Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres

Abstract

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 (39,360 bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 were inherited 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, for plasmid 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 DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.

Streptomyces species, a major source of antibiotics and pharmacologically active metabolites, are gram-positive, mycelial prokaryotes with high G+C content (29). They usually harbor conjugative circular and/or linear plasmids, propagating in autonomous and/or chromosomally integrating forms (17). Most known 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 plasmids (such as SLP1 and pSAM2 [3, 36, 38]), whereas SCP2 is larger, at 31,317 bp (14, 45). Replication of autonomous pSAM2 occurs by an RCR mechanism (13), but the locus and mechanism of autonomous replication of SLP1 are not clear. The replication locus of non-RCR plasmid SCP2 consists of two small rep genes and adjacent noncoding sequences to which is bound one of the Rep proteins (10, 14, 32), while its mode of replication—uni- or bidirectional—has not been determined.

In contrast to most eubacteria, Streptomyces species usually contain linear plasmids and linear chromosomes (15, 30, 34). Streptomyces linear plasmids vary in size between 12 and 1,700 kb (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 of adenoviruses and bacteriophage Φ29, which replicate by a mechanism of strand displacement (43), Streptomyces linear plasmids start replication from an internally located ori locus (46) and replication proceeds bidirectionally toward the telomeres (5). At least some Streptomyces linear plasmids can also propagate in circular mode when the telomeres are deleted (5, 46). The internally located replication locus of linear plasmid pSLA2 consists of rep1 (containing iterons) and rep2 (encoding a DNA helicase) (6). The minimal locus required for maintaining the replication of pSLA2 in circular mode cannot allow its propagation in linear mode unless it contains the rlrA locus (required for linear replication [40]). Such internally located replication loci, iterons plus rep genes, have also been identified in the linear plasmids SCP1, pSLA2-L, pSCL2, and SLP2 (16, 21, 41, 49, 50), suggesting that bidirectional replication is common for Streptomyces linear plasmids. This capacity of natural linear Streptomyces plasmids to propagate in circular mode raises questions about the ability of some naturally bidirectionally replicating circular plasmids to propagate in linear mode with artificially attached telomeres.

We describe here two newly detected Streptomyces circular plasmids, pFP11 and pFP1, of 35,139 bp and 39,360 bp, respectively, with novel iterons plus repA loci for replication. Unexpectedly, we found that plasmids containing the replication loci from pFP11 or SCP2 but not from pFP1, pIJ101, or SLP1 propagated in linear mode and were inherited stably when the telomeres of a linear plasmid were attached. These results suggest for the first time that SCP2 and pFP11 replicate in a bidirectional mode.

MATERIALS AND METHODS

Bacterial strains, plasmids, and general methods.

Twenty Streptomyces strains, 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 0.8 mmol zinc per kg soil) at a mine in Chenzhou Suburb, Hunan Province, China, and identified as Streptomyces species by PCR sequencing of the 16S rRNA genes, were provided by Ping Fang and Tongbin Chen (Institute of Geographic Sciences 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) was the source of plasmids SCP2 (45) and SLP1 (3), and Streptomyces rochei 7434-AN4 was the source for the telomere of linear plasmid pSLA2 (15). Streptomyces lividans 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 and Cohen (39) or Kieser (28). Plasmids pSP72 (Life Technologies, Inc.), pHZ132 (20), and pQC156 (40) were used as cloning vectors. Escherichia coli strain DH5α (supE44 ΔlacU169 φ80dlacZΔM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (Life Technologies, Inc.) was used as the cloning host. Plasmid isolation, transformation and transfection of E. coli, and PCR amplification followed the methods of Sambrook et al. (44).

TABLE 1.

Detection of circular plasmids among 100 Streptomyces 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)

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 plasmid DNA was ligated with isochizomer XbaI-digested cosmid pHZ132 (20) and introduced by transfection into E. coli to yield pFQ8. Similarly, pFP1 DNA was treated with BclI, BglII, HindIII, KpnI, NheI, SacI, XbaI, and XhoI, and BglII-partially digested pFP1 DNA was cloned into the BamHI site of pHZ132 to obtain pFQ3. Shotgun cloning and sequencing of pFQ3 and pFQ8 were performed with an Applied Biosystems Genetic Analyzer model 377 at the Chinese Human Genome Center in Shanghai. Analysis of Streptomyces protein coding regions 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 start codon. Sequence comparisons and protein domain searching were done with software from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). DNA secondary structures (e.g., direct repeats and inverted 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.

