pSV2 (Fig. 1) indicatinga high copy number of the extrachromosomal element in

Fig. 1. Comparison of pSV2 of S. violaceoruber SANK95570 with SCP1and SLP2 of S. coelicolor M171. PFGE gel with undigested DNA ofS. violaceoruber SANK95570 and S. coelicolor M171. Half the amountof S. violaceoruber PFGE-DNA was loaded on the gel to obtain compa-rable bands. Lane 1, S. violaceoruber SANK95570 DNA; lane 2 S. coe-licolor M171 DNA, lane 3, V-DNA HindIII digested. Running condi-tions: ramping 5 to 30 s, 24 h, 200 V, 14‡C. The ¢rst band within lanes1 and 2 represents large degraded DNA migrating with the same speedunder the applied conditions.

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comparison to the linear plasmid SLP2 of Streptomyceslividans 1326. Hybridization of the eluted and labeledpSV2 band against the ordered cosmid clones of SCP1[11] showed that both elements share signi¢cant homology(data not shown). To compare both linear replicons indetail we mapped pSV2. Ten thousand clones of a S. vio-laceoruber cosmid library were screened with gel eluted,labeled DNA of pSV2. More than 80% of the S. violaceo-ruber cosmid clones showed homology with pSV2. 24 ran-domly chosen pSV2 cosmids were used for further analysisand DNA of four randomly selected cosmids (pSV2-5, -11,-13, -19) was used to prepare labeled T3 and T7 probes ofthe insert ends that were hybridized against all 24 clones.Cosmid pSV2-19 was shown to possess a central positiondividing all analyzed 23 clones into either a T7 hybridizingor a T3 hybridizing group. To identify cosmids which

reach the plasmid termini closest, probes of the clonedterminal ends pE1 and pE2 (see below) were hybridizedagainst the 24 pSV2 cosmids. Cosmids pSV2-4 and -20that were aligned left and right of pSV2-19 hybridizedwith the terminal probes. A precise restriction map forHindIII, XbaI and EcoRI was generated on the basis ofthe selected cosmids which allowed calculating a size of100 kb for pSV2 (Fig. 2).

The three aligned pSV2 cosmids were labeled and hy-bridized against EcoRI restricted cosmid DNA of clonesof the ordered SCP1 library to locate the homology be-tween both replicons. This experiment showed that allSCP1 cosmids except SCP1-35 and -73 hybridized at leastwith one EcoRI fragment with the labeled DNA of thepSV2 cosmids (data not shown).

3.2. Cloning and sequence analysis of the pSV2 ends

Terminal pSV2 fragments were identi¢ed by the methodof Stoll and Cullum [14]. Total DNA of S. violaceoruberSANK95570 was digested either with BamHI or SalI andDNA fragments with terminal protein were selectively en-riched with the glass milk method as described in [14].BamHI restricted total DNA revealed two clearly visiblebands (1.35 and 4.2 kb). A 0.7-kb and a 1.2-kb fragmentcould be identi¢ed from SalI digested total DNA. All frag-ments were gel eluted and used for cross-hybridizationexperiments. This demonstrated that the 0.7-kb SalI frag-ment possesses homology with the 4.2 kb BamHI bandand that the 1.2-kb SalI fragment shares homologous re-gions with the 1.35-kb BamHI terminal band. Veri¢cationof the terminal fragments was done by hybridizing the twolabeled BamHI fragments against Southern blots of Bam-

Fig. 2. Restriction map of pSV2. A: Aligned minimal set of cosmidclones. Black circle represents T7 end, open circle represents T3 end.B: Alignment of HindIII fragments. C: Alignment of EcoRI fragmentswith enlarged view of the left and right terminal ends. Numbers indicatethe size of fragments in kb. Small black bars pE1 and pE2 indicate thecloned terminal fragments. E, EcoRI; X represents the single XbaI re-striction site of pSV2.

Fig. 3. Hybridization of BamHI digested total S. violaceoruber SANK95570 DNA with labeled DNA of pE1, pE2 and complete pSV2. A: BamHI di-gested S. violaceoruber DNA. Lanes 2^10, digested DNA not treated with proteinase K. Lanes 11^19 digested DNA treated with proteinase K. Lanes 1and 20, 1-kb ladder. B: Hybridization of the blotted ¢lters. Lanes 2, 3, 18 and 19 hybridized with the 1.35-kb left terminal BamHI fragment of pSV2.Lanes 8, 9, 10, 14, 15, 16, 17 hybridized with the 4.2-kb right terminal BamHI fragment of pSV2. Lanes 4^7 and 11^13 hybridized with labeled DNAof complete pSV2.

