Department of Medical Microbiology, Otto von Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany
Correspondence
Steffen Backert
Steffen.Backert{at}medizin.uni-magdeburg.de
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ABSTRACT |
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INTRODUCTION |
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Additional evidence arguing for a conjugative DNA transfer mechanism in H. pylori is based on the identification of two putative relaxase genes, here termed rlx1 and rlx2, in the H. pylori chromosome. These genes show significant homology particularly to the unique active sites of other known relaxases (Backert et al., 1998). Relaxases are present in all conjugative DNA-transfer systems (Pansegrau & Lanka, 1996
; Llosa et al., 2002
; Cascales & Christie, 2003
; Schroder & Lanka, 2005
). They play a key role in initiating conjugative plasmid DNA transfer by producing a single-stranded scission within oriT and binding to the 5' end of the cleavage site. Sequence alignment of relaxase proteins from different DNA transfer systems such as the mobilizable plasmids and agrobacterial Ti (tumour-inducing) plasmid show the presence of three conserved functional motifs (IIII). These motifs are usually located at the N-terminus of the proteins and are arranged in a specific order (Pansegrau & Lanka, 1996
). Motifs I and III are involved in catalysing the nicking closing reaction at specific DNA target sequences (oriT) whereas motif II is thought to function as a DNA recognition domain. Motif I carries the tyrosine residue that covalently attaches the relaxase through a transesterification reaction to the 5' terminus of the cleaved single-stranded DNA (Pansegrau & Lanka, 1996
). All of these motifs are well conserved in the two putative rlx genes identifed in H. pylori (Backert et al., 1998
). The relaxase protein, its DNA substrate and a number of accessory DNA-binding proteins form a specialized proteinDNA complex at the oriT, termed the relaxosome. The relaxosome is specifically coupled to the transmembrane type IV secretion system (T4SS) for subsequent transfer by the so-called coupling protein. The TraG protein of RP4 and TrwB of R388 are two well-characterized examples of coupling proteins (Cabezon et al., 1997
; Gomis-Ruth et al., 2001
; Hormaeche et al., 2002
; Schroder et al., 2002
; Schroder & Lanka, 2003
). The N-terminal regions of many coupling proteins have transmembrane
-helices which mediate membrane anchorage. Coupling proteins have conserved Walker motifs A and B postulated to be responsible for ATP hydrolysis to provide energy for the transfer process (Walker et al., 1982
; Cabezon et al., 1997
; Llosa et al., 2002
; Schroder et al., 2002
; Schroder & Lanka, 2003
). Tato et al. (2005)
demonstrated recently that the coupling protein TrwB indeed possesses a robust ATPase activity and a propensity to hexamerize, both of which can be stimulated in a DNA-dependent manner. Two open reading frames in H. pylori, namely HP0524 (a virD4 homologue) and HP1006, have been reported to share sequence similarity with traG-like coupling factors (Backert et al., 1998
).
The set of conjugative proteins that assemble the transmembrane conjugation pore belong to the T4SS family of macromolecular transporters. H. pylori possesses several T4SSs which show significant homology to those of conjugation machineries (Hofreuter et al., 2001; Backert et al., 2002
). The first T4SS identified in H. pylori was that encoded by the 40 kb cag pathogenicity island (cagPAI), consisting of up to 31 genes (Covacci & Rappuoli, 2000
). This T4SS represents a major disease-associated determinant for the delivery of virulence factors such as the CagA protein into host target cells. The second T4SS in H. pylori is the comB system, consisting of the comB4, comB7, comB8, comB9 and comB10 genes, which has been shown to mediate the uptake of naked DNA (Hofreuter et al., 2001
). Components of the comB system display significant primary sequence and structural similarity to basic components of a T4SS showing homology to the virB4, virB7, virB8, virB9 and virB10 genes of the Ti plasmid. The third T4SS gene cluster found in certain strains of H. pylori is a segment named tfs3 (type IV secretion system 3) (Kersulyte et al., 2003
). It encodes up to 16 ORFs, some of which are homologous to the genes virB4, virB7 to virB11 and virD4 in Agrobacterium tumefaciens. No specific function has yet been ascribed to the latter putative T4SS. It is also unclear whether the aforementioned T4SSs could play a direct role in conjugative DNA transfer in H. pylori.
