APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2008, p. 3368–3376 Vol. 74, No. 110099-2240/08/$08.00�0 doi:10.1128/AEM.00402-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Replication and Conjugation of Streptomyces CircularPlasmids pFP1 and pFP11 and Their Ability To Propagate

in Linear Mode with Artificially Attached Telomeres�†Ran Zhang,1 Ana Zeng,1 Ping Fang,2 and Zhongjun Qin1*

Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,300 Fenglin Road, Shanghai,1 and College of Environmental Sciences and Engineering, National Key Laboratory of

Pollution Control and Resources Reuse, Tongji University, Shanghai,2 People’s Republic of China

Received 17 February 2008/Accepted 27 March 2008

Many Streptomyces species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb).We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepAproteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 wereinherited extremely unstably; par and additional loci were required for stable inheritance. Surprisingly,plasmids containing replication loci from pFP11 or Streptomyces circular plasmid SCP2 but not from pFP1,SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached.These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, forplasmid transfer, a major functional traB gene (encoding a DNA translocase typical for Streptomyces plasmids)as well as, surprisingly, a putative traA gene (encoding a DNA nickase, characteristic of single-stranded DNAtransfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.

Streptomyces species, a major source of antibiotics and phar-macologically active metabolites, are gram-positive, mycelialprokaryotes with high G�C content (29). They usually harborconjugative circular and/or linear plasmids, propagating in au-tonomous and/or chromosomally integrating forms (17). Mostknown Streptomyces circular plasmids are small (8 to 14 kb)and include rolling-circle-replication (RCR) plasmids (e.g.,pIJ101, pJV1, pSG5, pSN22, pSVH1, pSB24.2, and pSNA1 [11,17]) as well as chromosomally integrating/autonomous plas-mids (such as SLP1 and pSAM2 [3, 36, 38]), whereas SCP2 islarger, at 31,317 bp (14, 45). Replication of autonomouspSAM2 occurs by an RCR mechanism (13), but the locus andmechanism of autonomous replication of SLP1 are not clear.The replication locus of non-RCR plasmid SCP2 consists oftwo small rep genes and adjacent noncoding sequences towhich is bound one of the Rep proteins (10, 14, 32), while itsmode of replication—uni- or bidirectional—has not been de-termined.

In contrast to most eubacteria, Streptomyces species usuallycontain linear plasmids and linear chromosomes (15, 30, 34).Streptomyces linear plasmids vary in size between 12 and 1,700kb (24, 31), with terminal inverted repeats of 0.04 to 180 kb (7,37), and the 5� ends are linked covalently to terminal proteins(1, 51). Unlike the terminal-protein-capped linear replicons ofadenoviruses and bacteriophage �29, which replicate by a

mechanism of strand displacement (43), Streptomyces linearplasmids start replication from an internally located ori locus(46) and replication proceeds bidirectionally toward the telo-meres (5). At least some Streptomyces linear plasmids can alsopropagate in circular mode when the telomeres are deleted (5,46). The internally located replication locus of linear plasmidpSLA2 consists of rep1 (containing iterons) and rep2 (encodinga DNA helicase) (6). The minimal locus required for main-taining the replication of pSLA2 in circular mode cannot allowits propagation in linear mode unless it contains the rlrA locus(required for linear replication [40]). Such internally locatedreplication loci, iterons plus rep genes, have also been identi-fied in the linear plasmids SCP1, pSLA2-L, pSCL2, and SLP2(16, 21, 41, 49, 50), suggesting that bidirectional replication iscommon for Streptomyces linear plasmids. This capacity ofnatural linear Streptomyces plasmids to propagate in circularmode raises questions about the ability of some naturally bi-directionally replicating circular plasmids to propagate in lin-ear mode with artificially attached telomeres.

We describe here two newly detected Streptomyces circularplasmids, pFP11 and pFP1, of 35,139 bp and 39,360 bp, re-spectively, with novel iterons plus repA loci for replication.Unexpectedly, we found that plasmids containing the replica-tion loci from pFP11 or SCP2 but not from pFP1, pIJ101, orSLP1 propagated in linear mode and were inherited stablywhen the telomeres of a linear plasmid were attached. Theseresults suggest for the first time that SCP2 and pFP11 replicatein a bidirectional mode.

