Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence

Iris Haug1, Anke Weissenborn2, Dirk Brolle3, Stephen Bentley4, Tobias Kieser5 and Josef Altenbuchner1

1 Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
2 Mikrobiologie/Biotechnologie, Eberhard-Karls-Unversität Tübingen, 72076 Tübingen, Germany
3 Team Leader Marketing Urology, Pfizer GmbH, PO Box 4949, 76032 Karlsruhe, Germany
4 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
5 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Correspondence
Josef Altenbuchner
Josef.Altenbuchner{at}po.uni-stuttgart.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the {lambda} integrase family of site-specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1·6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix–turn–helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcriptional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome.


The GenBank accession numbers for the DNA sequences reported in this paper are AL645771 and NC_003904.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomycetes are Gram-positive soil bacteria that are well known for the production of a huge variety of secondary bioactive metabolites, such as antibiotics, and for their complex life-cycle. Many plasmids have been found in Streptomyces spp. Most of them are self-transmissible fertility factors. Both linear and circular, low- and high-copy-number plasmids occur and some plasmids are integrated into the chromosomes. The first circular plasmid identified in Streptomyces coelicolor A3(2) was SCP2 (Schrempf et al., 1975), a conjugative low-copy-number plasmid of 31 kb. It is present in one to four copies per chromosome (Bibb et al., 1977) and is stably inherited, being retained by 99·5 % of spores after a single spore-to-spore cycle (Bibb et al., 1980).

SCP2*, a spontaneous derivative of SCP2, was first analysed by Bibb & Hopwood (1981). It has a 1000-fold increased chromosome-mobilizing ability and forms more clearly visible pocks compared to SCP2. Pocks are an indicator for the presence of plasmids with DNA transfer ability and can be seen as a region of delayed differentiation when a plasmid-bearing spore germinates in a lawn of a strain lacking the same plasmid. Spontaneous conversion from SCP2 to SCP2* and vice versa has been observed (Bibb et al., 1977). The two forms of the plasmid cannot be distinguished by restriction digestion and the molecular basis of the different phenotypes is unknown.

Three functional regions on SCP2 were identified: the transfer region, the stability region and the replication region. The transfer region was first studied by Lydiate et al. (1985), a minimal region for replication of 1·4 kb was identified by Larson & Hersherger (1986) and the stability functions were located on a 4·9 kb BamHI–SacI fragment (Bibb et al., 1980; Lydiate et al., 1985).

SCP2 and SCP2* are valuable cloning vectors because of their stability, low copy number and ability to accept large cloned fragments of exogenous DNA, including whole gene clusters as for, for example, the actinorhodin biosynthesis pathway (Malpartida & Hopwood, 1984). Here we report the complete sequence of SCP2*. The knowledge gained from the sequence data may be a first step to a better understanding of the replication, stability and transfer functions of this important low-copy-number circular plasmid and to the construction of improved SCP2-derived cloning vectors.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, vectors, media and growth conditions.
Escherichia coli JM109 (Yanisch-Perron et al., 1985) was the usual host for cloning experiments, plasmid constructions and plasmid DNA preparations. For transposon mutagenesis the strains E. coli JM108 F' lacI proAB+[ : : Tn5491] (Fischer et al., 1996) and E. coli HB101 (Boyer & Rouland-Dussoix, 1969) were used. Replication and plasmid stability of SCP2-derived plasmids were tested in Streptomyces lividans TK64 (Hopwood et al., 1983). SCP2* was isolated from S. coelicolor M107 (Bibb et al., 1977). The vector pJOE803 (Altenbuchner & Eichenseer, 1991) contains the thiostrepton-resistance gene from Streptomyces azureus (Thompson et al., 1980) inserted into the NruI site of pIC20H (Marsh et al., 1984). The positive selection vector pJOE2114 used for transposon mutagenesis and deletion formation is a pIC20R derivative described by Fischer et al. (1996). The cloning vector pDS100 contains a ThaI fragment with the lacZ{alpha}-fragment of pUC19 inserted between the BamHI and HindIII sites of pACYC177. E. coli strains were grown at 37 °C in L-broth or on Luria–Bertani (LB) agar plates supplemented with the appropriate antibiotics (ampicillin, 100 µg ml-1; streptomycin, 200 µg ml-1). S. lividans was grown at 30 °C on Hickey–Tresner (HT) agar plates (Pridham et al., 1957). Thiostrepton (kindly provided by Aventis) was used in agar plates at a concentration of 50 µg ml-1 and in liquid culture at 15 µg ml-1. For transformation, S. lividans was grown in YEME liquid medium and the protoplasts were regenerated on R2YE agar plates as described by Kieser et al. (2000). Transformants were selected by overlaying the plates after 12 h with 3 ml R2YE soft agar containing 400 µg ml-1 thiostrepton.