Our aim in this study was to determine whether H. pylori possesses intrinsic capability for conjugative plasmid transfer and to identify the genes that might be involved in this process. For this purpose, we cloned oriT-containing shuttle vectors without mob functions and studied their mobilization among clinical H. pylori strains. The functional involvement of the previously identified putative rlx (HP0996, HP1004) and traG (HP1006) homologues in plasmid transfer was examined in detail using mutagenesis and primer extension studies. Our findings reveal that the shuttle plasmids can be mobilized in a conjugation-like manner in H. pylori. This process is dependent on specific rlx and traG homologues carried on the chromosome but independent of the comB- and cagPAI-encoded T4SSs.
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METHODS |
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This 370 bp PstI fragment with the oriT sequence is included only in the oriT+ plasmids and not in the oriTones (Table 2). The mutant-2 gfp (green fluorescent protein-2) gene in a XbaI fragment was cloned downstream of the constitutive H. pylori flaA promoter at a unique NdeI site in the start codon ATG as described by Heuermann & Haas (1998)
. The PflaA gfp fusion cassette was then inserted into the ClaI and KpnI sites of pHel2, resulting in a Chlr, GFP-expressing E. coli/H. pylori shuttle pSB13 (Fig. 1a
). To construct a similar shuttle vector with a Kanr gene cassette, the catGC cassette in pSB13 was replaced by the aphA-3 gene, which was amplified by PCR from pILL600 with the following primers, which have BamHI sites for subcloning: 52F (5'-AGGATCCGTCCGAATTCAAACCCAGCGAACCA-3'), 52R (5'-AGGATCCGGAAGATCTTTTAGACATCTAAAT-3'). The resultant vector is pSB14 (Fig. 1b
). For genetic complementation experiments, the gfp gene (0·9 kb NdeISalI fragment) in pSB13 or pSB14 was replaced by rlx1 and rlx2 genes, respectively. The following primer pairs were used for amplification of rlx1 and rlx2 genes: Rlx1-18F (5'-CATATGAAACGCTCCCACTTAGAAAATG-3'), Rlx1-18R (5'-GGATCCGAACAAGAATAAACCCCTTTCAAAG-3'); Rlx2-19F (5'-TCCATATGGCGTTAGAAAAAAGTTATAG-3'), Rlx2-19R (5'-CGGGATCCACTTTTTAGACTATCTAAATGATC-3'). Correct insertion of the amplified fragments into pSB13/pSB14 was verified by standard RFLP and sequencing. To generate a Rlx1Y12L mutant, we used the Sculptor mutagenesis kit (Amersham Biotech). The generated plasmids were named pSB17, pSB18, pSB21 and pSB22, respectively (Table 2
). All E. coli/H. pylori shuttle plasmids were mobilized into H. pylori by using E. coli GC7 harbouring the conjugative plasmid pRK2013 as mobilizer (Heuermann & Haas, 1998
). The expression of rlx1 and rlx2 was examined by immunoblotting of proteins using polyclonal rabbit anti-Rlx1 or mouse anti-Rlx2 antibodies.
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Mating experiments on GC agar plates.