MATERIALS AND METHODS

Bacterial strains, plasmids, and general methods. Twenty Streptomycesstrains, including F11, F2, FR1, and FQ1 (Table 1), isolated from heavy metal-contaminated land (9.1 mmol arsenic, 5.1 mmol copper, 1.3 mmol lead, and 0.8mmol zinc per kg soil) at a mine in Chenzhou Suburb, Hunan Province, China,

* Corresponding author. Mailing address: Shanghai Institute ofPlant Physiology and Ecology, Shanghai Institutes for Biological Sci-ences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai200032, People’s Republic of China. Phone and fax: 86-21-54924171.E-mail: qin@sibs.ac.cn.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 4 April 2008.

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and identified as Streptomyces species by PCR sequencing of the 16S rRNAgenes, were provided by Ping Fang and Tongbin Chen (Institute of GeographicSciences and Natural Resources Research, Chinese Academy of Sciences).Eighty Streptomyces strains, isolated from varied soils of Yunnan Province,China, and identified by the standard procedures of actinomycete classification,were provided by Chenglin Jiang and Lihua Xu (Institute of Microbiology,Yunnan University). Streptomyces coelicolor A3(2) (SCP1, SCP2, and SLP1) wasthe source of plasmids SCP2 (45) and SLP1 (3), and Streptomyces rochei 7434-AN4 was the source for the telomere of linear plasmid pSLA2 (15). Streptomyceslividans ZX7 (str-6 rec-46 pro-2 dnd) (53) was the host for plasmid propagation.Streptomyces culture, pulsed-field gel electrophoresis, preparation of protoplasts,and transformation followed the methods of Kieser et al. (29). Thiostrepton,kanamycin, and apramycin were used to select tsr, kan, and apr, respectively.Isolation of linear or circular plasmid DNA followed the method of Qin andCohen (39) or Kieser (28). Plasmids pSP72 (Life Technologies, Inc.), pHZ132(20), and pQC156 (40) were used as cloning vectors. Escherichia coli strain DH5�(supE44 �lacU169 �80dlacZ�M15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (LifeTechnologies, Inc.) was used as the cloning host. Plasmid isolation, transforma-tion and transfection of E. coli, and PCR amplification followed the methods ofSambrook et al. (44).

Cloning and sequencing of Streptomyces circular plasmids pFP11 and pFP1.pFP11 DNA was digested with restriction endonucleases ClaI, EcoRI, HindIII,NheI, and XhoI to make a restriction map, and NheI-partially digested plasmidDNA was ligated with isochizomer XbaI-digested cosmid pHZ132 (20) andintroduced by transfection into E. coli to yield pFQ8. Similarly, pFP1 DNA wastreated with BclI, BglII, HindIII, KpnI, NheI, SacI, XbaI, and XhoI, and BglII-partially digested pFP1 DNA was cloned into the BamHI site of pHZ132 toobtain pFQ3. Shotgun cloning and sequencing of pFQ3 and pFQ8 were per-formed with an Applied Biosystems Genetic Analyzer model 377 at the ChineseHuman Genome Center in Shanghai. Analysis of Streptomyces protein codingregions was performed with FramePlot 3.0 beta (22) (http://watson.nih.go.jp/�jun/cgi-bin/frameplot-3.0b.pl), and ATG, GTG, or TTG was used as the startcodon. Sequence comparisons and protein domain searching were done withsoftware from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). DNA secondary structures (e.g., direct repeats andinverted repeats) were predicted with DNA folder (http://www.bioinfo.rpi.edu/applications/mfold/dna/form1.cgi) and Clone manager, version 9 (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi).

Identification of the loci of pFP11 and pFP1 for replication in Streptomyces. Toidentify a pFP11 locus for plasmid replication, Sau3AI-partially digested DNAwas ligated with BamHI-digested E. coli plasmid pQC156 containing Streptomy-ces selection marker tsr and introduced by transformation into S. lividans ZX7.pZR7 from one transformant, containing the pFP11.27 to pFP11.29c loci, wasidentified. To subclone the inserted sequence of pZR7, three fragments, 3.3-kbPstI, 2.1-kb EcoRI/PstI, and 2.7-kb SmaI/XhoI, were cloned into pQC156 toyield pZR15, pZR14, and pZR105, respectively, and then the 0.9-kb and 0.3-kbfragments of pFP11 were introduced into XhoI-digested pZR14 to yield pZR76and pZR97. These pFP11-derived plasmids were constructed in E. coli DH5�and introduced by transformation into S. lividans ZX7. To compare transforma-tion frequencies of plasmids in different experiments, we used 0.1 ng DNA ofStreptomyces plasmid pIJ702 (23, 27) each time and took 1 � 106 transformantsper g DNA as a control frequency.

