Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Thomas J. Hosted, Tim Wang and Ann C. Horan

New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-C321-MS3600, Kenilworth, NJ 07033, USA

Correspondence
Thomas J. Hosted
thomas.hosted{at}spcorp.com


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin- and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector.


Abbreviations: ApR, apramycin resistance (-resistant); HmR, hygromycin resistance (-resistant); RCR, rolling circle replicating; TP, terminally attached protein

The GenBank/EMBL/DDBJ accession number for the sequences reported in this article is AY498874.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Linear plasmids with 5' terminally attached proteins (TPs) and terminal inverted repeats (TIRs) have been found in bacteria, plants, filamentous fungi and yeasts, and often within actinomycetes (Griffiths, 1995; Hinnebusch & Tilly, 1993; Meinhardt et al., 1990; Rohe et al., 1992). Actinomycete linear plasmids pSLA1 (17 kb) and pSLA2 (17 kb) were first detected in the lankacidin-producer Streptomyces sp. 7434-AN4 and in Streptomyces rochei, respectively (Hayakawa et al., 1979; Hirochika & Sakaguchi, 1982; Hirochika et al., 1984). Both plasmids were shown to contain TIRs and TPs (Hirochika & Sakaguchi, 1982; Hirochika et al., 1984). With the advent of PFGE analysis, other actinomycete linear plasmids have been identified including Streptomyces lividans SLP2 (50 kb), Streptomyces clavuligerus pSCL1 (12 kb), Streptomyces avermitilis pSA1 and pSA2 (100 kb and 250 kb), Streptomyces rimosus pZG101 (387 kb), Streptomyces lasaliensis pKSL (520 kb) and Streptomyces coelicolor SCP1 (363 kb) (Chen et al., 1993; Evans et al., 1994; Gravius et al., 1994; Keen et al., 1988; Kinashi & Shimaji, 1987; Kinashi & Shimaji-Murayama, 1991; Kinashi et al., 1994). All these plasmids were found to contain TIRs and TPs. In addition, both pSLA2 and SLP2 were shown to contain an internal origin of replication for bi-directional replication of DNA (Chang & Cohen, 1994; Chang et al., 1996; Huang et al., 2003).

Unlike the chromosomes of most bacteria, which are circular in nature, the chromosomes of actinomycetes are linear (Lezhava et al., 1995; Lin et al., 1993). This was first demonstrated in S. lividans and has now been established for several other actinomycetes including S. coelicolor, Streptomyces griseus and Saccharopolyspora erythraea (formerly Streptomyces erythraeus) (Lezhava et al., 1995; Lin et al., 1993; Reeves et al., 1998). All actinomycete chromosomes characterized to date are quite large, 8–9 Mb, and contain TIRs, TPs and internal origins of replication (Lezhava et al., 1995; Lin et al., 1993; Redenbach et al., 1999; Shiffman & Cohen, 1992). Therefore, actinomycete linear chromosomes and linear plasmids share similar features including TIRs, TPs and internal origins of replication.

Actinomycetes contain additional extrachromosomal genetic elements including rolling circle replicating (RCR) plasmids such as pSAM2 and pIJ101 (Kendall & Cohen, 1988; Kieser et al., 1982; Pernodet et al., 1984). Actinomycete extrachromosomal elements have been shown to shuttle their own genes as well as chromosomal genes to other actinomycete hosts, providing a route for the exchange of genetic information (Bibb et al., 1981; Chen et al., 1993; Hopwood et al., 1985; Kinoshita-Iramina et al., 1997; Ravel et al., 2000). The large linear plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred intact from actinomycete isolates CHR3 and CHR28, respectively, to Streptomyces sp. TK24 (Ravel et al., 2000). Plasmids such as SCP1 and pPZG101 from S. rimosus can recombine at an internal location within the chromosome to form plasmid-prime replicons containing both plasmid and chromosome linear ends (Bibb et al., 1981; Gravius et al., 1994; Kinashi et al., 1992; Pandza et al., 1998; Yamasaki et al., 2000, 2001). These plasmid-prime replicons may facilitate the transfer of chromosomal genes by utilizing linear plasmid transfer functions (Volff & Altenbuchner, 2000).