Each mating experiment involved a donor (designated strain A) and a recipient (designated strain B) with mutually exclusive antibiotic resistance markers (Table 2). After 12 h growth on GC agar, bacteria of each strain were harvested and suspended in 3 ml BHI. Matings were assayed by mixing strains A and B. Aliquots (100 µl containing 1x109 H. pylori of each strain) of cultures of the parent strains as well as the resultant mixed culture were then plated immediately onto the respective GC agar plates as follows: plate 1, strain A alone; plate 2, strain B alone; plates 3 and 4, mixed culture of strains A and B. Plates 3 and 4 differed by the fact that plate 3 was supplemented with 300 µg DNase I ml1 (Roche) and 2 mM MgCl2. In some control experiments, bacteria were incubated in the presence of a 0·1 µm pore-size membrane (Millipore) that prohibited cell-to-cell contact between the two parental strains. After the plates were incubated overnight, bacteria were harvested in separate 1 ml aliquots of BHI. To check for spontaneous mutants as control, serial dilutions of the bacteria harvested from plates 1 or 2 were plated on GCSK or GCSC plates depending on whether the recipient strain was Kanr or Chlr, respectively. Serially diluted bacteria from plates 3 and 4 were inoculated as follows: 200 µl plated on a GC agar plate without antibiotics, and 250, 100 and 25 µl on three individual GCSK or GCSC plates. All plates were incubated for 96 h, after which the number of single colonies was counted. To ascertain the recipient origin of the progeny, single colonies from the GCSK or GCSC plates were subcultured on GC agar plates containing rifampicin (10 µg ml1) and either kanamycin (8 µg ml1) or chloramphenicol (4 µg ml1) depending on the recipient parent. In parallel, an aliquot was plated on a GC agar plate without antibiotics as viability control. In an additional control experiment to check the possibility of transfer of naked DNA by transformation, 2 units of Benzonase nuclease (Novagen) were used per plate. Benzonase is a genetically engineered endonuclease from Serratia marcescens which degrades specifically all forms of DNA and RNA (single stranded, double stranded, linear and circular) while having no proteolytic activity. One unit of the enzyme completely digests 37 µg nucleic acids in 30 min to 5'-monophosphate-terminated oligonucleotides 2 to 5 bases in length (Novagen).
DNA exchange using cell-free extracts, purified plasmid DNA or heat-inactivated bacteria.
Mating experiments using intact recipient H. pylori strain and either cell-free extracts, purified plasmid DNA (1 µg) or heat-inactivated donor H. pylori were carried out using procedures described above. Plasmid DNA was isolated from strain P1 as described by Hofreuter & Haas (2002). Cell-free extracts of 1x109 H. pylori were prepared by five freeze/thawing steps followed by passage through a 0·2 µm sterile filter (Roth). Heat-inactivated bacteria were prepared by incubation at 80 °C for 10 min. These experiments were done in the presence and absence of 300 µg DNase I ml1 and 2 mM MgCl2. After incubation for 12 h, progeny colonies were inoculated onto nonselective and selective plates exactly as described above.
Primer extension assay and DNA cycle sequencing.
For primer extension studies, the oriT-sequence-specific primer 333F (5'-GTATATTCCTTTTTCGCACG-3'), which maps to a site 88 nt upstream of the RP4 oriT, was 5'-end-labelled using the Ready-To-Go T4 polynucleotide kinase kit (Amersham Biosciences) and [-32P]ATP (0·37 MBq; Amersham Biosciences). Asymmetric PCR reactions with 333F were performed in a 30 µl reaction including 0·2 mM dNTPs, 2 mM MgCl2 in 1x PCR buffer in the presence of 0·5 µg total DNA purified from culture of the H. pylori transconjugant of interest. DNA polymerase was purchased from Qiagen. Cycling was done at 94 °C for 30 s, 80 °C for 30 s and 72 °C for 15 s for 35 cycles. Extension products were resolved in denaturing polyacrylamide gels (10 % polyacrylamide, 7 M urea) in 1x TBE buffer (10 mM Tris pH 8, 1 mM EDTA and 10 mM boric acid). To determine precisely the size of primer extension products, a sequencing reaction was done in parallel using the Sequenase 2.0 kit with [35S]dATP
S (1·85 MBq) and primer 333F according to the instructions of the manufacturer (Amersham Biosciences). The template DNA used was vector pSB14 containing the cloned oriT of RP4 (Fig. 1b
). After electrophoresis, the gel was fixed by incubation in 5 % acetic acid, dried on filter paper and subsequently exposed to X-ray films (Amersham Biosciences).
Randomly amplified polymorphic DNA (RAPD) PCR fingerprinting and fluorescence microscopy.