Similarly, to identify the replication locus of pFP1, pZR61, containing an

�11-kb pFP1 fragment, was obtained from a ZX7 transformant. Various pZR61fragments, including four partially digested fragments (6.1-kb, 4.1-kb, and 4-kbSau3AI and 1.7-kb XbaI), were cloned into BamHI- or XbaI-digested pQC156 toobtain pZR114, pZR115, pZR116, and pZR94, respectively, and the 6-kb NheI/Sau3AI fragment was cloned into the XbaI/BamHI sites of pHY1 (pSP72 con-taining the apramycin resistance gene) to yield pZR62, which was further di-gested with EcoRV to delete the small fragment and obtain pZR75.

Inheritance of plasmids in Streptomyces. Plasmids were introduced by trans-formation into strain ZX7, and thiostrepton-resistant colonies were inoculatedinto complete medium without thiostrepton. After 7 days of incubation at 30°C,spores were harvested, diluted in water, and plated equally on LB and LBmedium containing thiostrepton, and colony counts were made after 2 to 3 daysof incubation. The frequency of plasmid inheritance equals 100 multiplied by theratio of colonies on LB (thiostrepton) to colonies on LB.

Electrophoretic mobility shift assays. The pFP1 repA gene (bp 16819 to 18318)was cloned into the NdeI and HindIII sites of E. coli plasmid pET28b (Novagen,Inc.) to obtain pZR349 and then introduced into E. coli strain BL21(DE3)(Novagen, Inc.). IPTG (isopropyl--D-thiogalactopyranoside) (1 mM) was addedto a log-phase LB culture at 30°C for 2 h to induce overexpression of the clonedgene. The His6 tag RepA protein was eluted in buffer containing imidazole andwas purified to �90% homogeneity by Ni2� column chromatography accordingto the supplier’s instructions (Qiagen, Inc.). The 582-bp DNA sequence (bp15431 to 16012) containing the pFP1 iterons was amplified by PCR and insertedinto the EcoRV site of pBluescript SK (Stratagene, Inc.) to construct pZR365.The 582-bp DNA was released by treatment of pZR365 with XhoI/PstI and endlabeled with [�-32P]dCTP using DNA polymerase Klenow fragment. Similarly,the pFP11 repA gene (bp 25356 to 27074) was cloned into the NdeI and HindIIIsites of E. coli plasmid pET28b, and the 321-bp DNA sequence (bp 27614 to27934) containing the pFP11 iterons was amplified by PCR. A Micro-Spin S-300HR column (Amersham Pharmacia Biotech, Inc.) was employed to separate thelabeled DNA and free [�-32P]dCTP. The DNA-binding reaction was performedat room temperature for 10 min in buffer (10 mM Tris-HCl at pH 7.5, 25 mMKCl, and 10% glycerol). Salmon sperm DNA was used as the nonspecific com-petitor and unlabeled probe as the specific competitor. The reaction complexeswere separated on a prerun 5% native acrylamide gel in 0.5� Tris-borate-EDTAbuffer at 150 V for 2 h. A gel was dried and analyzed using a phosphorimager(Fuji, Inc.).

Detection of replication intermediates by Southern hybridization. Streptomy-ces RCR plasmid pIJ101-derived pIJ702 and pFP11- and pFP1-derived plasmidspZR7 and pZR116 were introduced by transformation into strain ZX7, and theirgenomic DNAs were isolated. Aliquots of DNA were either untreated (in TEbuffer [10 mM Tris-HCl, 1 mM EDTA, pH 8]) or treated with S1 nuclease (inbuffer [40 mM sodium acetate, pH 4.5, 0.3 M NaCl, and 2 mM ZnSO4]; Fer-mentas, Inc.) to remove single-stranded DNA and then electrophoresed onagarose gels. The gels were soaked in neutral pH buffer (0.5 M Tris-HCl, pH 7.5,1 M NaCl) or alkaline solution (0.4 M NaOH) for 15 min and then transferredonto nylon membranes. Southern hybridization with the [�-32P]dCTP-labeledStreptomyces tsr gene as the probe was performed at 65°C overnight (8). Themembranes were analyzed with a phosphorimager.