In this study, we have characterized a 42 kb portion of the linear plasmid pSLV45 from Streptomyces lavendulae IMRU 3455 and have shown pSLV45 derivatives containing HmR to be self-transmissible to S. lavendulae IMRU 467. Sequence analysis of an internal region of pSLV45 has revealed genes encoding proteins predicted to be involved in plasmid transfer, partitioning and regulation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, media, culture conditions, transformation, conjugation and molecular genetic protocols.
Bacterial and derived strains used in this study are listed in Table 1. Actinomycete culture conditions, conjugation protocols and molecular techniques were performed as described previously (Alexander et al., 2003).


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Table 1. Bacterial strains and plasmids used in this study

 
Preparation of DNA inserts for PFGE analysis.
Mycelial cells were prepared for PFGE analysis according to the method of Beyazova & Lechevalier (1993). DNA plugs were equilibrated with PFGE running buffer and subjected to PFGE using a PFGE-DR III Pulsed-Field Electrophoresis System (Bio-Rad). PFGE 1·0 % pulsed-field-grade agarose gels (Bio-Rad) in 0·5x TBE buffer at 14 °C were electrophoresed at 6 V cm–1 with a 60 s switch time, 120° angle for 15 h followed by a 90 s switch time for 9 h. Ladders of linear lambda concatemers (Bio-Rad) were used as molecular mass markers and gels were stained with SYBR Green I (Molecular Probes).

Isolation and cloning of S. lavendulae IMRU 3455 chromosomal and pSLV195 and pSLV45 cosmids.
Cosmid libraries of S. lavendulae IMRU 3455 and IMRU 467 were prepared in pSupercos I according to the manufacturer's recommendations (Stratagene). Cosmid libraries and dot-blot screening filters were prepared as described previously (Hosted et al., 1993). pSLV45 DNA was isolated by excision from a PFGE agarose gel, equilibrated with BamHI digestion buffer, digested with BamHI, ethanol-precipitated and used as a DNA probe against the S. lavendulae IMRU 3455 cosmid library to isolate the pSLV45-containing cosmids pSPRX604 and pSPRX600. pSLV195 DNA was isolated from a PFGE agarose gel as described above and digested with BamHI; a 500 bp BamHI fragment was cloned into pSPRH900b to yield pSPRX592. The 500 bp BamHI insert from pSPRX592 was used to probe against the S. lavendulae IMRU 467 cosmid library to isolate the pSL195-containing cosmids pSPRX607 and pSPRX608. All cosmids were used as probes against a PFGE gel Southern blot of S. lavendulae IMRU 3455 to confirm pSLV45 or pSLV195 hybridizing sequences. The sequence of a 42 kb internal fragment of pSLV45 cosmid pSPRX604 was determined.

Generation of donor strains SPW680 and SPW681, containing the pSLV45 derivatives pSLV45.680 and pSLV45.681.
A 2·2 kb and a 1·3 kb BamHI fragment from pSPRX600 were ligated into the pSPRH900b insertion vector (ApR) to yield pSPRX620 and pSPRX621 (Hosted et al., 2001). The pSPRX620 and pSPRX621 BamHI inserts were subcloned into pSPRH826b (HmR) to yield pSPRX681 and pSPRX680, respectively (Hosted et al., 2001). An HmR marker was inserted into pSLV45 by introduction of pSPRX680 and pSPRX681 into S. lavendulae IMRU 3455 through conjugation from Escherichia coli ET12567 (Flett et al., 1997). HmR exconjugants containing pSPRX680 and pSPRX681 inserted into pSLV45 were isolated and confirmed by Southern hybridization to yield donor strains SPW680 and SPW681, containing the pSLV45 derivatives pSLV45.680 and pSLV45.681, respectively.

Recipient strain generation.
To counterselect against the donor strains during actinomycete mating experiments, S. lavendulae IMRU 467 was tagged with an ApR marker by introduction of the pSPRH910 integrating plasmid by conjugation from E. coli ET12567 (Flett et al., 1997). ApR exconjugants were obtained to yield the recipient strain SPW910.

Hybridization and Southern blot analysis.
Southern blots were performed as described previously (Alexander et al., 2003). To demonstrate transfer of plasmids pSLV45.680 and pSLV45.681 from donor strains SPW680 and SPW681, a Southern blot was prepared from parental, donor, recipient and exconjugant chromosomal DNA digested with BamHI. This Southern blot was probed with a 1·3 kb BamHI fragment from pSPRX621 (pSLV45 DNA), an HmR gene fragment PCR-amplified with primers PRX807 (5'-GCGAGAGCACCAACCCCGTAC-3') and PRX808 (5'-GGCGGCCCGGGGCGTCAG-3'), and a 1·4 kb BamHI S. lavendulae IMRU 3455 chromosomal fragment from pSPRX624.