DNA manipulations.
For DNA manipulations, including DNA restriction, ligation, agarose gel electrophoresis, fragment isolation from gels, transformation of E. coli and plasmid isolation, standard methods were used as described in Sambrook et al. (1989) and Kieser et al. (2000). Nested deletions in SCP2 restriction fragments for subcloning and for DNA sequencing were generated by transposon mutagenesis using the Tn1721-derived Tn5491 as described by Fischer et al. (1996). In principle, fragments of interest were inserted into pJOE2114 and the plasmids brought into JM108 F'lacI proAB+[ : : Tn5491]. The transformants were mated with HB101 at 30 °C in liquid culture and the bacteria plated on LB agar plates containing ampicillin and streptomycin. This selected the transfer of the non-mobilizable pJOE2114 derivates into HB101 via insertion of Tn5491 into pJOE2114 and cointegrate formation. Because of the EcoRI sites in the vector and inverted repeats of the transposon and a universal primer site at one end of the transposon, plasmids resolved from the cointegrate could be used directly for DNA sequencing or for subcloning.

DNA sequencing and annotation.
DNA sequences were obtained from a Pharmacia ALF (automated laser fluorescence sequencer), using the AutoRead Sequencing Kit and the Repro Gel Long System from Amersham Pharmacia Biotech and oligonucleotides from MWG Biotech. The sequences were aligned with LASERGENE 99 software (DNASTAR). The sequence was annotated as described previously (Cole et al., 1998), using ARTEMIS (Rutherford et al., 2000) to collate data and facilitate annotation.

Determination of plasmid stability.
The plasmids were brought into S. lividans TK64 and transformants were selected with thiostrepton. After sporulation on the R2YE regeneration plates, spores were harvested and serial dilutions plated on HT agar plates without antibiotic selection to obtain single colonies at 30 °C. After sporulation, the colonies were replica-plated to HT agar plates with and without antibiotic and the percentage of antibiotic-sensitive colonies was determined (first generation of spores). A second round of growth and sporulation without antibiotic was achieved by plating 107 spores from the regeneration plate first on HT agar plates without antibiotic and after harvest of spores in serial dilutions on HT agar plates as described above (second generation of spores).

Vector construction for plasmid stability testing.
The vector pFIS86 was constructed by inserting the SCP2* replication region (SalI/XhoI fragment) from pEI16 (Volff et al., 1996) as a PstI/BglII fragment between the PstI and BamHI sites of pJOE803 (Altenbuchner & Eichenseer, 1991). The complete SCP2* plasmid was cleaved with EcoRI and inserted into the EcoRI site of the E. coli plasmid pDS100 to give pAWE1. Part of the stability region (mrpA, parA) was obtained from pAWE1 as a BglII/SacI fragment and inserted between the EcoRV sites of pJOE2114 to give pJOE2950. The new plasmid was used for transposon mutagenesis as described and EcoRI fragments containing mrpA and/or parA were inserted into the EcoRI site of pFIS86 to give pFIS87, pFIS88 and pFIS112, respectively. The complete SCP2* stability region was obtained from pAWE1 as a BamHI/SacI fragment and again inserted between the EcoRV sites of pJOE2114 to give pFIS120. The genes mrpA, parA and parB together were obtained from pFIS120 as an EcoRI fragment and inserted into pFIS86 to give pFIS123. The fragment containing parA and parB alone was obtained from pFIS120 as an AscI/EcoRI fragment, inserted blunt-ended into the SmaI site of pIC20R and from there as an EcoRI fragment into pFIS86 to give pFIS209. The parS sequence from S. coelicolor was synthesized as a self-complementary oligonucleotide (S3386: 5'-GATCGTTTCACGTGAAAC-3') and inserted into the BamHI site of pFIS86 (pFIS226) and pFIS87 (pFIS221). Before transformation of S. lividans the plasmids were cut with HindIII and ligated without pIC vector, as shown in Fig. 2, to give pFIS96 (pFIS86), pFIS97 (pFIS87), pFIS98 (pFIS88), pFIS127 (pFIS112), pFIS128 (pFIS123), pFIS212 (pFIS209), pFIS226 (pFIS220) and pFIS227 (pFIS221).