Propagation of circular plasmids in linear mode in Streptomyces. By using asimilar strategy (39), pZR131, containing two functional 381-bp pSLA2 telo-meres and the Streptomyces tsr and melC genes of plasmid pIJ702, was con-structed. A 3.8-kb EcoRI/XbaI fragment from pZR7 containing the pFP11 rep-lication locus was cloned into pZR131 to obtain pZR173. A PCR fragment(primers 5�-TCTAGAGTTGCTCATGCCGCCACC-3� and 5�-AAGCTTGTGTGGACCATGCTGCCT-3�) containing the rlrA and rorA genes (required forlinear replication) of linear plasmid pSLA2 was cloned into XbaI-treatedpZR173 to yield pZR181. An SCP2 fragment (bp 28683 to 30305) was clonedinto EcoRI-digested pZR131 to yield pZR156. DraI-linearized pZR181,pZR173, and pZR156 DNAs were introduced by transformation into ZX7 andplasmids were isolated. Aliquots of DNA were either untreated (in TE buffer) ortreated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage �exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gelat 3 V/cm for 6 h.

Transfer functions of the tra genes of pFP11 and pFP1 in Streptomyces lividans.Plasmid pFQ8 contained full-length pFP11 and vector pHZ132. The rep gene ofpSG5 on pHZ132 was deleted by PCR targeting (primers 5�-ATGATCAACAGGACCCAGAAATGGCACGAGCCCGGGATTATTCCGGGGATCCGTCGACC-3� and 5�-CGCGACCGCTGTGTCGACGCGGAGGCAGTCTCCGGGGTGTGTAGGCTGGAGCTGCTTC-3� [12]) and then deleting kan in strainBT340 to obtain pAZ109, followed by further replacement of pFP11 traB (bp17787 to 21580) with the kan gene (primers 5�-TCAGGCCGTGAGGCCGTACTTCGCCTGGACCGTGCGGAGATTCCGGGGATCCGTCGACC-3� and

TABLE 1. Detection of circular plasmids among 100Streptomyces strains

Strain no. Size(s) (kb) of plasmid(s)detected (plasmid name�s )

44006 .................................................................10 (pRC1)44014 .................................................................6 (pRC2)44018 .................................................................10 (pRC3)44292 .................................................................13 (pRC4)44529 .................................................................6 (pRC5), 80 (pRC6)44554 .................................................................11 (pRC7)44571 .................................................................85 (pRC8)44583 .................................................................8 (pRC9)FR1....................................................................40 (pFP4)FQ1 ...................................................................39 (pFP1)F2.......................................................................34 (pFP3), 85 (pFP2)F11.....................................................................27 (pFP12), 35 (pFP11)

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FIG. 1. Identification and characterization of a pFP11 locus for replication. (A) Identification of a pFP11 locus for replication in Streptomyces.Plasmids were constructed in E. coli and introduced by transformation into strain ZX7. Positions of these cloned fragments on pFP11 andtransformation frequencies are shown. Iterons are indicated by striped boxes, replication genes by filled arrowheads, and other relevant genes byopen arrowheads. (B) Iterons of pFP11. Possible iteron sequences and AT-rich regions (underlined) from bp 27649 to 27893 on pFP11 are shown.Direct-repeat (DR) and inverted-repeat (IR) sequences are indicated by arrows. (C) Detection of the binding activity of the pFP11 replicationprotein with its iteron DNA. Electrophoretic mobility shift assays were employed to detect DNA-binding activity between the RepA protein anditeron DNA. The amount of DNA probe for each lane was 5 ng; the probe was also used as the specific competitor and salmon sperm DNA asthe nonspecific competitor, and the numbers equal the addition (n-fold) in excess of 5 ng. DNA-protein complexes are indicated.

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5�-ATGAGCTACCGCGAGGAGCGCCGCGCCGACGAGGCGGCCTGTAGGCTGGAGCTGCTTC-3�) to obtain pAZ110. By use of cosmid vectorpHAQ31 (containing E. coli ColE1 ori, two cos sequences, and the Streptomycestsr and melC genes [our unpublished data]), SphI-digested full-length pFP1 wascloned to obtain pAZ107, followed by further replacement of pFP1 traB (bp22465 to 29662) with the kan gene (primers 5�-ATGAGCGAGGCAACCACCTACCGCGGCCGCGAGCAGATGATTCCGGGGATCCGTCGACC-3� and5�-CTCGGCGGCGTCCTGCGCCTCCTTGATCAGCTCCGCGATTGTAGGCTGGAGCTGCTTC-3�) to yield pAZ114. Plasmids pAZ107, pAZ109,pAZ110, and pAZ114 were introduced by transformation into S. lividans TK21(no SLP2 and SLP3) and mated with �10-fold excess spores of TK21 containingpSET152 (apr) integrated in the chromosome. After incubation at 30°C for 4days, spores were harvested, diluted in water, and plated equally on LB (thio-strepton), LB (apramycin), and LB (thiostrepton plus apramycin). The frequencyof plasmid transfer equals 100 multiplied by the ratio of colonies on LB (thio-strepton plus apramycin) to colonies on LB (thiostrepton).