To identify a pFP11 locus for plasmid replication, Sau3AI-partially digested DNA was ligated with BamHI-digested E. coli plasmid pQC156 containing Streptomyces selection marker tsr and introduced by transformation into S. lividans ZX7. pZR7 from one transformant, containing the pFP11.27 to pFP11.29c loci, was identified. To subclone the inserted sequence of pZR7, three fragments, 3.3-kb PstI, 2.1-kb EcoRI/PstI, and 2.7-kb SmaI/XhoI, were cloned into pQC156 to yield pZR15, pZR14, and pZR105, respectively, and then the 0.9-kb and 0.3-kb fragments of pFP11 were introduced into XhoI-digested pZR14 to yield pZR76 and pZR97. These pFP11-derived plasmids were constructed in E. coli DH5α and introduced by transformation into S. lividans ZX7. To compare transformation frequencies of plasmids in different experiments, we used 0.1 ng DNA of Streptomyces plasmid pIJ702 (23, 27) each time and took 1 × 10⁶ transformants per μg 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 pZR61 fragments, including four partially digested fragments (6.1-kb, 4.1-kb, and 4-kb Sau3AI and 1.7-kb XbaI), were cloned into BamHI- or XbaI-digested pQC156 to obtain pZR114, pZR115, pZR116, and pZR94, respectively, and the 6-kb NheI/Sau3AI fragment was cloned into the XbaI/BamHI sites of pHY1 (pSP72 containing the apramycin resistance gene) to yield pZR62, which was further digested with EcoRV to delete the small fragment and obtain pZR75.

Inheritance of plasmids in Streptomyces.

Plasmids were introduced by transformation into strain ZX7, and thiostrepton-resistant colonies were inoculated into complete medium without thiostrepton. After 7 days of incubation at 30°C, spores were harvested, diluted in water, and plated equally on LB and LB medium containing thiostrepton, and colony counts were made after 2 to 3 days of incubation. The frequency of plasmid inheritance equals 100 multiplied by the ratio 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-β-d-thiogalactopyranoside) (1 mM) was added to a log-phase LB culture at 30°C for 2 h to induce overexpression of the cloned gene. The His₆ tag RepA protein was eluted in buffer containing imidazole and was purified to ∼90% homogeneity by Ni²⁺ column chromatography according to the supplier's instructions (Qiagen, Inc.). The 582-bp DNA sequence (bp 15431 to 16012) containing the pFP1 iterons was amplified by PCR and inserted into 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 end labeled with [α-³²P]dCTP using DNA polymerase Klenow fragment. Similarly, the pFP11 repA gene (bp 25356 to 27074) was cloned into the NdeI and HindIII sites of E. coli plasmid pET28b, and the 321-bp DNA sequence (bp 27614 to 27934) containing the pFP11 iterons was amplified by PCR. A Micro-Spin S-300 HR column (Amersham Pharmacia Biotech, Inc.) was employed to separate the labeled DNA and free [α-³²P]dCTP. The DNA-binding reaction was performed at room temperature for 10 min in buffer (10 mM Tris-HCl at pH 7.5, 25 mM KCl, and 10% glycerol). Salmon sperm DNA was used as the nonspecific competitor and unlabeled probe as the specific competitor. The reaction complexes were separated on a prerun 5% native acrylamide gel in 0.5× Tris-borate-EDTA buffer at 150 V for 2 h. A gel was dried and analyzed using a phosphorimager (Fuji, Inc.).

Detection of replication intermediates by Southern hybridization.

Streptomyces RCR plasmid pIJ101-derived pIJ702 and pFP11- and pFP1-derived plasmids pZR7 and pZR116 were introduced by transformation into strain ZX7, and their genomic DNAs were isolated. Aliquots of DNA were either untreated (in TE buffer [10 mM Tris-HCl, 1 mM EDTA, pH 8]) or treated with S1 nuclease (in buffer [40 mM sodium acetate, pH 4.5, 0.3 M NaCl, and 2 mM ZnSO₄]; Fermentas, Inc.) to remove single-stranded DNA and then electrophoresed on agarose 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 transferred onto nylon membranes. Southern hybridization with the [α-³²P]dCTP-labeled Streptomyces tsr gene as the probe was performed at 65°C overnight (8). The membranes were analyzed with a phosphorimager.

Propagation of circular plasmids in linear mode in Streptomyces.

