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Journal of Bacteriology, October 2006, p. 6851-6857, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00873-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the Genetic Components of Streptomyces lividans Linear Plasmid SLP2 for Replication in Circular and Linear Modes

Mingxuan Xu,1 Yingmin Zhu,1 Ran Zhang,1 Meijuan Shen,1 Weihong Jiang,1 Guoping Zhao,1* and Zhongjun Qin1,2*

Shanghai Institute of Plant Physiology, Shanghai Institutes of Biological Sciences, The Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032 People's Republic of China,1 Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-51202

Received 17 June 2006/ Accepted 18 July 2006


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ABSTRACT
 
The nucleotide sequence of Streptomyces lividans linear plasmid SLP2 consists of 50,410 bp (C. H. Huang, C. Y. Chen, H. H. Tsai, C. Chen, Y. S. Lin, and C. W. Chen, Mol. Microbiol. 47:1563-1576, 2003). Here we report that the basic SLP2 locus for plasmid replication in circular mode resembles that of Streptomyces linear plasmids pSLA2 and SCP1 and comprises iteronsSLP2 and the adjacent repSLP2 gene. More efficient replication additionally required the 47-bp sequence between bp 581 and 628 upstream of the iterons. Replacement of either the iterons or the rep gene of SLP2 by the corresponding genes of pSLA2 or SCP1 still allows propagation in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that these plasmids share similar replication mechanisms. To replicate SLP2 in linear mode, additional SLP2 loci—either mtapSLP2/tpgSLP2 or mtapSLP2/ilrASLP2—were required. IlrASLP2 protein binds specifically to the iteronsSLP2 in vitro. Interactions were detected between these SLP2-borne replication proteins (MtapSLP2, TpgSLP2, and IlrASLP2) and the telomeric replication proteins (TpgL, TapL, and TpgL) of the S. lividans chromosome, respectively, but the SLP2 proteins failed to interact. These results suggest that SLP2 recruits chromosomally encoded replication proteins for its telomere replication.


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INTRODUCTION
 
Unlike most eubacteria, Streptomyces species usually contain linear chromosomes and plasmids (9, 16, 17). The linear plasmids are 12 to 1,700 kb long (16, 27). Their telomeres contain inverted repeat sequences from 44 bp (7) to 180 kb (19), and their 5' telomeric ends are linked covalently to terminal proteins (1, 28). Unlike linear replicons of adenoviruses and bacteriophage {phi}29, which also contain terminal proteins linking covalently to 5' telomeric DNA ends and undergo replication by a mechanism of strand displacement (23), replication of Streptomyces linear plasmids starts at centrally located loci (5, 25) and continues bidirectionally towards the telomeres—leaving an ~280-nucleotide 3' single-strand overhang as an intermediate (5). This is converted to a double strand by a postulated "folding back" of multiply short palindromes on the telomere extension (20). The chromosomal telomere-associated protein (TapL, encoded by tapL) binds to the palindromes II/III then to recruit telomere terminal protein (TpgL, encoded by tpgL) (1, 2). Neither tapL nor tpgL homologous genes are carried by the linear plasmids pSLA2 and pSCL1 (1, 2), and the mechanism of recruitment or activation of these chromosomal telomere proteins for plasmid telomere replication is unknown.

The centrally located loci of Streptomyces linear plasmids can also maintain propagation in circular mode when the telomeres are deleted (5, 8, 22, 25). The centrally located locus for replication of linear plasmid pSLA2 is composed of the iterons located within the essential genes rep1 (encoding DNA-binding protein) and rep2 (DNA helicase) (6). Experimental evidence shows that the replication origin of linear SCP1 plasmids contains a rep2pSLA2-like gene and its adjacent regions contain different iteron sequences (22). Similar loci are also indicated in linear plasmids pSCL1 and SLP2 (11, 27). The extent of functional similarity of these individual replicating components, and consequently the extent to which mechanisms of replication are similar among linear plasmids, is not known.