Nucleotide sequence accession numbers. The GenBank accession numbers forthe complete nucleotide sequences of pFP11 and pFP1 are AY943952 andAY943953, respectively.

RESULTS

Characterization of two newly detected plasmids, pFP11 andpFP1. While investigating natural circular plasmids of Strepto-myces, we detected 15 such plasmids from 12 strains among 100newly isolated Streptomyces strains (Table 1). Strains F11, F2,FR1, and FQ1, isolated from heavy metal-contaminated land,harbored six large circular plasmids from 27 to �85 kb in size.The 80- to �85-kb plasmids have not been cloned, and twoother plasmids, �35-kb pFP11 from strain F11 and �39-kbpFP1 from FQ1, were cloned into E. coli plasmid pHZ132 andsequenced.

The complete nucleotide sequence of pFP11 consisted of35,159 bp with 69.4% G�C content, slightly lower than that ofa typical Streptomyces genome [e.g., 72.1% for S. coelicolorA3(2) (see reference 2)]. Thirty-seven open reading frames(ORFs) were predicted; 12 resembled genes of known func-tion, eight were hypothetical genes, and 17 were unknowngenes (see Table S1 in the supplemental material). In contrastto the chromosomal dnaA gene (encoding 656 amino acids) ofStreptomyces coelicolor (2, 4), pFP11 contained a truncateddnaA (pFP11.35c, encoding only 74 amino acids). No othergenes on pFP11 resembled any known genes involved in rep-lication. Interestingly, there were two tra genes (traA and traB,encoding DNA nickase and translocase). As in Streptomycessmall plasmid pIJ101, a korA gene was transcribed divergentlyfrom a gene cluster including traB, spdB, and other genes,suggesting its negative transcriptional regulation of the genecluster (25, 48).

The complete nucleotide sequence of pFP1 consisted of39,360 bp with 72.1% G�C content. Forty-three ORFs were

predicted, including 13 resembling genes of known function,seven hypothetical genes, and 23 unknown genes (see Table S2in the supplemental material). No genes on pFP1 resembledany known genes involved in replication. As in pFP11, pFP1contained traA, traB, and korA and divergently transcribedtraB, spdB, and other genes.

New loci for replication and inheritance. To identify apFP11 locus for plasmid replication, DNA fragments wereshotgun cloned into E. coli plasmid pQC156, and plasmidpZR7 containing a 3.8-kb pFP11 sequence was obtained bytransformation into Streptomyces lividans ZX7. Defining thepZR7 sequence for replication (Fig. 1A), pZR97—containingpFP11.27 and an adjacent 244-bp iteron sequence—couldpropagate in ZX7, but deletion of pFP11.27 (i.e., pZR15) orthe iterons (pZR14) abolished propagation in ZX7, suggestingthat both pFP11.27 and the iterons were needed for plasmidreplication. A fragment containing pFP11.34 to pFP11.36 wascloned into pQC156 and introduced by transformation intoZX7, but no transformants were obtained, suggesting that thetruncated dnaA gene (pFP11.35c) was not required for plasmidreplication. These results indicated that the locus for pFP11replication consisted of pFP11.27 (bp 25356 to 27074, encodingan ATP/GTP binding protein, designated pFP11 repA) anditerons (bp 27649 to 27893, containing three direct repeats, twopairs of inverted repeats, and one AT-rich sequence) (Fig. 1B).

The inheritance of pFP11-derived plasmids in ZX7 sporeswas determined. As shown in Table 2, a plasmid containing theminimal iterons and pFP11 repA was extremely unstable(0.00006%); additional loci, including pFP11.28 and pFP11.29and pFP11.31c to pFP11.32c (parA and parB), were requiredfor stable inheritance of plasmids.

To identify a pFP1 locus for plasmid replication, pZR61,containing an �11-kb pFP1 fragment, was obtained from aZX7 transformant. pZR115 or pZR116, containing pFP1.14c(bp 16819 to 18318, encoding a hypothetical protein, desig-nated pFP1 repA) and the adjacent 534-bp iteron region (bp15458 to 15991, containing two pairs of direct repeats and fourpairs of inverted repeats) (Fig. 2B), could propagate in ZX7(Fig. 2A).