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HI digested total DNA of S. violaceoruber SANK95570treated with and without proteinase K (Fig. 3). ProteinaseK treated DNA revealed two bands in the size of thelabeled BamHI fragments. In contrast non-proteinase Ktreated DNA did not reveal clear signals in the expectedsize indicating retardation of the protein bound terminalfragments. Final veri¢cation that the glass milk selectedDNA bands correspond to the ends of the plasmid wasobtained by sequence analysis as described below.

The 0.7-kb SalI fragment (pE1) and the 1.35-kb BamHIfragment (pE2) were successfully cloned in SalI, EcoRVand in BamHI, EcoRV restricted pBluescript KS+ andsequence data were obtained from di¡erent clones. Se-quence analysis of pE1 and pE2 clones showed the pres-ence of 426-bp-long terminal inverted repeats (TIRs) (Fig.4). The TIRs are perfectly homologous between both endsup to nucleotide (nt) position 317. From nt positions 318to 426 the ends are nearly identical, di¡ering in 13 posi-tions. Seven palindromes were found in the ¢rst 157 bp ofthe pSV2 ends, resembling structures already found attermini of several investigated Streptomyces linear repli-cons [16^18]. Five additional palindromes are present be-tween nt positions 213 and 380 that are separated from the¢rst block of palindromes by a region without any signi¢-cant repeat structures. In addition, three imperfect in-verted repeats were found within the TIR that mightform a superpalindrome as described for di¡erent Strepto-myces termini [16,18].

Homology between the pSV2 ends and the termini ofthe S. coelicolor chromosome was identi¢ed. The ¢rst 41bp of pSV2 including palindromes I and II are nearlyidentical with that of the S. coelicolor chromosome termi-ni, di¡ering at only one position. Additional blocks ofhomology between the pSV2 ends and the S. coelicolorchromosome termini occur in regions 230^288 nt and301^426 nt that are part of the TIRs of pSV2. Outsidethe TIRs a short homologous DNA region (427^492 nt)between the left end of pSV2 represented by pE2 and theS. coelicolor ends exists. A second block of homologoussequences between pE2 and the S. coelicolor TIR starts atnt position 654 and extends until the end of the clonedfragment.

Hybridization experiments of labeled pE1 and pE2DNA against the DNA of pSV2 cosmids 20 and 4 re-vealed signals and indicated that both cosmids reachedclose to the termini of the linear plasmid. Sequencing ofthe T7 ends of cosmids pSV2-20 and -4 veri¢ed the hy-bridization experiments. The T7 end of cosmid insertpSV2-4 overlaps with the left TIR and starts at nt position232. The T7 end of cosmid pSV2-20 overlaps with theright pSV2 TIR starting at nt position 404. These resultsdemonstrate in addition that the cloned end fragmentsderived from pSV2.

The distribution of sequence similarities in the di¡erentreplicons of S. coelicolor A3(2) and S. violaceoruberSANK95570 are intriguing. The termini of the linear plas-

Fig. 4. Terminal sequences of pSV2 and comparison with that of the S. coelicolor A3(2) chromosome end. The ¢gure shows the terminal 771 nucleoti-des of the right pSV terminus (pE1), the terminal 905 nucleotides of the left pSV2 terminus (pE2) and the ¢rst 880 nucleotides of the S. coelicolor chro-mosomal TIR (www.sanger.ac.uk). Solid arrows with latin numbers indicate palindromic sequences in S. violaceoruber (top of sequence) and S. coelicol-or A3(2) (below sequence). Broken arrows show small inverted repeat structures that might form putative superpalindromic structures. Sequences inblack ¢elds indicate homology between all three sequences. Gray ¢elds show sequence identities between two sequences.

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mid SCP1 do not show any structural similarities with theS. coelicolor chromosome ends neither with any describedactinomycete termini reported until now and may use ex-clusively its own terminal protein (Stoll and Cullum, per-sonal communication). On the other hand, the linear plas-mid pSV2 shows homology with SCP1 and the S.coelicolor chromosome termini, possibly re£ecting a dy-namic exchange of DNA in the genomes of S. violaceor-uber SANK95570 and S. coelicolor A3(2). Further se-quence analysis of pSV2 and comparison with thecomplete sequence of SCP1 will provide more detailed in-sight into the evolution of the S. violaceoruber genomestructure.