RAPD fingerprinting and fluorescence microscopy were carried out as described below to verify the successful transfer of plasmid DNA from the donors to the recipients. For RAPD fingerprinting, chromosomal DNA was prepared from parental and recombinant H. pylori strains using a genomic DNA preparation kit (Qiagen). RAPD-PCR was carried out in 25 µl containing 20 ng genomic H. pylori DNA, 3 mM MgCl2, 30 pmol primer, 250 µM dNTPs and 1 unit Taq polymerase in 1x reaction buffer (Qiagen). The following RAPD primers were used: 5'-CCGGATCCGTGATGCGGTGCG-3' (D9355) or 5'-GGTTGGGTGAGAATTGCACG-3' (D14307). The cycling conditions have been described previously (Akopyanz et al., 1992). The results were analysed by agarose gel electrophoresis using standard procedures. The expression of gfp encoded on plasmid pSB13 or pSB14 in the donor and recipient strains was verified using the following procedures. The bacteria were harvested and fixed with 4 % paraformaldehyde for 20 min at room temperature on glass coverslips. The fluorescence intensity of the specimen was examined using a DMIRE-2 fluorescence microscope. The results of RAPD fingerprinting and the fluorescence micrographs were provided to the reviewers for assessment.
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RESULTS |
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The following observations further support the notion that mobilization of pSB13 between H. pylori strains occurred by a conjugation-like mechanism rather than transformation or transduction. First, matings carried out in 1 ml liquid BHI medium routinely produced significantly fewer recombinants as compared to matings performed on solid agar medium (Table 3). Second, no plasmid DNA transfer was detected when parents were separated by a 0·1 µm pore-size membrane that prohibited cell-to-cell contact or when the donor strain was heat-inactivated. These observations imply that exchange of pSB13 requires tight cell-to-cell contact between viable strains, a feature which is characteristic of bacterial conjugation. Third, the transfer frequency dropped only marginally in the presence of DNase I or Benzonase nuclease. Since conjugation allows DNA to be transferred in a DNase-insensitive manner whereas uptake of naked DNA by transformation is DNase-sensitive, these results argue against the possibility of plasmid transfer by transformation. Fourth, no or a very low number of recombinants were obtained when the recipients were incubated with a cell-free lysate of the donor or when the recipient strains were incubated with purified pSB13 isolated from strain P1 (Table 3
), further arguing against DNA transfer by transformation or transduction. Thus, the observations presented above provide multiple lines of evidence indicating that the transfer of pSB13 between the various H. pylori strains occurred by a conjugation-like mechanism requiring RP4 oriT and tight cell-to-cell contact between two viable parent strains.
Conjugative plasmid transfer in H. pylori is independent of the comB system, cagPAI and recA
Since our pSB13 and pSB14 shuttle vectors do not carry mob genes, the genes involved in their transfer must be carried on the H. pylori chromosome. The comB and cagPAI systems are two T4SSs in H. pylori which have been studied intensively (Covacci & Rappuoli, 2000; Hofreuter et al., 2001
; Backert et al., 2002
). Given that both of them share significant homology with T4SSs of conjugative origin, we examined whether the cagPAI and the comB system play a role in the mobilization of the pSB vectors. As shown in Table 4
, disruption of comB10 in both donor and recipient H. pylori had only a minor effect on the frequency of pSB14 mobilization, although the mutation abolished uptake of free DNA by tranformation (Hofreuter et al., 2001
and data not shown). Thus, mobilization of pSB14 among H. pylori strains appeared to occur in a transformation (comB)-independent manner. P1 wild-type and the virD4, virB7 and virB11 isogenic mutants alike gave rise to similar mobilization frequency (Table 4
, top). Given that the cagPAI-encoded T4SS was likely to be dysfunctional in the virB11, virB7 and virD4 mutants (Covacci & Rappuoli, 2000
), this observation suggests that the cagPAI also did not play a significant role in the transfer of pSB14. In contrast to the requirement of RecA function in the natural transformation of H. pylori, the conjugative transfer of pSB14 in H. pylori was RecA-independent. This was shown by the fact that inactivation of recA in the recipient strains did not affect the number of transconjugants obtained (Table 4
, top).