RepA proteins bind specifically to their iterons in vitro. Tosee if there was an interaction between the RepA protein andthe iteron sequence, electrophoretic mobility shift assays forDNA-protein complex formation were performed. The pFP11RepA protein was incubated with an [�-32P]dCTP-labeled321-bp DNA sequence (bp 27614 to 27934, containing threedirect repeats and two pairs of inverted repeats) and thenelectrophoresed and autoradiographed. As shown in Fig. 1C,

TABLE 2. Inheritance of pFP11-derived linear and circular plasmids in Streptomyces ZX7

Plasmid Cloned pFP11 loci in pQC156 DNAconformation

Frequency ofinheritance (%)

pZR97 pFP11.27 (repA), iterons Circular 0.00006pZR105 pFP11.27 (repA), iterons, pFP11.28 Circular 0.3pZR7 pFP11.27 (repA), iterons, pFP11.28 and pFP11.29 Circular 0.1pZR138 pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, pFP11.31c to pFP11.32c (parAB) Circular 61pZR173 pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, telomeres (pSLA2) Linear 17pZR181 pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, telomeres (pSLA2), rlrA and

rorA (pSLA2)Linear 30

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FIG. 2. Identification and characterization of a pFP1 locus for replication. Panel descriptions are the same as those in the legend for Fig. 1,except that in panel C the amount of DNA probe for each lane was 2.5 ng.

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the “shift” DNA bands were visualized by the addition ofRepA protein, indicating that the RepA protein could bind tothe DNA probe to form a DNA-protein complex. Formation ofthis complex was inhibited almost completely by adding athreefold excess of unlabeled probe but was affected only par-tially by adding a 40- or 400-fold excess of salmon sperm DNAas the nonspecific competitor, indicating that the binding ofpFP11 RepA with iteron DNA was highly specific.

Similarly, the “shift” DNA bands were detected on the gel bythe addition of pFP1 RepA protein (Fig. 2C). Formation ofthis DNA-protein complex was inhibited by adding a 60-foldexcess of unlabeled probes but was not affected by adding600-fold excess salmon sperm DNA, indicating that the bind-ing of pFP1 RepA protein with the [�-32P]dCTP-labeled582-bp iteron DNA (bp 15431 to 16012, containing two pairs ofdirect repeats and four pairs of inverted repeats) of pFP1 wasspecific.

No single-stranded circular DNA was detected as a replica-tion intermediate. Full-length single-stranded circular DNA isa replication intermediate for all RCR bacterial plasmids butnot for theta-type replicating plasmids (9, 26). To investigatepossible replication intermediates of pFP11 and pFP1,genomic DNAs of S. lividans ZX7 containing plasmid pZR7 orpZR116 were electrophoresed on agarose gels and transferredin neutral or alkaline buffer onto nylon membranes for South-ern hybridization. As shown in Fig. 3, the Streptomyces RCRplasmid pIJ101-derived pIJ702 contained single-strandedDNA as the replication intermediate, to be removed by S1nuclease digestion, and no single-stranded circular pZR7 orpZR116 DNA was detected, suggesting that replication ofpFP11 and pFP1 is theta type (either uni- or bidirectional).

A plasmid containing the replication locus of pFP11, but notthat of pFP1, propagates in linear mode when the telomeres ofa linear plasmid are attached. The replication loci of bothpFP11 and pFP1 comprise repA plus iterons, resembling thoseof bidirectionally replicating Streptomyces linear plasmids (6,16, 21, 41, 49, 50). To see if pFP11 or pFP1 could also replicate

in linear mode when the telomeres of a linear plasmid wereattached, we constructed pZR181 (Fig. 4), containing the rep-lication locus of pFP11, two 381-bp functional telomeres oflinear plasmid pSLA2 (39), and the rlrA and rorA genes re-quired for replication in linear mode (40). DraI-linearizedpZR181 DNA from E. coli was introduced by transformationinto ZX7. Surprisingly, transformants were obtained at a fre-quency of 2 � 104/g DNA. Genomic DNA was isolated, anda 9.4-kb plasmid DNA band was detected on an agarose gel. Asshown in Fig. 4, this band was resistant to treatment by �exonuclease but sensitive to E. coli exonuclease III, suggestingthat it was a double-stranded linear DNA with free 3� butblocked 5� ends.

We also constructed a pFP11-derived plasmid, pZR173 (Fig.4), containing two telomeres but without rlrA and rorA. Trans-formants were obtained at a frequency of 9 � 103/g DNA inZX7. A 7.4-kb linear plasmid band was seen (Fig. 4). Theseresults indicated that, unlike with Streptomyces linear plasmidpSLA2 (40), the replication locus of pFP11 alone was capableof maintaining plasmid replication in linear mode.