By using a similar strategy (39), pZR131, containing two functional 381-bp pSLA2 telomeres and the Streptomyces tsr and melC genes of plasmid pIJ702, was constructed. A 3.8-kb EcoRI/XbaI fragment from pZR7 containing the pFP11 replication 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 for linear replication) of linear plasmid pSLA2 was cloned into XbaI-treated pZR173 to yield pZR181. An SCP2 fragment (bp 28683 to 30305) was cloned into EcoRI-digested pZR131 to yield pZR156. DraI-linearized pZR181, pZR173, and pZR156 DNAs were introduced by transformation into ZX7 and plasmids were isolated. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 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 of pSG5 on pHZ132 was deleted by PCR targeting (primers 5′-ATGATCAACAGGACCCAGAAATGGCACGAGCCCGGGATTATTCCGGGGATCCGTCGACC-3′ and 5′-CGCGACCGCTGTGTCGACGCGGAGGCAGTCTCCGGGGTGTGTAGGCTGGAGCTGCTTC-3′ [12]) and then deleting kan in strain BT340 to obtain pAZ109, followed by further replacement of pFP11 traB (bp 17787 to 21580) with the kan gene (primers 5′-TCAGGCCGTGAGGCCGTACTTCGCCTGGACCGTGCGGAGATTCCGGGGATCCGTCGACC-3′ and 5′-ATGAGCTACCGCGAGGAGCGCCGCGCCGACGAGGCGGCCTGTAGGCTGGAGCTGCTTC-3′) to obtain pAZ110. By use of cosmid vector pHAQ31 (containing E. coli ColE1 ori, two cos sequences, and the Streptomyces tsr and melC genes [our unpublished data]), SphI-digested full-length pFP1 was cloned to obtain pAZ107, followed by further replacement of pFP1 traB (bp 22465 to 29662) with the kan gene (primers 5′-ATGAGCGAGGCAACCACCTACCGCGGCCGCGAGCAGATGATTCCGGGGATCCGTCGACC-3′ and 5′-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 containing pSET152 (apr) integrated in the chromosome. After incubation at 30°C for 4 days, spores were harvested, diluted in water, and plated equally on LB (thiostrepton), LB (apramycin), and LB (thiostrepton plus apramycin). The frequency of plasmid transfer equals 100 multiplied by the ratio of colonies on LB (thiostrepton plus apramycin) to colonies on LB (thiostrepton).

Nucleotide sequence accession numbers.

The GenBank accession numbers for the complete nucleotide sequences of pFP11 and pFP1 are AY943952 and AY943953, respectively.

RESULTS

Characterization of two newly detected plasmids, pFP11 and pFP1.

While investigating natural circular plasmids of Streptomyces, we detected 15 such plasmids from 12 strains among 100 newly 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 two other plasmids, ∼35-kb pFP11 from strain F11 and ∼39-kb pFP1 from FQ1, were cloned into E. coli plasmid pHZ132 and sequenced.

The complete nucleotide sequence of pFP11 consisted of 35,159 bp with 69.4% G+C content, slightly lower than that of a typical Streptomyces genome [e.g., 72.1% for S. coelicolor A3(2) (see reference 2)]. Thirty-seven open reading frames (ORFs) were predicted; 12 resembled genes of known function, eight were hypothetical genes, and 17 were unknown genes (see Table S1 in the supplemental material). In contrast to the chromosomal dnaA gene (encoding 656 amino acids) of Streptomyces coelicolor (2, 4), pFP11 contained a truncated dnaA (pFP11.35c, encoding only 74 amino acids). No other genes on pFP11 resembled any known genes involved in replication. Interestingly, there were two tra genes (traA and traB, encoding DNA nickase and translocase). As in Streptomyces small plasmid pIJ101, a korA gene was transcribed divergently from a gene cluster including traB, spdB, and other genes, suggesting its negative transcriptional regulation of the gene cluster (25, 48).

The complete nucleotide sequence of pFP1 consisted of 39,360 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 S2 in the supplemental material). No genes on pFP1 resembled any known genes involved in replication. As in pFP11, pFP1 contained traA, traB, and korA and divergently transcribed traB, spdB, and other genes.

New loci for replication and inheritance.