The minimal locus required for maintaining the replication of pSLA2 in circular mode cannot allow its propagation in linear mode unless it also contains a new plasmid locus, rlrApSLA2 (required for linear replication) (21). Plasmids containing rlrApSLA2 are detrimental for propagation in circular mode, the effect of which can be reversed by an adjacent and divergently transcribed locus, rorA pSLA2 (rlrA override), which resembles korA (kilA override) of Streptomyces circular plasmid pIJ101 (14, 26). rlrApSLA2 and rorApSLA2 increase inheritance and copy number of pSLA2 circular plasmids, suggesting that they may affect the origin locus (e.g., iterons) (21). Although rorApSLA2-homologous genes are found in linear plasmids SCP1, pSLA2-L, and SLP2, no rlrApSLA2-homologous loci of the plasmids are found (3, 11, 18), suggesting that they carry a distinct locus that enables replication in linear mode.

SLP2 is a large (50,410-bp) linear plasmid of Streptomyces lividans (7, 10, 11). Here we report that the basic locus of SLP2 for replication in circular mode consists of the iteronsSLP2 and repSLP2, while efficient replication requires an additional sequence upstream of the iteronsSLP2. Combination of the iterons and rep genes of linear plasmids SLP2, pSLA2, and SCP1 enables propagation in Streptomyces. Replication of SLP2 in linear mode required both the SLP2-borne genes mtapSLP2 and either tpgSLP2 or ilrASLP2, while interactions between these SLP2 proteins and the S. lividans chromosomal telomeric proteins were also detected. These results suggest that structurally distinct linear plasmids of different Streptomyces species have highly conserved replication modes and functions.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and general methods. Escherichia coli strain DH5{alpha} (Life Technologies, Inc.) and plasmid pSP72 (Promega) or pBluescript KS (Strategene) were used as cloning host and vector. E. coli plasmid isolation, transformation, and PCR amplification followed the methods of Sambrook et al. (24). Streptomyces lividans ZX7 was the host for propagating plasmids, and S. lividans strains 1326 and TK20 were the native hosts for linear plasmid SLP2 (15). Streptomyces culture, plasmid isolation, preparation of protoplasts, and transformation followed the methods of Kieser et al. (15). To compare the transformation frequencies of plasmids at different times in the experiments, we used 0.1 ng plasmid DNA of pIJ702 as a control each time.

Cloning, sequencing, and analysis of the DNA fragment of the SLP2 portion of pQC542. The 0.8-kb SLP2 telomeres from pLUS450 (containing the 2.6-kb chromosomal telomeres; kindly provided by Carton Chen) were cloned into pSP72 to yield pQC154. pQC177 was obtained by ligating three DNA fragments: a 3.3-kb fragment of XbaI/HpaI-cleaved pQC154; a 1.2-kb fragment of XbaI/SspI-cleaved pQC154; and a 2.6-kb fragment of XbaI-cleaved pQC98 (containing the melC/tsr genes). The restriction endonuclease BclI-linearized pQC177 DNA was ligated with the isoschizomer Sau3AI-digested partially genomic DNA of S. lividans 1326 (harboring plasmids SLP2 and SLP3 [10]) and transformed into a plasmid-free host, S. lividans ZX7. An ~30-kb plasmid DNA band from one transformant was detected, and restriction endonuclease analysis indicated that it contained an ~23-kb portion of SLP2 (15). The SLP2 portion of plasmid pQC542 DNA was sequenced via a "shotgun" strategy by the Chinese Human Genome Center in Shanghai. Streptomyces open reading frames (ORFs) were predicted (4, 13; see also the website http://watson.nih.go.jp/~jun/cgi-bin/frameplot-3.0b.pl).

RT-PCR assays of the expressed RNAs in Streptomyces. Total RNA of Streptomyces sp. strain TK20 (harboring plasmid SLP2) was prepared by the standard procedure (15). Approximately 1 µg of RNA was reversely transcribed into cDNA by using the RevertAid first-strand cDNA synthesis kit (MBI Fermentas). Then, 2 µl of product was subjected to PCR amplification. The pair of primers for reverse transcription-PCR (RT-PCR) amplification of the predicted mtap was 5'-CTGACTCATATGGAGCTAGCCTTGTCTGACCT-3' and 5'-CTGACTGAATTCGCTACTCCACTTCGTCTCCG-3'. PCR conditions were 94°C for 3 min and then 94°C for 35 s, 63°C for 50 s, and 72°C for 1 min for 30 cycles.