A pZR173-like plasmid, pZR279 (data not shown), contain-ing the replication locus of pFP1, was constructed, but notransformants were obtained after introduction of DraI-linear-ized pZR279 into ZX7.

The pFP11-derived linear plasmids are inherited more sta-bly in Streptomyces. The frequency of inheritance of artificiallylinearized pFP11 plasmids was determined. As shown in Table2, pFP11-derived linear plasmid pZR173 was inherited morestably through spores than circular plasmid pZR7 (17% versus0.1%) in ZX7. A circular plasmid containing E. coli pSP72 (i.e.,pZR138) was inherited stably, suggesting no influence of the E.coli sequence on pFP11 plasmid inheritance. In contrast toresults for pSLA2-derived plasmids (40), the rlrA and rorAgenes did not significantly increase inheritance of the pFP11-derived linear plasmid (i.e., pZR181).

A plasmid containing telomeres and the replication locusfrom Streptomyces circular plasmid SCP2, but not from plas-mids pIJ101 or SLP1, propagates in linear mode. We furtherinvestigated if other naturally circular Streptomyces plasmidscould replicate in linear mode when telomeres were attached.

FIG. 3. Detection of possible replication intermediates by South-ern hybridization. Aliquot DNAs were electrophoresed on agarose gelsand then soaked with neutral pH buffer or alkaline solution for transferon nylon membranes. Southern hybridizations were performed. Dou-ble-stranded and single-stranded circular pIJ702 as positive controlsare indicated. CCC, covalently closed circular; SS, single strand.

FIG. 4. Plasmids containing the replication locus of pFP11 or SCP2and telomeres of pSLA2 propagated in linear mode in Streptomyces.Plasmids were identified, and aliquots of DNA were tested with nonuclease (TE), E. coli exonuclease III (Exo III), or bacteriophage �exonuclease (� exo) and electrophoresed. Chromosome (Chr) andlinear plasmid DNA bands are indicated.

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Replication loci from Streptomyces circular plasmid SCP2, thechromosomally integrating/autonomous plasmid SLP1, and theRCR plasmid pIJ101 were employed to construct pZR173-likeplasmids pZR156, pZR215, and pQC13 (Z. Qin and S. N.Cohen, unpublished data), respectively. DraI-linearized plas-mid DNAs were introduced by transformation into ZX7.Transformants were obtained at a frequency of 8 � 103/gDNA from pZR156, but none were obtained from pZR215 orpQC13 (data not shown). As for pZR173, a 5.1-kb linear con-formation of pZR156 was verified (Fig. 4).

The traB (DNA translocase) but not the traA (DNA nickase)gene functions in plasmid transfer in Streptomyces lividans.Interestingly, as shown in Table S1 in the supplemental mate-rial, two ORFs of pFP11, pFP11.6c (designated pFP11 traA)and pFP11.20c (pFP11 traB), resembled plasmid transfer genestraA (DNA relaxase or nickase) of Agrobacterium tumefaciensTi plasmid and traB (DNA translocase) of Streptomyces plas-mid pSG5, a typical Streptomyces tra gene. To investigate if thetra genes functioned for transfer, plasmids were introduced bytransformation into S. lividans TK21 and the yielding strainswere mated with TK21 containing the apr gene. As shown inFig. 5, full-length pFP11 transferred at a high frequency (0.86),but replacement of traB with kan abolished detectable transfer,suggesting that traB was a major functional gene for plasmidtransfer and that traA might not be functional. Similar resultswere obtained for pFP1 (Fig. 5).

DISCUSSION

Previous studies showed that 17 strains harbored linear plas-mids among 100 Streptomyces strains (52). Here we showedthat 12 strains among the same population of strains containedcircular plasmids. Streptomyces strains F11, F2, FR1, and FQ1,isolated from heavy metal-contaminated land, harbored sixlarge circular plasmids, including pFP11 and pFP1 character-ized here, as well as five large linear plasmids (i.e., F2, 360 kb;F11, 500 kb; and FR1, 54, 100, and 450 kb [52]), suggesting that

distribution of plasmids among Streptomyces strains in naturalhabitats is not random.