Actinomycete mating protocol.
S. lavendulae IMRU 3455 donor strains SPW680 and SPW681 were mated with the S. lavendulae IMRU 467 recipient strain SPW910 as follows. Donor and recipient cultures were grown for 3 days in trypticase soy broth (TSB; BRL, Bethesda, MD, USA) medium, harvested by centrifugation and resuspended; the number of c.f.u. was determined by dilution plating. Cells were plated onto ASI (Baltz & Matsushima, 1983) agar as 1 : 10, 1 : 1 and 10 : 1 mixtures; the plates were allowed to dry, incubated overnight at 28 °C and overlaid with R2 modified agar containing apramycin and hygromycin (Baltz & Matsushima, 1983). Exconjugants arising in 2–4 days were patched onto ASI plates, then grown in TSB medium for further analysis.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification and characterization of the S. lavendulae IMRU 3455 and S. lavendulae IMRU 467 linear plasmids
PFGE analysis of undigested DNA insert plugs identified two linear plasmids designated pSLV195 (195 kb) and pSLV45 (45 kb) in S. lavendulae IMRU 3455, and one linear plasmid designated pSLV300 (300 kb) in S. lavendulae IMRU 467 (Fig. 1). PFGE analysis using different pulse times showed that the relative migration of pSLV195, pSLV45 and pSLV325 remained constant relative to the linear lambda concatemers (data not shown). This indicates that the plasmids are linear rather than circular, since migration rates of circular plasmids change depending on the switch time used during electrophoresis (Kalkus et al., 1990; Kinashi & Shimaji-Murayama, 1991).



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Fig. 1. SYBR Green I-stained pulsed-field agarose gel of S. lavendulae IMRU 3455 (lane 1), S. lavendulae IMRU 467 (lane 2) and lambda ladder (lane M). pSLV45, pSLV195 and pSLV300 are indicated by arrows.

 
Sequence analysis and deduced functions of pSLV45 proteins
An internal 42 kb segment of cosmid pSPRX604 was sequenced and ORFs with a bias toward G or C in the third codon position, characteristic of actinomycetes, were identified (Wright & Bibb, 1992). ORF designations, predicted protein coding size, proposed function, representative BLAST homologue, plasmid homologues, and identity and similarity values for genes described in this work are shown in Table 2.


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Table 2. Properties of the ORFs found on pSLV45

 
Duplicated proteins
SLV.3 and SLV.8.
SLV.3 and SLV.8 encode proteins of 74 and 75 aa, respectively, and have similarity to the 112 aa protein SCP1.108 from the S. coelicolor linear plasmid SLP1. They appear to be recently duplicated proteins that contain near identity at the amino acid level (88 % identity over a 58 aa region) with several amino acid substitutions clustered at the C terminus. At the nucleotide level, the coding regions have diverged more widely yet contain 16 synonymous substitutions at the third codon position, which retains the original encoded amino acid.

SLV.4 and SLV.9.
SLV.4 and SLV.9 encode proteins of 110 and 113 aa, respectively, and have similarity to CAC36629.1, a 201 aa protein from S. coelicolor. They are also duplicated proteins that contain a high degree of identity at the amino acid level (76 % identity and 82 % positives over a 107 aa region) and several amino acid substitutions clustered at the C terminus. At the nucleotide level, the coding regions have 22 synonymous substitutions at the third codon position.

Plasmid mobilization genes
SLV.15.
SLV.15 encodes a protein of 846 aa with similarity to the putative ATP/GTP-binding protein SCP1.146 from the S. coelicolor linear plasmid SCP1 (Redenbach et al., 1996). As with the SCP1 protein SCP1.146, SLV.15 has weak but full-length similarity to Q9L4H0 (EMBL accession no. AJ243106), a conjugative protein from integrative element ICESt1 of Streptococcus thermophilus.