3.3. Veri¢cation of the presence of pSV1 in S. violaceoruberby PFGE

PFGE analysis was applied to verify the presence of thepostulated circular plasmid pSV1 of S. violaceoruberSANK95570. PFGE-DNA was digested with di¡erentrare cutter enzymes such as EcoRV, HindIII, VspI, XbaIand separated by PFGE. The gels were blotted and the¢lters were hybridized with the labeled DNA of an inter-nal fragment of the mmr gene that was supposed to residewithin the complete cluster on the putative circular plas-mid (Fig. 5). DNA digested with EcoRV, HindIII andXbaI revealed a single hybridization band of 15, 20 and155 kb, respectively. In contrast, VspI restricted DNAshowed only strong hybridization signals with DNA inthe gel slot. Large circular molecules do not enter PFGEgels, which suggests the presence of a large circular plas-

mid in S. violaceoruber including restriction sites for Hin-dIII, EcoRV and XbaI but not for VspI.

To visualize pSV1 in an agarose gel, PFGE-DNA wasdigested with VspI and all generated restriction fragmentswere separated by PFGE, leaving only undigested pSV1DNA in the slot. The blocks were excised from the gelslots, digested with XbaI and separated by PFGE, reveal-ing a 155 kb band. The gel was blotted and the ¢ltersubsequently hybridized with labeled mmr DNA. The155-kb band lighted up with the mmr probe, suggestingthat pSV1 is a circular molecule of at least 155 kb (datanot shown).

3.4. Overlapping cosmids and restriction map of pSV1

A thousand clones of the S. violaceoruber SANK95570cosmid library that did not hybridize against gel elutedand labeled pSV2 were screened with labeled mmr DNA.Seven clones were obtained sharing a similar restrictionpattern. From clone 3G1, one of the mmr positive cos-mids, T3 and T7 probes corresponding to the insert endswere generated. The labeled transcripts were hybridizedagainst the S. violaceoruber library. Six positive cloneswere selected, analyzed and one clone overlapping withthe left end of 3G1 and one overlapping with the rightend of 3G1 were chosen for an additional round of chro-mosome walking. After ¢ve walking steps the cosmids 5B2and 6E4 were identi¢ed which were overlapping and clos-ing the alignment to form a circular map (Fig. 6). Usingthe set of overlapping cosmids a restriction map with sitesfor EcoRI, EcoRV, HindIII and XbaI was generated.These results con¢rmed that pSV1 corresponds to a175.35-kb large circular molecule and veri¢es the pub-lished observation by [6,7]. The size di¡erence of 20 kb

Fig. 6. Restriction map of pSV1. The aligned pSV1 cosmids are indi-cated as bold lines. Sizes of individual restriction fragments (kb) areshown inside the circle. The black ¢lled circle marks the location of themmr resistance gene in the 5.8-kb EcoRI^EcoRV fragment.

Fig. 5. PFGE gel of restricted S. violaceoruber DNA and hybridizationof the blot with a mmr probe. A: PFGE gel with restricted S. violaceor-uber DNA. Running conditions: ramping 5^30 s, 24 h, 200 V at 14‡C.Lane 1, S. violaceoruber DNA VspI restricted; lane 2, XbaI; lane 3,HindIII; lane 4, EcoRV; lane 5, V-concatemers. B: Hybridization of the¢lter with a Dig-labeled mmr probe.

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obtained for pSV1 by PFGE analysis is due to the di¡er-ent mobility of GC rich PFGE fragments which lead tounderestimations of the PFGE bands [19].

DNA of the aligned pSV1 cosmids was labeled and hy-bridized against the ordered clones of SCP1. All pSV1clones revealed homology to the SCP1 clones 35 and 73(data not shown). The cosmids 3G1 and 4A10 showed themost intensive hybridization patterns, with SCP1-73 and-35 indicating most likely the location of the methyleno-mycin biosynthesis cluster. Cosmids 6A8, 5B2 and 6E4,which do not overlap with 3G1 and 4A10, provided ho-mology with single EcoRI bands of SCP1, which are cov-ered by SCP1-35 and -45. These results suggest that someDNA repetitions exist in pSV1.

pSV1 is to our knowledge one of the largest circularStreptomyces plasmids reported. The fact that it alsocontains homologous regions with the plasmid SCP1 ofS. coelicolor A3(2) con¢rms the already mentioned dynam-ic exchange of DNA between S. violaceoruber SANK-95570 and S. coelicolor A3(2).

Acknowledgements

We thank John Cullum for his constant support andDavid Hopwood for the gift of the strain S. violaceoruberSANK95570. K.S. greatly acknowledges a fellowship ofthe Landesgraduierten-Stipendium of the State Rhein-land-Pfalz. Many thanks to Bernhard Thiele and RolfKlein for sequencing support.

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