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To investigate whether the putative relaxase genes rlx1 and/or rlx2 play a role in plasmid mobilization, we first overexpressed them under the control of the constitutively active flaA promoter in wild-type H. pylori strain P1 and examined their effects on transfer frequency. Overexpression of rlx1 but not that of rlx2 led to an increase in the frequency of pSB plasmid transfer by approximately one order of magnitude (Fig. 2a, Table 4
), suggesting that rlx1 but not rlx2 is important for the mobilization. In support of this hypothesis, inactivation of rlx1 but not rlx2 abolished the plasmid transfer (Fig. 2b
, Table 4
). Complementation of rlx1 restored the frequency of transfer to a wild-type level (Fig. 2b
, Table 4
). Western blot analysis of bacterial cell lysates using anti-Rlx1 or anti-Rlx2 antisera revealed that while both Rlx1 and Rlx2 were expressed in H. pylori P1, only the expression level of Rlx1 correlated strictly with a transfer-positive phenotype (Fig. 2c
). The Western blots also showed that (i) deletion of rlx1 was non-polar with respect to expression of rlx2 and vice versa, and (ii) the genetic complementation was functional.
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Mapping of the Rlx1 nicking site in the oriT sequence of RP4
Relaxases catalyse the cleavage of a specific phosphodiester bond at the nic site within the related oriT to initiate single-stranded DNA transfer during conjugation (Pansegrau & Lanka, 1996). To map the nic site produced by H. pylori Rlx1 during the transfer of pSB14, we performed primer extension assays, a sensitive technique for detecting DNA discontinuity and characterizing nic sites generated in vivo (Pansegrau et al., 1990
; Liu & Haggard-Ljungquist, 1994
; Llosa et al., 1995
; Backert et al., 1997
). As shown in Fig. 3
, wild-type bacteria but not the rlx1 mutant produced a single band corresponding to an 86 nt primer extension product that terminates at the specific site 5'-TCC/TGCCC-3' within the oriT sequence in the pSB14 template. This suggests that the sequence 5'-TCC/TGCCC-3' is the nic site for Rlx1 in this oriT sequence. A functional rlx1 gene was required for the nicking activity, as no primer extension product was obtained with a donor expressing the Rlx1-Y12L mutation (Fig. 3
, lane 4). The fact that the rlx2 gene mutant also gave rise to an 86 nt primer extension product supports the notion that rlx2 is nonessential for conjugative transfer of pSB14 plasmids between H. pylori strains. Interestingly, the deduced nic site 5'-TCC/TGCCC-3' in our primer extension studies corresponds to a site 2 nt upstream of the nic site 5'-TCCTG/CCC-3' of TraI in the pRP4 system (Pansegrau & Lanka, 1996
). Relaxases from different organisms have been reported to exhibit different DNA sequence specificities. Future studies are necessary to define in detail the sequence specificity of Rlx1 and to identify the possible cognate oriT sites in the H. pylori genome.
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DISCUSSION |
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The T4SS encoded by the cagPAI or tfs3 appeared to be unnecessary for the conjugative DNA transfer observed because, firstly, the H. pylori strains examined do not encode the full set of tfs3 genes (our unpublished data), and secondly, mutations of the various cagPAI genes did not block plasmid transfer. Instead, we found a gene cluster in the so-called plasticity zone (Tomb et al., 1997; Alm et al., 1999
) that encodes relaxase homologues (Rlx1 and Rlx2) and a TraG-like protein, which are typical components of conjugative T4SS systems (Pansegrau & Lanka, 1996
; Cabezon et al., 1997
; Llosa et al., 2002
; Cascales & Christie, 2003
; Schroder & Lanka, 2005
; Tato et al., 2005
). Their involvement in plasmid transfer among H. pylori strains was supported by the following findings from our mutagenesis experiments: (i) a traG knock-out mutant was defective in plasmid DNA transfer; (ii) likewise, a rlx1 knock-out mutant was completely deficient in plasmid transfer; (iii) transfer frequency was enhanced by overexpression of rlx1; and (iv) genetic complementation of rlx1 in the rlx1 mutant strain restored the plasmid transfer to wild-type levels. Taken together, our findings support a model in which plasmid DNA mobilization in H. pylori depends on the Rlx1 homologue acting in concert with a TraG-like protein encoded by HP1006.