Construction of plasmids containing various pQC542 fragments for linear DNA replication. Plasmids pQC654, pQC682, and pQC684 were obtained by deletions of the 7.7-kb MscI, 7.5-kb MluI, and 4.9-kb NheI fragments of pQC542, respectively. Plasmids pQC656 and pQC653 were obtained by ligating the 15.8-kb AscI and 7.7-kb MluI fragments with pQC177. Deletions of the 7.5-kb MluI fragment of pQC656 and the 0.36-kb NheI fragment of pQC653 were performed to obtain pQC704 and pQC730, respectively. Further deletions of the 1.6-kb StuI-HindIII fragments of pQC653 and pQC730 were made to obtain pQC699 and pQC742, respectively. Plasmids pQC546 and pXQ25 were obtained by ligating the 3.7-kb Sau3A fragment and the 3.0-kb PCR-amplified fragment (containing ORFs pQC542.19 and pQC542.20) of pQC542 with pQC177 treated with BclI, respectively. Plasmids pQC709, pQC734, and pQC743 were obtained by ligation of the 1.1-kb NheI fragment of pQC542 with pQC546 treated with XbaI, with pXQ25 treated with XbaI, and deletion of a 0.36-kb NheI fragment of pQC709, respectively. These plasmid DNAs were isolated from E. coli, linearized by DraI, and introduced by transformation into Streptomyces sp. strain ZX7.

Yeast two-hybrid assays for detecting interactions between SLP2 proteins and chromosomal telomeric proteins. The four SLP2-borne replication genes (ilrASLP2, mtapSLP2, tpgSLP2, and repSLP2) and two chromosomal telomeric genes (tapL and tpgL) were PCR amplified individually and cloned into both the DNA-binding domain of "bait" vector pGBKT7 and into the GAL4 activation domain of "target" vector pGADT7. The pairs of bait and target plasmids were cointroduced by transformation into the recipient yeast strain AH109. Yeast cells were streaked on SD medium lacking leucine and tryptophan (SD/-Leu/-Trp) and SD medium lacking leucine, tryptophan, and histidine but containing 3 mM 3-amino-1,2,4-triazole (SD/-Leu/-Trp/-His/+3AT). Colonies grown on SD/-Leu/-Trp/-His/+3AT were inoculated into the corresponding liquid broth to determine ß-galactosidase activity as described in the protocols supplied by Clontech, Inc.

Electrophoretic mobility shift assays (EMSA). The ilrASLP2 gene was cloned into the NdeI and HindIII sites of E. coli plasmid pET28b (Novagen) to obtain pXQ165 and introduced into E. coli strain BL21(DE3) containing plasmid pQC630 (Z. Qin and S. N. Cohen, unpublished), which was constructed by ligating pACYC184 with the tRNA genes for the rarely used arginine and proline codons. The overexpressed IlrASLP2 protein was purified by Ni2+ column chromatography (QIAGEN). The 249-bp DNA sequence containing the iteronsSLP2 was amplified by PCR with a pair of primers (5'-AGGTAACGGCGAAGCAAAAC-3' and 5'-GGGGTAGGGGAGGAGGAATC-3') and inserted into the EcoRV site of pBluescript KS to construct pXQ160. The 249-bp DNA was released by treatment of pXQ160 with EcoRI and XhoI and end labeled with [{alpha}-32P]dCTP by using DNA polymerase Klenow fragment. The DNA-binding reaction was performed at room temperature for 30 min in buffer (10 mM Tris at pH 7.5, 25 mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol, 50 µg/ml bovine serum albumin, and 5% glycerol). The reaction complexes were separated on prerun 5% native acrylamide gels in 0.5x Tris-borate-EDTA buffer at 150 V for 2 h. The gel was dried and exposed to X-ray film at –80°C.