Like Streptomyces plasmid SCP2 RepI, which binds to itsadjacent noncoding DNA sequence (10), both pFP11 andpFP1 RepA proteins were found to bind to their iteron se-quences. The repA gene of pFP11 resembles SCO4617 of Strep-tomyces integrated/autonomous plasmid SLP1 (expectationvalue of 2 � 10�60; identity of 175/491 [35%]). A plasmidcontaining SCO4617 and an adjacent noncoding sequence (bp5040367 to 5043247 of the Streptomyces coelicolor chromo-some) plus the impA sequence (bp 5049286 to 5050825 [47])propagates in Streptomyces lividans autonomously (our unpub-lished data), suggesting that SCO4617 is the SLP1 replicationlocus. pFP1 repA resembles a gene of unknown function,SAP1_35 (expectation value of 2 � 10�4; identity of 93/359, or25%), of Streptomyces linear plasmid SAP1. Similarly, a plas-mid containing SAP1_35 and an adjacent 1.1-kb noncodingsequence replicates as a circular plasmid in Streptomyces livi-dans (our unpublished data), suggesting that SAP1_35 is theSAP1 replication locus. These results suggest that rep plusnoncoding sequence or rep plus iterons may be common rep-lication loci for both non-RCR circular and linear plasmids inStreptomyces.

Previous studies have shown that plasmids containing theinternally located loci of Streptomyces linear plasmids can alsopropagate in circular mode (6, 16, 21, 41, 46, 49, 50). We foundthat attachment of the 381-bp telomere sequence of linearplasmid pSLA2 to the replication loci from Streptomyces nativecircular plasmid pFP11 or SCP2 permits plasmids to propagatein linear mode with stable inheritance. These results suggestrelationships between some Streptomyces naturally linear andcircular plasmids.

Replication of RCR-type plasmids has to be unidirectional,while that of theta-type plasmids can be either uni- or bidirec-tional (9, 26). Given the model of bidirectional replication ofStreptomyces linear replicons (5), it is reasonable that plasmidscontaining telomeres and a replication locus of the RCR-type

FIG. 5. Transfer of pFP11 and pFP1 with or without traB genes among Streptomyces lividans species. Plasmids containing traAB or traA(indicated by arrows) of pFP11 and pFP1 were introduced by transformation into strain TK21. After mating with excess TK21 (apr) occurred,spores were harvested and plated equally on LB containing different antibiotics. Transconjugants are colonies with a phenotype of resistance toboth thiostrepton and apramycin. Numbers of colonies on selection plates and transfer frequencies are shown.

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pIJ101 could not propagate in linear mode. This inability oftheta-type plasmid pFP1 or SLP1 with artificially attached telo-meres to replicate in linear mode does not exclude its ability topropagate in linear mode, since one or more elements neces-sary for linear replication might be missing. Plasmids withreplication loci from pFP11 or SCP2 propagate in linear mode,indicating that their replication is bidirectional. These resultssuggest a simple way to test some bidirectional replication oftheta-type circular plasmids in Streptomyces.

Like other Streptomyces plasmids, pFP11 and pFP1 containa major functional traB (DNA translocase) gene for plasmidtransfer. The proteins encoded by such genes are DNA motorsthat move double-stranded DNA molecules, providing con-vincing evidence that DNA transfer as described for Strepto-myces differs fundamentally from the mechanism of single-strand transfer promoted by F and other gram-negativeplasmids (18, 42). Unexpectedly, there is another traA generesembling traA (DNA nickase or relaxase) of Agrobacteriumtumefaciens Ti and traI of Escherichia coli F-like plasmids. Thisappears to be the first time that a traA-like gene on a plasmidin Streptomyces has been reported. For F, formation of a rel-axosome requires TraI and two other proteins (TraY and in-tegration host factor [19]); plasmid-borne mating proteinstrack along the single-stranded DNA and push it through thetype IV secretion system (33, 35). Our data show that pFP11and pFP1 contain traA but that deleting traB abolishes transferamong Streptomyces lividans strains. By incubating the purifiedTraA protein of pFP11 with pFP11 DNA, no sequence-specificnicking activity was detected (our unpublished data). Theseresults suggest that traA might be a pseudo-gene or anothergene from the original hosts of pFP11 or that both traA andtraB may be required for pFP11 traA to function in plasmidtransfer.

ACKNOWLEDGMENTS

We thank Stanley Cohen, David Hopwood, Zixin Deng, ChenglinJiang, Lihua Xu, and Tongbin Chen for strains and plasmids. We aregrateful to David Hopwood for critical reading of and useful sugges-tions on the manuscript.

These investigations were supported by grants from the NationalNature Science Foundation of China (30325003 and 30770045), theNational 863 Project (2007AA021503), and the Chinese Academy ofSciences Project (KSCX2-YW-G-014) to Z. Qin.

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