To identify a pFP11 locus for plasmid replication, DNA fragments were shotgun cloned into E. coli plasmid pQC156, and plasmid pZR7 containing a 3.8-kb pFP11 sequence was obtained by transformation into Streptomyces lividans ZX7. Defining the pZR7 sequence for replication (Fig. 1A), pZR97—containing pFP11.27 and an adjacent 244-bp iteron sequence—could propagate in ZX7, but deletion of pFP11.27 (i.e., pZR15) or the iterons (pZR14) abolished propagation in ZX7, suggesting that both pFP11.27 and the iterons were needed for plasmid replication. A fragment containing pFP11.34 to pFP11.36 was cloned into pQC156 and introduced by transformation into ZX7, but no transformants were obtained, suggesting that the truncated dnaA gene (pFP11.35c) was not required for plasmid replication. These results indicated that the locus for pFP11 replication consisted of pFP11.27 (bp 25356 to 27074, encoding an ATP/GTP binding protein, designated pFP11 repA) and iterons (bp 27649 to 27893, containing three direct repeats, two pairs of inverted repeats, and one AT-rich sequence) (Fig. 1B).

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 and transformation frequencies are shown. Iterons are indicated by striped boxes, replication genes by filled arrowheads, and other relevant genes by open 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 replication protein with its iteron DNA. Electrophoretic mobility shift assays were employed to detect DNA-binding activity between the RepA protein and iteron 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 as the nonspecific competitor, and the numbers equal the addition (n-fold) in excess of 5 ng. DNA-protein complexes are indicated.

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

TABLE 2.

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

Plasmid	Cloned pFP11 loci in pQC156	DNA conformation	Frequency of inheritance (%)
pZR97	pFP11.27 (repA), iterons	Circular	0.00006
pZR105	pFP11.27 (repA), iterons, pFP11.28	Circular	0.3
pZR7	pFP11.27 (repA), iterons, pFP11.28 and pFP11.29	Circular	0.1
pZR138	pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, pFP11.31c to pFP11.32c (parAB)	Circular	61
pZR173	pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, telomeres (pSLA2)	Linear	17
pZR181	pFP11.27 (repA), iterons, pFP11.28 and pFP11.29, telomeres (pSLA2), rlrA and rorA (pSLA2)	Linear	30

To identify a pFP1 locus for plasmid replication, pZR61, containing an ∼11-kb pFP1 fragment, was obtained from a ZX7 transformant. pZR115 or pZR116, containing pFP1.14c (bp 16819 to 18318, encoding a hypothetical protein, designated pFP1 repA) and the adjacent 534-bp iteron region (bp 15458 to 15991, containing two pairs of direct repeats and four pairs of inverted repeats) (Fig. 2B), could propagate in ZX7 (Fig. 2A).

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.

RepA proteins bind specifically to their iterons in vitro.

To see if there was an interaction between the RepA protein and the iteron sequence, electrophoretic mobility shift assays for DNA-protein complex formation were performed. The pFP11 RepA protein was incubated with an [α-³²P]dCTP-labeled 321-bp DNA sequence (bp 27614 to 27934, containing three direct repeats and two pairs of inverted repeats) and then electrophoresed and autoradiographed. As shown in Fig. 1C, the “shift” DNA bands were visualized by the addition of RepA protein, indicating that the RepA protein could bind to the DNA probe to form a DNA-protein complex. Formation of this complex was inhibited almost completely by adding a threefold excess of unlabeled probe but was affected only partially by adding a 40- or 400-fold excess of salmon sperm DNA as the nonspecific competitor, indicating that the binding of pFP11 RepA with iteron DNA was highly specific.

Similarly, the “shift” DNA bands were detected on the gel by the addition of pFP1 RepA protein (Fig. 2C). Formation of this DNA-protein complex was inhibited by adding a 60-fold excess of unlabeled probes but was not affected by adding 600-fold excess salmon sperm DNA, indicating that the binding of pFP1 RepA protein with the [α-³²P]dCTP-labeled 582-bp iteron DNA (bp 15431 to 16012, containing two pairs of direct repeats and four pairs of inverted repeats) of pFP1 was specific.

No single-stranded circular DNA was detected as a replication intermediate.

Full-length single-stranded circular DNA is a replication intermediate for all RCR bacterial plasmids but not for theta-type replicating plasmids (9, 26). To investigate possible replication intermediates of pFP11 and pFP1, genomic DNAs of S. lividans ZX7 containing plasmid pZR7 or pZR116 were electrophoresed on agarose gels and transferred in neutral or alkaline buffer onto nylon membranes for Southern hybridization. As shown in Fig. 3, the Streptomyces RCR plasmid pIJ101-derived pIJ702 contained single-stranded DNA as the replication intermediate, to be removed by S1 nuclease digestion, and no single-stranded circular pZR7 or pZR116 DNA was detected, suggesting that replication of pFP11 and pFP1 is theta type (either uni- or bidirectional).