Nucleotide sequence accession number. The GenBank accession number of the SLP2 portion of pQC542 is DQ410885.


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RESULTS
 
Identification of SLP2 loci for basic and efficient replication in circular mode. The complete nucleotide sequence of SLP2 reveals a locus containing the "three pairs of long repeats" (iteronsSLP2 from bp 7896 to 8080) and an adjacent gene, SLP2.8 (designated repSLP2), encoding DNA helicase (11). To investigate the capacity for replication of this locus in Streptomyces, we employed pQC156 (21), a derivative of E. coli plasmid pSP72 bearing the Streptomyces melC/tsr markers, to clone various lengths of SLP2 fragments containing the locus. The resulting plasmid DNAs were introduced by transformation into Streptomyces lividans ZX7 (using 0.1 ng plasmid pIJ702 DNA as a control each time). As shown in Fig. 1, pZR245 containing the iteronsSLP2 and repSLP2 was able to propagate in ZX7, although the transformation frequency was very low. When plasmids (e.g., pXQ20, pXQ57, and pXQ55) contained the 47-bp sequence between bp 581 and 628 upstream of the iteronsSLP2, the transformation frequency increased ~1,000 times compared to that of pXQ58, indicating that this sequence affected the efficiency of replication. Like plasmid pSLA2 (21), plasmid (e.g., pXQ20) containing the basic locus of replication propagated in strain ZX7 at a low copy number (data not shown) and was inherited unstably (~1%).


Figure 1
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  		FIG. 1. Identification of sequence requirements for SLP2 replication in circular mode. Plasmids pXQ20, pXQ21, pXQ53, pXQ55, pXQ57, pXQ58, and pZR245 were obtained by ligating pQC156 with specific PCR-amplified fragments of the SLP2 origin. Nucleotides of the fragments are indicated by numbers, starting from the first nucleotide (8080) of the SLP2 iterons; the upstream nucleotides are shown as negative numbers. The iteronsSLP2 are indicated by open boxes, and the direction of transcription is shown by arrowheads. Plasmid DNAs were isolated from E. coli and introduced by transformation into strain ZX7. Transformation frequencies are shown.

Combinations of the rep genes and iterons of the origins of Streptomyces linear plasmids SLP2, pSLA2, and SCP1 still propagate. As seen in Fig. 2, reversing the native orientation of the iteronsSLP2 and repSLP2 (e.g., pXQ153) did not affect transformation, indicating that the two components could be separated. To investigate whether combinations of these similar components from Streptomyces linear plasmids pSLA2, SCP1, and SLP2 were functional, the rep1 (containing ~0.3-kb iterons) and rep2 genes of pSLA2, the iterons (plus the 491-bp upstream region) and rep of SCP1, and the iterons (plus the 628-bp upstream region) and rep of SLP2 were amplified individually by PCR and then paired and cloned into pQC156. The resulting plasmids were introduced by transformation into strain ZX7, as shown in Fig. 2; they were able to propagate in ZX7, although the transformation frequencies were ~1,000 times lower than that of pXQ20 (containing the native SLP2 origin).


Figure 2
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  		FIG. 2. Combination of the origin components of plasmids SLP2, pSLA2, and SCP1 for replication. The PCR-amplified individual fragments (iterons or reps of SLP2, pSLA2, and SCP1) (see text) were digested with restriction enzymes HindIII (abbreviated as Hi) and XbaI (Xb). These fragments were ligated into pQC156. The resulting plasmid DNAs were isolated from E. coli and introduced by transformation into strain ZX7. Transformation frequencies are shown. The iterons are indicated by open boxes, and the direction of transcription is shown by arrowheads.