Results were also obtained in this study which provide preliminary insights into the molecular mechanism by which Rlx1 might function as a relaxase in H. pylori. Tyrosine residue Y22 in motif I in TraI plays a key role in initiating conjugative plasmid DNA transfer of plasmid RP4 by producing a single-stranded scission within oriT and binding to the 5' end of the cleavage site (Pansegrau & Lanka, 1996). Analogous to the functional role of Y22 of TraI, the conserved residue Y12 in Rlx1 appeared to be essential for the relaxase function, as expression of Rlx1-Y12L in H. pylori abolished plasmid transfer. Consistent with this finding, the Rlx1-Y12L mutant did not produce the nicking product in our primer extension study. The Rlx1 nicking site in the oriT sequence of RP4 was identified and mapped to the sequence 5'-TCC/TGCCC-3', indicating that Rlx1 is able to process the oriT of RP4. It has been noted that relaxases that act on IncP-like transfer origins share three conserved motifs at their N-termini (Pansegrau & Lanka, 1996
). Since Rlx1 contains all three of the conserved relaxase motifs (Backert et al., 1998
), the ability of Rlx1 to act on the RP4 oriT (which has an IncP origin) is not totally surprising. However, it is interesting to note that the Rlx1 nicking site determined lies 2 nt upstream of the cleavage site produced by the TraI relaxase (5'-TCCTG/CCC-3'). The reason for this discrepancy is as yet unclear. Identification of the cognate oriT in H. pylori is one of the most important aims in future studies and may shed light into the sequence specificity of Rlx1. It will also be of interest to see if transfer of plasmids in H. pylori using a cognate oriT of Rlx1 would occur more efficiently.
A recent view of the conjugation machinery is that it consists of a DNA-processing system linked to a T4SS. The former consists of relaxases, accessory proteins and DNA, whereas the latter forms a conjugation pore through which the DNA/protein complexes of conjugative machineries are transferred across the membrane barriers (Llosa et al., 2002; Schroder & Lanka, 2003
, 2005
). Interestingly, the mosaic nature of the conjugative machineries is often accompanied by the organization of the corresponding genes in a gene cluster. For instance, in the well-characterized RP4 conjugation system, the genes traM, traL and traJ, which encode components of the conjugation pore, lie in a gene cluster with the rlx and traG genes, which encode DNA-processing and coupling functions (Pansegrau & Lanka, 1996
). In H. pylori, the genes flanking rlx or traG have no significant homology to any T4SS components, such as the tra or virB genes, although they contain smaller stretches which are homologous to genes encoding conjugative proteins (our unpublished data). This could imply that the H. pylori conjugative apparatus may have some atypical features unique to this organism. The fact that HP1006 shows homology with only the C-terminal portion of TraG tends to support this hypothesis. Future work is required to identify the genes involved in forming the conjugation pore and to further characterize the relaxosome and coupling process in H. pylori conjugation. It is also intriguing that although H. pylori contains two putative rlx genes, only rlx1 appears to be essential for the conjugative transfer of pSB13 or pSB14. It will also be important to investigate the role of rlx2. We have indications that rlx2 rather than rlx1 is involved in mobilization of chromosomal DNA among H. pylori strains (our unpublished data).
In conclusion, in this study we have identified novel components of a chromosomally encoded conjugative apparatus in H. pylori which mediates plasmid DNA transfer among strains and is independent of the three known T4SSs in H. pylori, namely comB, cagPAI and tfs3. The genes in H. pylori which encode the T4SS dedicated to forming a conjugation pore are yet to be defined. An understanding of how Helicobacter exploits its conjugative transfer system during the course of infection will provide important insights into the evolutionary strategies of this group of clinically important pathogens. It is plausible that this phenomenon may help the bacteria to adapt to environmental stress in the acidic milieu of the stomach, where free chromosomal DNA is short-lived, and may contribute to the heterogeneity of H. pylori strains. In addition, transfer of genetic materials via a conjugative mechanism could be a means to overcome the interstrain restriction barrier, assuming that the single-stranded DNA being transferred is less likely to be recognized by conventional restriction enzymes (Lin et al., 2001; Nobusato et al., 2000
; Aras et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 3 June 2005;
revised 26 August 2005;
accepted 2 September 2005.
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