SLV.20.
SLV.20, designated traSLVA, encodes a putative protein of 527 aa with strong similarity to FtsK, DNA segregation ATPase FtsK/SpoIIIE-related proteins involved in cell division, chromosome partitioning and translocation of double-stranded DNA (Grohmann et al., 2003). This suggests that TraSLVA may be involved in DNA segregation or conjugative DNA mobilization functions for pSLV45. The C terminal portion of TraSLVA (aa 454–523) has strong similarity (52 % identity and 59 % positives) to the SCP1.194 hypothetical protein from Streptomyces coelicolor on the linear plasmid SCP1 (Redenbach et al., 1996).

SLV.19.
The deduced protein of SLV.19, designated RegA, has strong similarity to AAC73053.1, a putative repressor protein from the Rhodococcus sp. X309 plasmid pSOX (Denis-Larose et al., 1998). Because of its linkage and orientation to traSLVA, RegA may act to regulate DNA transfer.

SLV.28.
The gene product of SLV.28, designated TraSLVB, encodes a 734 aa protein with very strong similarity to the TraJ transfer protein of the self-transmissible RCR plasmid pSG5 from Streptomyces ghanaensis and TraJ from the Amycolatopsis methanolica RCR plasmid pMEA300 (Muth et al., 1995; Vrijbloed et al., 1995a). These TraJ transfer proteins are phylogenetically distinct from TraJ proteins found on many actinomycete RCR-type plasmids (Muth et al., 1995). TraSLVB also contains similarity to the FtsK/SpoIIIE family (aa 361–529) of proteins and a DEAD box motif found within many plasmid transfer proteins but not found on other TraJ proteins (Grohmann et al., 2003).

SLV.31.
SLV.31, designated traSVR, encodes a 276 aa protein very similar to the GntR family of repressor proteins. These proteins are found on many actinomycete RCR-type plasmids and have been shown to regulate plasmid transfer functions (Grohmann et al., 2003). TraSVR had the strongest similarity to the KorSAF regulator protein (EMBL accession no. AF014839) from the RCR plasmid pFQ31 of Frankia sp. ArI3. Its linkage with traSLVB suggests that it may regulate the transfer process of pSLV45, which would be similar to other GntR regulatory proteins in actinomycete plasmids (Grohmann et al., 2003).

Numerous Streptomyces plasmids contain plasmid transfer proteins with similarity to the SpoIII/FtsK family of septal DNA translocator proteins shown to be involved in plasmid transfer (Brolle et al., 1993; Hagege et al., 1993; Kieser et al., 1982; Maas et al., 1998; Pettis & Cohen, 1994; Vrijbloed et al., 1995b). pSLV45 contains two proteins, TraSLVA and TraSLVB, that showed significant similarity to SpoIII/FtsK proteins. This indicates that TraSLVA and TraSLVB may also be involved in plasmid transfer functions. Interestingly, TraSLVB contained high similarity to the TraJ RCR plasmid transfer proteins from pSG5 and pMEA300 and low similarity to the S. lividans linear plasmid SLP2 TraSLP2. In addition to TraSLVA and TraSLVB, pSLV.45 contains SLV.15 with homology to the Lactococcus lactis TraE protein that has been shown to be involved in the pMRC01 RCR plasmid transfer complex (Dougherty et al., 1998).

The S. lividans linear plasmid SLP2 contains TtrA, a newly described plasmid transfer protein, which contains a DEAD box helicase motif similar to that found in TraSLVB (Huang et al., 2003). Mutation of the SLP2-encoded ttrA gene abolished SLP2 transfer, and mutation of either copy of ttrA abolished chromosomal mobilization (Huang et al., 2003). While no homologue of ttrA was found on pSLV45, it is possible that either the S. lavendulae chromosome or pSLV195 contains a TtrA homologue utilized in pSLV45 transfer. Alternatively, the pSLV45 TraSLVB protein may provide the DEAD motif helicase functions required for plasmid transfer.

Restriction modification and methylation genes
SLV.6.
SLV.6 encodes a protein of 813 aa with very strong similarity to AAK47145.1, a type I restriction modification system adenine methylase from Mycobacterium tuberculosis CDC 1551. Type I restriction systems have both restriction and modification activities in one heteromeric enzyme and function as a defence mechanism protecting bacterial cells from foreign DNA. SLV.6 contains both M and S type I restriction regions (Cooper & Dryden, 1994).

SLV.37.
SLV.37 encodes a 187 aa protein with similarity to methyltransferases such as CobL from Rhodococcus erythropolis (Nagy et al., 1995).