Construction and characterization of SLP2-derived linear plasmid pQC542. The Streptomyces large linear plasmid SLP2 is only detected by using pulsed-field gel electrophoresis (7). To investigate its essential function of replication in linear mode, we employed plasmid pQC177 (Fig. 3A), which contained SLP2 telomeres and the melC/tsr markers, to clone ~23-kb SLP2 in Streptomyces to yield plasmid pQC542 (Fig. 3A) (see Materials and Methods). DraI-linearized (removing an ~0.7-kb E. coli fragment) pQC542 DNA was introduced into strain ZX7, and a ~27-kb band from ZX7 transformants was detected on the gel. It was sensitive to treatment with E. coli exonuclease III but resistant to {lambda} exonuclease (Fig. 3B), suggesting that pQC542 can propagate in linear mode in strain ZX7.


Figure 3
Figure 3
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  		FIG.3. Identification of SLP2-borne loci required for linear plasmid replication. (A) Schematic maps of pQC177 and pQC542. See Materials and Methods for details on construction of the plasmids. The telomeres are indicated by open arrowheads. The ~23-kb SLP2 fragment of pQC542 is indicated by a bold arc. (B and E) Determination of the linearity of pQC542 and pQC709. DraI-linearized plasmid DNA was introduced by transformation into Streptomyces. Native chromosomal and plasmid DNA was isolated from ZX7 transformants by a nondenaturing method (20) and electrophoresed in a 0.5% agarose gel for 16 h. Aliquots of the DNA were treated with Tris-EDTA, 10 units of {lambda} exonuclease, or 100 units of E. coli exonuclease III. Linear plasmid DNA bands of ~27 kb and ~9 kb are shown. Chr, chromosome; L, linear plasmid. (C) RT-PCR examination of transcriptional activity of the predicted mtap gene of pQC542. PCR amplifications of the TK20 RNAs, DNA, and cDNA by using primers for mtapSLP2 were performed (see Materials and Methods) and electrophoresed in a 1.5% agarose gel at 100 V for 2 h. (D) Identification of SLP2-borne loci for linear plasmid replication. Plasmids containing various fragments of pQC542 were constructed (see Materials and Methods) and introduced by transformation into ZX7. The iteronsSLP2 are indicated by a dotted box, and repSLP2, ilrASLP2, tpgSLP2, and mtapSLP2 are indicated by filled boxes. Abbreviations: As, AscI; Ms, MscI; Ml, MluI; Nh, NheI; St, StuI; Hi, HindIII. (F) Positions of the replication loci on SLP2 and pQC542. The slash mark indicates the unpredicted mini-ORF (mtap) of SLP2 reported by Huang et al. (11). The iterons are indicated by open boxes, and the direction of transcription is shown by arrowheads.

The SLP2 portion of pQC542 was sequenced independently and consisted of 22,816 bp—highly resembling the corresponding sequence from nucleotides 2573 to 25674 in the 50,410-bp complete nucleotide sequence of plasmid SLP2 published by Huang et al. (11), except there were five single-nucleotide polymorphisms and SLP2 had an additional 287-bp sequence (from nucleotides 3680 to 3967) which was missing in pQC542. An unpredicted mini-ORF from nucleotides 8794 to 9057 in SLP2 (Fig. 3F) encoded a small protein of 88 amino acids, resembling (48% identity; expectation value of 2 x 10–10) the N-terminal fragment of the S. lividans chromosomal telomere-associated protein (TapL, 739 amino acids) (2). This ORF, verified to be transcribed in vivo by RT-PCR examination (Fig. 3C) (see Materials and Methods), was designated mtapSLP2 (mini-tap).

In addition to the origin, the SLP2-borne genes mtapSLP2 and either tpgSLP2 or ilrASLP2 are required for plasmid replication in linear mode. To define the loci for linear plasmid replication, various lengths of the pQC542 fragment were cloned into pQC177, and the resulting plasmids (see Materials and Methods) were DraI linearized and introduced into ZX7. As shown in Fig. 3D, the linearized plasmids pQC709, containing the additional loci mtapSLP2 and SLP2.13 (designated ilrASLP2, for involved in linear SLP2 replication), and pQC699, containing the other additional loci (mtapSLP2 and tpgSLP2), were able to propagate in ZX7. The linear conformations of pQC709 (Fig. 3E) and pQC699 (data not shown) in ZX7 were verified. These results indicated that two alternative sets of loci, mtapSLP2/ilrASLP2 and mtapSLP2/tpgSLP2 (Fig. 3F), were required for SLP2 replication in linear mode.