FIG. 3.

Detection of possible replication intermediates by Southern hybridization. Aliquot DNAs were electrophoresed on agarose gels and then soaked with neutral pH buffer or alkaline solution for transfer on nylon membranes. Southern hybridizations were performed. Double-stranded and single-stranded circular pIJ702 as positive controls are indicated. CCC, covalently closed circular; SS, single strand.

A plasmid containing the replication locus of pFP11, but not that of pFP1, propagates in linear mode when the telomeres of a linear plasmid are attached.

The replication loci of both pFP11 and pFP1 comprise repA plus iterons, resembling those of 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 were attached, we constructed pZR181 (Fig. 4), containing the replication locus of pFP11, two 381-bp functional telomeres of linear plasmid pSLA2 (39), and the rlrA and rorA genes required for replication in linear mode (40). DraI-linearized pZR181 DNA from E. coli was introduced by transformation into ZX7. Surprisingly, transformants were obtained at a frequency of 2 × 10⁴/μg DNA. Genomic DNA was isolated, and a 9.4-kb plasmid DNA band was detected on an agarose gel. As shown in Fig. 4, this band was resistant to treatment by λ exonuclease but sensitive to E. coli exonuclease III, suggesting that it was a double-stranded linear DNA with free 3′ but blocked 5′ ends.

FIG. 4.

Plasmids containing the replication locus of pFP11 or SCP2 and telomeres of pSLA2 propagated in linear mode in Streptomyces. Plasmids were identified, and aliquots of DNA were tested with no nuclease (TE), E. coli exonuclease III (Exo III), or bacteriophage λ exonuclease (λ exo) and electrophoresed. Chromosome (Chr) and linear plasmid DNA bands are indicated.

We also constructed a pFP11-derived plasmid, pZR173 (Fig. 4), containing two telomeres but without rlrA and rorA. Transformants were obtained at a frequency of 9 × 10³/μg DNA in ZX7. A 7.4-kb linear plasmid band was seen (Fig. 4). These results indicated that, unlike with Streptomyces linear plasmid pSLA2 (40), the replication locus of pFP11 alone was capable of maintaining plasmid replication in linear mode.

A pZR173-like plasmid, pZR279 (data not shown), containing the replication locus of pFP1, was constructed, but no transformants were obtained after introduction of DraI-linearized pZR279 into ZX7.

The pFP11-derived linear plasmids are inherited more stably in Streptomyces.

The frequency of inheritance of artificially linearized pFP11 plasmids was determined. As shown in Table 2, pFP11-derived linear plasmid pZR173 was inherited more stably through spores than circular plasmid pZR7 (17% versus 0.1%) 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 to results for pSLA2-derived plasmids (40), the rlrA and rorA genes did not significantly increase inheritance of the pFP11-derived linear plasmid (i.e., pZR181).

A plasmid containing telomeres and the replication locus from Streptomyces circular plasmid SCP2, but not from plasmids pIJ101 or SLP1, propagates in linear mode.

We further investigated if other naturally circular Streptomyces plasmids could replicate in linear mode when telomeres were attached. Replication loci from Streptomyces circular plasmid SCP2, the chromosomally integrating/autonomous plasmid SLP1, and the RCR plasmid pIJ101 were employed to construct pZR173-like plasmids pZR156, pZR215, and pQC13 (Z. Qin and S. N. Cohen, unpublished data), respectively. DraI-linearized plasmid DNAs were introduced by transformation into ZX7. Transformants were obtained at a frequency of 8 × 10³/μg DNA from pZR156, but none were obtained from pZR215 or pQC13 (data not shown). As for pZR173, a 5.1-kb linear conformation 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 material, two ORFs of pFP11, pFP11.6c (designated pFP11 traA) and pFP11.20c (pFP11 traB), resembled plasmid transfer genes traA (DNA relaxase or nickase) of Agrobacterium tumefaciens Ti plasmid and traB (DNA translocase) of Streptomyces plasmid pSG5, a typical Streptomyces tra gene. To investigate if the tra genes functioned for transfer, plasmids were introduced by transformation into S. lividans TK21 and the yielding strains were mated with TK21 containing the apr gene. As shown in Fig. 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 plasmid transfer and that traA might not be functional. Similar results were obtained for pFP1 (Fig. 5).