Terminal binding protein, partitioning and origin of replication
SLV.22.
A 212 aa protein encoded by SLV.22, designated ParSLV, is very similar, over a 204 aa region, to the ParAF partitioning protein (AAB88413.1) from the Frankia sp. ArI3 RCR plasmid pFQ11 and the ParF protein from Salmonella enterica subsp. enterica serovar Newport RCR plasmid TP228 (Lavire et al., 2001; Hayes, 2000). Therefore, ParSLV may be involved in partitioning and stable segregation of the linear plasmid pSLV45.

SLV.23.
This gene encodes a putative protein of 283 aa with N-terminus similarity to the S. coelicolor SCP2, SCP2.04c, protein (CAD11997.1) that is linked to the partitioning genes ParA and ParAB and the transfer region of S. coelicolor SCP2. No other proteins with any significant similarity were identified by BLAST analysis.

SLV.25.
SLV.25, designated tpgSV45, encodes a putative protein of 185 aa with very strong similarity (52 % identity, 62 % positives), over its entire length, to TpgR2, a recently described 5' terminal binding protein from S. rochei (Bao & Cohen, 2001). This protein falls into the class of TPs that include TpgR1, TpgR2 and Tpgr3 from S. rochei, TpgL from S. lividans, TpgC from S. coelicolor and TpgSLP2 from the 50 kb linear plasmid SCP2 of S. lividans (Bao & Cohen, 2001, 2003; Yang et al., 2002). The tpgR1 gene mapped to the S. rochei chromosome and the tpgR2 and tpgr3 genes mapped to the 206 kb and 100 kb linear plasmids within S. rochei (Bao & Cohen, 2001). The TP homologue TpgSV2, identified on pSLV45, is most probably also involved in protein-primed DNA synthesis for plasmid replication.

In addition to TP, a second protein, telomere-associated protein (TAP), linked to chromosomally encoded streptomycete tpg genes, has recently been shown to be essential for plasmid and chromosomal DNA replication in a linear form (Bao & Cohen, 2003). No TAP protein homologue was located on pSLV45. Presumably, a TAP protein is located on either pSLV195 or the S. lavendulae IMRU 3455 chromosome and supplies this function for pSLV45 replication. In addition, pSLV.680 and pLV.681, which transferred as intact plasmids, would need a TAP protein to be supplied from either pSLV.300 or the S. lavendulae IMRU 467 chromosome.

SLV.26.
This gene encodes a 518 aa protein with similarity at the N terminus (aa 29–221) to a ParB-like protein, SCP1.140, from the S. coelicolor linear plasmid SCP1.

SLV.21.
SLV.21 encodes a putative 114 aa protein with no homology to database proteins. Downstream of SLV.21 is a 1·16 kb segment of DNA with no apparent coding regions. Within this segment are four regions of high A+T content and a number of direct and inverted repeats including GGCGCTTGGCCTGG-24 bp-CCAGGCGAAGAGCC and ACCTCGATGCCCGC-6 bp-ACCTCTATGCCCTC. These features are similar to a region in S. coelicolor plasmid SCP1 thought to be involved in plasmid replication (Redenbach et al., 1999). In addition, the SLP1.194 protein that SLV.20 has similarity to also precedes the S. coelicolor SCP2 replication region. This region downstream of SLV.21 and preceding parSLV-containing AT-rich regions and indirect repeats may represent the pSLV.45 origin of replication.

Terminal regions
No coding regions could be detected following SLV.37 in a 0·85 kb segment. However, a number of indirect repeats (IRs) including a large perfect IR (AAAGGGCCCTGGCCTCAC 4 bp GTGAGGCCAGGGCCCTTT) and several short perfect IRs including CCCCGCCAGGGC-16 bp-GCCCTGGCGGGG and GTGATGGCCGG-26 bp-CCGGCCATCAC were found. These IRs are similar to those located in the terminal portions of linear plasmids. Therefore, this region may represent a portion of the terminal region of pSLV45.

Determination of the self-transmissible nature of pSLV45.680 and pSLV45.681
An HmR marker was placed onto pSLV45 by homologous insertion of pSPRX680 or pSPRX681 onto pSLV45, to generate donor strains SPW680 and SPW681, respectively. PFGE analysis of donor strains SPW680 and SPW681 showed an increase in the size of the pSLV45 staining band consistent with insertion of pSPRX680 and pSPRX681 into pSLV45 (Fig. 2). Southern analysis also confirmed the presence of the HmR marker in SPW680 and SPW681 (Fig. 3).