IlrASLP2 binds specifically to the iteronsSLP2 in vitro. Like the organization of rlrA-rorA of plasmid pSLA2, the rorASLP2-adjacent and divergently transcribed gene was ilrASLP2. It was suggested that the action of the RlrA protein of plasmid pSLA2 might be at the plasmid iterons’ structure (21). To investigate if there was an interaction between the IlrA protein of SLP2 and the iteronsSLP2 sequence, electrophoretic mobility shift assays for DNA-protein complex formation were employed. Purified IlrASLP2 protein was incubated with [{alpha}32-P]dCTP-labeled 249-bp DNA of the core sequence of iteronsSLP2 and then electrophoresed and autoradiographed. As shown in Fig. 4, the IlrASLP2 protein could bind to the DNA probe to form a DNA-protein complex. Formation of this complex was competed by adding 10x or 25x unlabeled probe DNA but was unaffected by the addition of 100x nonspecific DNA, suggesting that the binding reaction of the IlrASLP2 protein and iteronsSLP2 DNA was specific.


Figure 4
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  		FIG. 4. Detection of the binding activity of the IlrASLP2 protein with iteronsSLP2 DNA. EMSA were employed to detect DNA-binding activity between purified IlrASLP2 protein and the 249-bp DNA-containing iteronsSLP2 (see Materials and Methods). For concentrations of protein, "1" equals 1.4 x 10–6 nmol; for concentrations of DNA probe, referring to the concentration of specific competitor, "1" equals 1.3 x 10–4 nmol; the concentration of free DNA probe for each lane was 1.3 x 10–4 nmol; for concentrations of nonspecific competitor (salmon sperm DNA), "1" equals 20 ng. DNA-protein complexes are indicated.

Detection of interactions of SLP2 replication proteins and chromosomal telomeric proteins. To investigate if there were interactions of SLP2-borne replication proteins and chromosomal telomeric proteins, yeast two-hybrid assays were employed. The four SLP2 genes (ilrASLP2, mtapSLP2, tpgSLP2, and repSLP2) and two chromosomal genes (tapL and tpgL) were PCR amplified individually to use in construction of both the bait and target plasmids and were cotransformed into yeast (see Materials and Methods). As shown in Table 1, some protein-protein interactions, including IlrASLP2 with TapL, TpgSLP2 with TapL, and MtapSLP2 with TpgL, were detected. Unlike chromosomal TapL and TpgL (2), no interactions were observed between SLP2-borne replication proteins MtapSLP2 and TpgSLP2. Interaction of the telomeric proteins TapL and TpgL is required for telomere replication (2), suggesting that the chromosomal TpgL and TapL may be involved in replication of plasmid SLP2 telomeres.


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  		TABLE 1. Detection of protein-protein interactions among SLP2 replication proteins and chromosome telomeric proteins by yeast two-hybrid assay


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DISCUSSION
 
Previous experiments showed that the centrally located loci for replication of Streptomyces linear plasmids pSLA2, SCP1, and pSLA2-L in circular mode consisted of the different iterons and the rep genes (6, 8, 22). Huang et al. (11) proposed that the replication loci of SLP2 containing tpgSLP2, repSLP2 (DNA helicase), and the complex repeat sequences (iteronsSLP2) are at kb 7.8 to 8.1 of SLP2. Here we show that the basic SLP2 locus for circular plasmid replication consisted of iteronsSLP2 and the adjacent repSLP2 gene. We found that plasmids containing combinations of the iterons and rep genes of SLP2, pSLA2, and SCP1 still propagate in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that the mechanisms of initiating replication are similar and implying that interaction of replication proteins with the origins (e.g., iterons) does not depend strictly on the DNA sequences.