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Fig. 2. Construction of plasmids to identify stability functions. Top: the construction of pFIS96 from pFIS86 via deletion of the E. coli vector is shown. The fragments below contain the stability region with various deletions (dotted lines). These fragments were all in the same orientation inserted into pFIS86 and used to transform S. lividans after deletion of the E. coli vector DNA, as shown for pFIS86.

 
Vector constructions to determine the minimal replication region.
Plasmid pJOE4038 contains the stability region of SCP2* which was obtained from pAWE1 as a BamHI/SphI fragment and inserted between the BamHI and SphI sites of pJOE803. The replication region of SCP2* was isolated from pAWE1 as an EcoRI/SalI fragment and inserted between the EcoRV sites of pJOE2114. After transposon mutagenesis, the replication region, now containing deletions on one or the other side of the EcoRI–SalI fragment was isolated again as an EcoRI fragment and inserted into the EcoRI site of pJOE4038. In addition, the minimal replication region was amplified from pAWE1 using the primer pairs S3241/S3242 (pJOE4132), S3241/S3244 (pJOE4133) and S3242/S3243 (pJOE4134), inserted into the pCR-BluntII-TOPO-vector (Invitrogen) and from there as an EcoRI fragment into the EcoRI site of pJOE4038 (primer S3241, 5'-AAAAGGATCCTGACCGTCTGGCACATAG-3'; S3242, 5'-AAAAGGATCCTCAGCCACAGAAAGGCA-3'; S3243, 5'-AAAAGGATCCTTCTACGGGCATGGTCG-3'; S3424, 5'-AAAAGGATCCGTAGAAGCTAAGAGCGG-3').


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
DNA sequence analysis of SCP2*
SCP2* was isolated from S. coelicolor M107, cleaved with EcoRI and inserted into the EcoRI site of the E. coli plasmid pDS100 to give pAWE1. From pAWE1, various overlapping restriction fragments were subcloned into pJOE2114 and sequenced using primer-walking and transposon-tagging strategies. The fragments were assembled to a continuous DNA sequence of 31 317 bp. It should be noted that parts of SCP2* had already been sequenced. These were the region containing Tn5714 (GenBank accession no. AF395846), kindly provided by J. McCormick for comparison, the traA gene essential for conjugation of SCP2* (accession no. X72857 and a corrected version in X90546) and the minimal region necessary for replication of SCP2* (accession no. E01115 and an extended version in AJ414671). The entire sequence (accession no. AL645771) was annotated using the program ARTEMIS (Rutherford et al., 2000). In this way, 34 ORFs were identified, most of them encoding proteins of unknown function. A physical map of SCP2* is shown in Fig. 1. According to this map, 27 ORFs are in the anticlockwise orientation and seven are in the clockwise orientation. The features of the ORFs are listed in Table 1.



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Fig. 1. Restriction map of SCP2*. The replication, transfer and stability functions as well as the two transposable elements are indicated by open boxes. Identified ORFs are shown as arrows. Open arrows indicate genes with unknown functions, and black arrows genes with putative or proven functions in replication, transfer, stability or transposition. The scale on the map corresponds to 5 kb between divisions. For SalI and SphI (in parentheses), only the restrictions sites relevant for plasmid constructions are shown.