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Fig. 2. SYBR Green I-stained pulsed-field agarose gel of S. lavendulae IMRU 3455(pSLV45 and pSLV195) (lane 1), IMRU 3455(pSLV45.680) (lane 2), IMRU 3455(pSLV45.681) (lane 3), IMRU 467(pSLV300) (lane 4) (SPW910 ApR recipient strain), IMRU 467(pSPR910) (lane 5), SPW910 exconjugate SPW100-A containing pSLV.680 (lane 6), SPW910 exconjugate SPW100-B containing pSLV.680 (lane 7), SPW910 exconjugate SPW101-A containing pSLV.681 (lane 8), SPW910 exconjugate SPW101-B containing pSLV.681 (lane 9) and lambda ladder (lane M). Arrows indicate positions of pSLV45, pSLV195, pSLV45.680, pSLV45.681 and pSLV300.

 


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Fig. 3. Southern analysis of BamHI-digested DNA hybridized with (a) the 1·3 kb pSLV45 BamHI fragment or (b) the hyg gene fragment. S. lavendulae IMRU 3455(pSLV45 and pSLV195) (lane 1), IMRU 3455(pSLV45.680) (lane 2), IMRU 3455(pSLV45.681) (lane 3), IMRU 467(pSLV300) (lane 4) (SPW910 ApR recipient strain), IMRU 467(pSPR910) (lane 5), SPW910 exconjugate SPW100-A containing pSLV.680 (lane 6) and SPW910 exconjugate SPW101-A containing pSLV.681 (lane 7). Arrows indicate hybridizing fragments discussed in the text.

 
Donor strains SPW680 and SPW681 were mated separately with the S. lavendulae IMRU 467 recipient strain SL910, to obtain exconjugants SPW100-A, SPW100-B (pSLV45.680) and SPW101-A, SPW101-B (pSPL2.681). The results of these experiments indicated that both pSLV45.680 and pSLV45.681 transfer at a frequency of 5x10–5 per recipient in mycelial mating experiments. When probed with a 1·3 kb BamHI pSLV45 DNA fragment, a Southern blot of BamHI-digested DNA of S. lavendulae IMRU 3455 and IMRU 467, donor strains SPW680 and SPW681, recipient strain SPW910 and the SPW100-A and SPW101-A exconjugants showed hybridization of the probe to the DNA of S. lavendulae IMRU 3455, SPW680, SPW681, SPW100-A and SPW101-A (Fig. 3). The hybridization of the probe to the 1·3 kb band in the SPW100-A and SPW101-A lanes indicates the transfer of pSL2.680 and pSL2.681 into SPW910. No hybridization was seen in the lanes corresponding to S. lavendulae IMRU 467 or recipient strain SPW910. Further evidence of transfer of pSPRX680 and pSPRX681 was obtained from the same Southern blot probed with an HmR gene probe (marker on pSLV45.680 and pSLV45.681). A 4·4 kb hybridizing fragment was detected in the lanes corresponding to donor strains SPW680 and SPW681, and exconjugant strains SPW100-A and SPW101-A, indicating transfer of pSPRX680 and pSPRX681 into SPW910 (Fig. 3). No hybridization was seen in the lanes corresponding to S. lavendulae parental strains IMRU 3455 and IMRU 467 and the recipient strain SPW910.

Transfer of intact self-replicating linear plasmids
To determine whether pSLV45.680 and pSLV45.681 had transferred as intact self-replicating linear plasmids, undigested PFGE DNA plugs of parental, donor, recipient strains and exconjugants SPW100-A, SPW100-B, SPW101-A and SPW101-B were subjected to PFGE analysis (Fig. 2). Results of this experiment showed the appearance of a SYBR Green I staining band migrating at the predicted molecular mass for pSLV45.680 and pSLV45.681 (approx. 45 kb) (Fig. 2). This band also co-migrates with pSLV45.680 and pSLV45.681 in the SPW680 and SPW681 donor strain lanes (Fig. 2). The results indicate that pSLV45.680 and pSLV45.681 have transferred as intact stable linear plasmids and are compatible with the pSLV325 plasmid found within S. lavendulae IMRU 467.

Determination of the transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Dr Martha Beyazova for providing the S. lavendulae IMRU 3455 culture and assistance with PFGE.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 16 December 2003; revised 16 February 2004; accepted 16 February 2004.



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