We found that that in order to replicate efficiently, the 47-bp sequence between bp 581 and 628 upstream of the iteronsSLP2 is required. This sequence, along with the iteronsSLP2, may be embedded in a putative ORF (pQC542.19, from bp 8673 to 7891 of SLP2, encoding a hypothetical protein of 443 amino acids). However, the RT-PCR experiment showed that this region was not transcribed (unpublished data). Thus, the 581 to 628 bp upstream of the iteronsSLP2 may contain cis-acting regulatory elements, rather than a gene, for SLP2 replication.

The minimal origin of replication of pSLA2 in circular mode cannot propagate in linear mode when the telomeres are attached; a plasmid-borne gene, rlrApSLA2, is required for linear DNA replication (21). The detrimental effect of rlrApSLA2 on maintenance of circular plasmids can be reversed by its adjacent and divergently transcribed rorApSLA2 gene (21). Although no sequence similarity to rlrApSLA2 was observed, ilrASLP2 (SLP2.13), which we identified here as being involved in linear replication, is significantly similar to unknown genes of linear plasmids SCP1 and SAP1 (28% identity with SCP1.92 and 31% identity with SAP1.39c [3, 12]). Interestingly, all have adjacent and divergently transcribed genes, i.e., SLP2.12c, SCP1.91c, and SAP1.38, encoding proteins resembling RorApSLA2 (40%, 27%, and 42% identity, respectively), suggesting that they may have similar functions in linear plasmid replication.

rlrApSLA2 and rorApSLA2 can also maintain plasmids to propagate in high copy number, suggesting that they may act at the origin (e.g., iterons) (21). Our data show that the IlrASLP2 protein binds specifically to iteronsSLP2, supporting the suggestion of action of RlrApSLA2. Unlike rlrApSLA2, ilrASLP2 alone cannot enable plasmids containing the SLP2 origin and telomeres to replicate in linear mode; instead, either of the pairs ilrASLP2/mtapSLP2 and mtapSLP2/tpgSLP2 is required, indicating two alternative pairs of SLP2 genes involved in linear plasmid replication.

To initiate chromosomal telomere replication, the chromosomal telomeric protein TapL recruits TpgL and then binds to the single-stranded telomere DNA (2), suggesting that interaction of TapL and TpgL is crucial for telomere replication. Our results (Table 1) show interactions between the plasmid replication proteins MtapSLP2 and TpgSLP2 and chromosomal telomere proteins TpgL and TapL, respectively, but no interaction between plasmid MtapSLP2 and TpgSLP2 is detected, indicating that MtapSLP2 and TpgSLP2 might not accomplish SLP2 telomere replication by themselves. In other words, although the plasmid TpgSLP2 and chromosomal TpgL display high homology (70% identity; expectation value, 1 x 10–48) and are of similar size, chromosomal TpgL, not plasmid TpgSLP2, together with chromosomal TapL may function at replication of the SLP2 telomere. The replication at the SLP2 telomere may require SLP2-borne proteins to activate or recruit chromosomal TpgL/TapL proteins.


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ACKNOWLEDGMENTS
 
We thank Stanley N. Cohen, in whose laboratory these experiments were initiated, for support and suggestions, David Hopwood for Streptomyces lividans strains, and Carton Chen for the plasmid pLUS450 containing a chromosomal telomere. We are also grateful to David Hopwood, Stanley Cohen, and Christine Miller for critical reading and useful suggestions on the manuscript.

These investigations were supported by National Institutes of Health grant AI08619 to S.N.C. and by grants from the National and Shanghai Nature Science Foundation of China (30170019, 30270030, 30325003, and 0202ZA14096), the Chinese Academy of Sciences project KSCX2-SW-329-3, and National "863" projects (2005AA227020) to Z.Q.


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FOOTNOTES
 
* Corresponding author. Mailing address: Shanghai Institute of Plant Physiology, Shanghai Institutes of Biological Sciences, The Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032 People's Republic of China. Phone: 86 (21) 54924171. Fax: 86 (21) 54924171. E-mail for Z. Qin: qin{at}sibs.ac.cn. E-mail for G. Zhao:gpzhao{at}sibs.ac.cn. Back


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Journal of Bacteriology, October 2006, p. 6851-6857, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00873-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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