 

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Table 1. Properties of the ORFs found on SCP2*

 
Transposable elements on SCP2*
Two transposable elements were identified on SCP2* by DNA sequence analysis. IS1648 was first identified on the chromosome of S. coelicolor M145, where it is present in three copies (Bentley et al., 2002). IS1648 is bounded by 32 bp imperfect inverted repeats. No flanking direct repeats are present in the SCP2* copy. IS1648 belongs to the IS3 family controlled by programmed ribosomal frameshifting (Mahillon & Chandler, 1998). Indeed, there are two genes, SCP2.01 and SCP2.02, encoded by IS1648 with a Pfam match to Transposase 6 family proteins. The second ORF is in a -1 phase to the first ORF and the first ORF contains, at its C-terminal end, the heptanucleotide AAAAAAG representing the typical slippery codons for translational shifting.

There are two copies of Tn5714 present in S. coelicolor A3(2): one is on the linear plasmid, SCP1 (accession no. NC_003903) and one is on SCP2. The transposon has a length of 3512 bp. It is bounded by 28 bp imperfect inverted repeats and flanked by a 3 bp duplicated target sequence. The putative gene products encoded by the three genes SCP2.10c, SCP2.11c and SCP2.12c of Tn5714 are most similar to those of S. lividans Tn4811 (Chen et al., 1992). SCP2.10c shows Pfam matches to the Transposase 13 and Transposase 6 families and presumably is the transposase of Tn5714. The function of the two other ORFs is unknown. Tn5714 is flanked by the ORFs SCP2.09c and SCP2.13c of unknown functions, which might have been a single gene disrupted by insertion of Tn5714.

The stability region of SCP2*
The region necessary for stable inheritance of SCP2* was originally localized on a 4·9 kb BamHI/SacI fragment (Bibb et al., 1980; Lydiate et al., 1985) lying between the two transposable elements. Three genes resembling known plasmid stability functions were identified in this fragment. SCP2.07c encodes a protein (371 aa) belonging to the {lambda} integrase family. Site-specific recombinases of the {lambda} integrase or resolvase type are known as plasmid and chromosome stability factors. They are believed to resolve plasmid and chromosome dimers or multimers arising during the replication cycle into monomers by site-specific recombination and thereby ensure a correct segregation of the molecules into daughter cells (Nordström, 1989). The second gene, SCP2.05c, encodes a protein (310 aa) similar to the parA genes involved in active partition of chromosomes and low-copy-number plasmids. All known plasmid-encoded par systems specify three components: a cis-acting centromere-like nucleotide sequence (parS) and two trans-acting proteins that bind in a complex to parS (Bignell & Thomas, 2001). The proteins are encoded by two genes in an autoregulated operon. In all cases analysed so far, the parA gene, encoding an ATPase, lies upstream in the operon. Two types of partitioning ATPases are known: the Walker-type and actin-like ATPases. SCP2.05c belongs to the Walker-type ATPases. It shares high identity in amino acid sequence with the Caulobacter crescentus (36·2 % in 257 aa) and S. coelicolor (36·1 % in 263 aa) chromosomal ParA proteins. The parB genes are less conserved. SCP2.04c encodes a 325 aa protein with low similarity to E. coli ZipA, a protein involved in septation of bacterial cells. SCP2.04c is translationally coupled to the upstream gene, SCP2.05c, by overlapping start and stop codons, and might have the function of parB. Tentatively, SCP2.07c was called mrpA, for multimer resolution protein, SCP2.05c was called parA and SCP2.04c was called parB. In addition, two small ORFs, SCP2.6 and SCP2.8, encoding putative proteins of 109 and 61 aa, were identified in the stability region. The deduced protein sequences showed no similarity to aa sequences deposited in the protein databases and were not further investigated. To measure the contribution to plasmid stability of the mrpA, parA and parB genes, a shuttle vector was constructed consisting of the E. coli vector pIC20H, a thiostrepton-resistance gene and the SCP2* replication region, giving rise to pFIS86. The putative stability genes were then inserted into pFIS86 in different combinations (Fig. 2) and the stability of the plasmids was determined in S. lividans TK64. This was done by plating spores on agar plates without antibiotic selection and replica-plating the sporulated colonies on plates containing the antibiotic. The stability of the shuttle vectors in general was very low in S. lividans. For example, with pFIS87 containing mrpA inserted into pFIS86, only 16 % of the replica-plated colonies grew under selection (data not shown). Therefore the shuttle vectors were cut with HindIII, the E. coli vector DNA was removed and the experiments repeated with the new plasmids. This improved the stability of the plasmids considerably (Table 2). Stability was highest (96 %) in the construction containing all three genes (pFIS128), and mrpA alone (pFIS97) gave around 80 % stability. Thus the mrpA function seems to have the highest impact on stability. The presence of parA alone had no effect; however, together with parB a stabilizing effect of parA could be detected (compare parA parB with parA alone, and compare mrpA parA parB with mrpA parA). This is in agreement with the role of the two genes as an active partitioning system for SCP2. A parA, parB, parS system was recently described for the S. coelicolor chromosome (Kim et al., 2000). To see if it could complement the SCP2 par system, the parS sequence of the S. coelicolor chromosome was synthesized as a self-complementary oligonucleotide and inserted into pFIS86 together with mrpA. Indeed, there seems to be some stabilization of SCP2 by parS in S. lividans which is closely related to S. coelicolor (compare pFIS227 and pFIS97). On the other hand, no effect was seen with parS alone in pFIS86 (pFIS226). In S. coelicolor, there are 18 completely conserved parS sites on the chromosome, so maybe more sites are necessary to achieve the same effect as that seen with the SCP2 parAB genes in pFIS128.