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 to both thiostrepton and apramycin. Numbers of colonies on selection plates and transfer frequencies are shown.

DISCUSSION

Previous studies showed that 17 strains harbored linear plasmids among 100 Streptomyces strains (52). Here we showed that 12 strains among the same population of strains contained circular plasmids. Streptomyces strains F11, F2, FR1, and FQ1, isolated from heavy metal-contaminated land, harbored six large circular plasmids, including pFP11 and pFP1 characterized 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 natural habitats is not random.

Like Streptomyces plasmid SCP2 RepI, which binds to its adjacent noncoding DNA sequence (10), both pFP11 and pFP1 RepA proteins were found to bind to their iteron sequences. The repA gene of pFP11 resembles SCO4617 of Streptomyces integrated/autonomous plasmid SLP1 (expectation value of 2 × 10⁻⁶⁰; identity of 175/491 [35%]). A plasmid containing SCO4617 and an adjacent noncoding sequence (bp 5040367 to 5043247 of the Streptomyces coelicolor chromosome) plus the impA sequence (bp 5049286 to 5050825 [47]) propagates in Streptomyces lividans autonomously (our unpublished data), suggesting that SCO4617 is the SLP1 replication locus. pFP1 repA resembles a gene of unknown function, SAP1_35 (expectation value of 2 × 10⁻⁴; identity of 93/359, or 25%), of Streptomyces linear plasmid SAP1. Similarly, a plasmid containing SAP1_35 and an adjacent 1.1-kb noncoding sequence replicates as a circular plasmid in Streptomyces lividans (our unpublished data), suggesting that SAP1_35 is the SAP1 replication locus. These results suggest that rep plus noncoding sequence or rep plus iterons may be common replication loci for both non-RCR circular and linear plasmids in Streptomyces.

Previous studies have shown that plasmids containing the internally located loci of Streptomyces linear plasmids can also propagate in circular mode (6, 16, 21, 41, 46, 49, 50). We found that attachment of the 381-bp telomere sequence of linear plasmid pSLA2 to the replication loci from Streptomyces native circular plasmid pFP11 or SCP2 permits plasmids to propagate in linear mode with stable inheritance. These results suggest relationships between some Streptomyces naturally linear and circular plasmids.

Replication of RCR-type plasmids has to be unidirectional, while that of theta-type plasmids can be either uni- or bidirectional (9, 26). Given the model of bidirectional replication of Streptomyces linear replicons (5), it is reasonable that plasmids containing telomeres and a replication locus of the RCR-type pIJ101 could not propagate in linear mode. This inability of theta-type plasmid pFP1 or SLP1 with artificially attached telomeres to replicate in linear mode does not exclude its ability to propagate in linear mode, since one or more elements necessary for linear replication might be missing. Plasmids with replication loci from pFP11 or SCP2 propagate in linear mode, indicating that their replication is bidirectional. These results suggest a simple way to test some bidirectional replication of theta-type circular plasmids in Streptomyces.

Like other Streptomyces plasmids, pFP11 and pFP1 contain a major functional traB (DNA translocase) gene for plasmid transfer. The proteins encoded by such genes are DNA motors that move double-stranded DNA molecules, providing convincing evidence that DNA transfer as described for Streptomyces differs fundamentally from the mechanism of single-strand transfer promoted by F and other gram-negative plasmids (18, 42). Unexpectedly, there is another traA gene resembling traA (DNA nickase or relaxase) of Agrobacterium tumefaciens Ti and traI of Escherichia coli F-like plasmids. This appears to be the first time that a traA-like gene on a plasmid in Streptomyces has been reported. For F, formation of a relaxosome requires TraI and two other proteins (TraY and integration host factor [19]); plasmid-borne mating proteins track along the single-stranded DNA and push it through the type IV secretion system (33, 35). Our data show that pFP11 and pFP1 contain traA but that deleting traB abolishes transfer among Streptomyces lividans strains. By incubating the purified TraA protein of pFP11 with pFP11 DNA, no sequence-specific nicking activity was detected (our unpublished data). These results suggest that traA might be a pseudo-gene or another gene from the original hosts of pFP11 or that both traA and traB may be required for pFP11 traA to function in plasmid transfer.