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Table 2. Stability of SCP2-derived plasmids in S. lividans TK64

 
The transfer region on SCP2*
Most Streptomyces plasmids are self-transmissible. The genetic organization of the tra genes usually is much simpler than in Gram-negative bacteria, reflecting the absence of sex pili. In pIJ101, for example, there is just one gene responsible for transfer of the plasmid between donor and recipient (traA), three ORFs for presumed transfer through septa in the recipient hyphae (spdA,B; kilB) and the regulatory genes korA and korB (Pettis & Cohen, 1994). The traA gene (SCP2.20c), essential for transfer of SCP2*, has been described by Brolle et al. (1993). There is a series of 10 ORFs upstream of, and in the same orientation as, traA (SCP2.21c–SCP2.32c) that might be involved in transfer and spreading. By transposon mutagenesis (Brolle et al., 1993) three regions were identified differing in transfer functions. Insertions in traA abolished transfer, pock formation and chromosomal mobilization. Transposition into a region of SCP2 containing SCP2.22c to SCP2.25c resulted in loss of pock formation and a low frequency of transfer and chromosomal mobilization. Insertion into a region corresponding to SCP2.27c led to the loss of the spreading function. According to the TMpredict program, the deduced protein from SCP2.27c contains four transmembrane-spanning regions and shows low similarity to the spread gene spdB2 of Streptomyces phaeochromogenes plasmid pJV1 (Servin-Gonzales et al., 1995). Upstream of SCP2.32c there is another gene in the opposite orientation belonging to the gntR transcription regulator family with a helix–turn–helix motif at the N-terminal end (aa position 15–74). The deduced protein shows significant similarity to negative regulators of plasmid transfer functions, for example TraR of pJV1 (Servin-Gonzales et al., 1995) and KorSA of Streptomyces ambofaciens pSAM2 (Hagege et al., 1993) and might be the regulatory gene of the SCP2 transfer genes. It should also be mentioned that there were two insertions between traA and the SCP2.27c region that had no effect on transfer (Brolle et al., 1993). Since the sites of insertions were mapped only by restriction enzyme analysis it is not clear if there are genes in this region without recognizable phenotypes or if the transposons were inserted into intergenic regions. Furthermore, some of the insertions may have had polar effects on transcription of traA downstream of the integration site.

The replication region of SCP2*
The work of Larson & Hershberger (1984) located the origin of SCP2* replication on a 5·9 kb EcoRI/SalI fragment. Deletion of a 1·3 kb KpnI/SalI fragment led to a 10-fold increase in plasmid copy number. The minimal replication region was narrowed down further to a 1·4 kb fragment, leading to a copy number about 1000 times higher compared to the wild-type plasmid (Larson & Hershberger, 1986). The sequence of this minimal replication region (GenBank accession no. E01115) corresponds to bp position 28 708–30 101 of SCP2. Only one complete ORF (SCP2.39c, 161 aa) could be identified in this region. The deduced protein contains a putative helix–turn–helix motif at aa positions 66–87 and shows some similarity to the ReplI protein (Hiratsu et al., 2000) of the Streptomyces rochei pSLA2-L plasmid (27 % identity in 147 aa). A second ORF (SCP2.38c, 136 aa), downstream of SCP2.39c and with the same orientation, is truncated. Interestingly, the deduced protein shows significant similarity to that of SCP2.39c (32 % identity in 118 aa overlap) and to a short region of the tetracycline repressor of class D (accession no. P09164), but no helix–turn–helix motif was found. The putative replication origin upstream of SCP2.39c shows no features such as long inverted or direct repeats or low G+C content as is sometimes found in plasmid origins (Chattoraj, 2000).

Larson & Hershberger (1986) postulated a negative replication regulator in the region between the SalI site at base pair 25 523 and SCP39c because of the increase in the copy number in deletion mutants. In this region, only one putative gene was identified with the potential to bind to DNA (SCP2.35) and which had previously been suggested as regulating the SCP2 tra functions. The experiments by Larson & Hershberger (1986) were done with plasmids containing no plasmid-stabilization functions, which might have influenced the results. Therefore the 5·9 kb EcoRI/SalI was inserted into the vector pJOE2114 to generate deletions by the transposon-tagging strategy as used for DNA sequencing. The fragments were inserted into a vector consisting of the E. coli plasmid pIC20H, a thiostrepton-resistance gene and a SacI/BamHI fragment containing the stability region. The plasmids were then used to transform S. lividans TK64. The results are shown in Fig. 3. Deletions from the SalI site up to bp position 27 927 gave a high number of transformants, as did deletions from the EcoRI site down to bp position 30 110. It was shown previously that deletion of the small 157 bp BclI fragment (bp positions 29 949–30 106) abolished replication of SCP2 (Larson & Hershberger, 1986). This defines one border of the replication origin. To see if SCP2.39c was sufficient for replication, three fragments were generated by PCR containing the regions from bp 28 478 to 30 089 (pJOE4132), from bp 28 949 to bp 30 089 (pJOE4134) and from bp 28 476 to bp 29 850 (pJOE4133). No transformants were obtained with pJOE4133, as expected, and a high number of transformants were obtained from pJOE4132, which contained the two putative genes SCP2.38c and SCP39c as well as a putative transcriptional terminator with a typical hairpin structure downstream of SCP2.38c. Very few transformants were obtained with pJOE4134 and the colonies obtained on the R2YE regeneration plate selected with thiostrepton could not be further propagated under antibiotic selection. This means that, for stable replication, both ORFs are necessary, together with a 650 bp non-coding region. Therefore, SCP2.39c and SCP2.38c were named repI and repII. Plasmids with a complete replication region were isolated from the various transformants and analysed on agarose gels. No significant difference in DNA yield was observed. To quantify the DNA, E. coli was transformed with the plasmid DNA and the number of transformants counted. The yields of DNA obtained from different colonies of one transformation and under different growth conditions differed considerably (more than 10-fold) and were as high as between different plasmids.



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Fig. 3. Identification of the SCP2 minimal replication region. Various fragments obtained by transposon mutagenesis or PCR of the SCP2* replication region were inserted into the E. coli–S. lividans shuttle vector pJOE4038 and used to transform S. lividans TK64 (Yes, high numbers of transformants; No, none, or very few, unstable transformants). The scale below the restriction map indicates the bp position of the SalI/EcoRI fragment on the SCP2 map.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
SCP2* is one of the few circular plasmids isolated from streptomycetes which is not replicating by a rolling-circle mechanism. So far, it is not known if the plasmid replicates in a uni- or bidirectional way. Presumably, the origin of replication is located in an essential, non-coding 650 bp region upstream of repI. The repII gene shows high similarity to repI at the aa sequence level and perhaps originated from repI by gene duplication but had no helix–turn–helix motif, in contrast to RepI. The minimal replication region described by Larson & Hershberger (1986) ended somewhere in the middle of the repII gene, so in their plasmid constructions a functional repII was not essential for replication. They observed ultra-high plasmid levels without repII, which might point to a possible role for RepII in controlling the activity of RepI, perhaps by protein–protein interactions. Over-replication and the additional stabilization by the SCP2 stability region of pJOE4134 lacking repII might have been lethal to the cells, in contrast to the plasmids of Larson & Hershberger (1986), which contained no stabilizing genes and perhaps were just tolerated.

Besides a coordinated replication of chromosomes and plasmids, three major stability mechanisms were identified so far for low-copy-number plasmids (Nordström, 1989). Sometimes, all three mechanisms are realized in a plasmid. One is a suicide system like the hok/sok system of plasmid R1 which kills cells that have lost the plasmids (Gerdes et al., 1990). There is no indication of such a system in SCP2*. On the other hand, the corresponding genes are usually very small and there is no common consensus DNA or protein sequence, which makes it difficult to recognize them. Another stability mechanism is the resolution of concatemers accidentally formed by RecA during replication of the plasmids. Resolution of plasmid dimer molecules by site-specific recombinases ensures that each daughter cell receives at least one copy of the plasmid. Most such recombinases found on plasmids and chromosomes belong to the {lambda} integrase family, like mrpA found on SCP2*. The third stability mechanism is the active distribution of plasmids into daughter cells by a centromere-like sequence, parS, and a pair of proteins called ParA and ParB. ParB recognizes the parS sequence and in a complex with ParA, the plasmid is bound to so far unknown structures of the cell either at the cell poles or at the mid-cell position before and at the one-quarter and three-quarter positions of the cell after replication. The parA gene of SCP2 could easily be recognized by the Walker motifs typical of ATPases in the parA gene product. The role of SCP2.4c as a parB gene, on the other hand, was only surmised from its location immediately downstream of parA and its effect on SCP2 stability. A stabilization was seen only when both genes were present, as expected from a par system. There are two further very small ORFs in the stability region, i.e. SCP2.06 and SCP2.08. It is not known if these putative genes are transcribed and if they really play any role in plasmid stability.

The plasmid-stability mechanisms in Streptomyces are important for the distribution of the plasmids into spores. It is not clear if they play any role during vegetative growth of the multinucleate substrate mycelium, since segregation into the spores is the only easy method for testing plasmid stability.

The third functional region on SCP2* is responsible for DNA transfer. The two important functions identified so far by transposon mutagenesis were the traA gene, which was already sequenced, and the spreading function located around SCP2.27c. It is not clear if any of the many other ORFs between traA and SCP2.27c and upstream of SCP2.27c play any function in plasmid transfer or chromosomal DNA mobilization. Another open question is the difference between SCP2* and SCP2 which leads to a reversible change in mobilization of chromosomal DNA. In mating experiments the conversion of SCP2 to SCP2* was found in up to 6 % of recombinants and the reversion to SCP2 in 0·8 % of strains with SCP2* (Bibb et al., 1977). This frequency is too high for point mutations. Maybe there are small deletions or DNA duplications at short direct repeats leading to the change in chromosomal mobilization, which were not seen in the restriction digests of the plasmids. Another plausible explanation would be an integration of SCP2 into the chromosome, as in the formation of E. coli Hfr strains, but unlike F there might be one copy of SCP2 integrated and additional copies still autonomously replicating. This would explain why SCP2 plasmid DNA can be isolated from cells with the SCP2* phenotype. The two transposable elements on SCP2 might play an important role in integration, either by homologous recombination between the IS1648 on SCP2 and one of the three copies on the chromosome, or by true transposition of SCP2 into the chromosome via IS1648 or Tn5714.


   ACKNOWLEDGEMENTS
 
We are grateful to Professor Ralf Mattes and Professor Wolfgang Wohlleben for helpful discussions and support, Professor D. A. Hopwood for encouragement and critical reading of the manuscript, and Dr J. McCormick for sharing unpublished sequence data.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 21 May 2002; revised 13 October 2002; accepted 17 October 2002.



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