Centro de Investigaciones Biológicas, CSIC, Velázquez, 144, E-28006 Madrid, Spain1
Author for correspondence: Manuel Espinosa. Tel: +34 915611800. Fax: +34 915627518. e-mail: mespinosa{at}cib.csic.es
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ABSTRACT |
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Keywords: plasmid RP4, streptococcal pMV158 transfer, mobilization protein, oriT
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INTRODUCTION |
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Many small multicopy plasmids, like those replicating by the rolling circle mechanism, may carry a cassette of gene(s) involved in their mobilization by larger plasmids. Such is the case of the streptococcal plasmid pMV158 (Burdett, 1980 ), in which the MobM protein is involved in pMV158 mobilization mediated by plasmids of the Inc18 family, like pIP501 or pAMß1 (Priebe & Lacks, 1989
). In vitro, purified MobM protein cleaves the 5'-GpT-3' dinucleotide (between pMV158 coordinates 3591 and 3592) in the sequence 5'-AGTGTG
TTA-3' (
being the site of cleavage) within the plasmid oriT (Guzmán & Espinosa, 1997
). The DNA-binding sequence of protein MobM has been shown to span 28 nt within the in vitro defined oriT, a DNA region that includes an inversely repeated sequence, termed IR2 (Grohmann et al., 1999
). In addition, the 5' and 3' ends of the nick introduced by MobM have been determined i) by in vitro assays with purified protein (Guzmán and Espinosa, 1997
) and ii) in exponentially growing Streptococcus pneumoniae cells harbouring pMV158 (Grohmann et al., 1997
). In vitro cleavage of DNA from pMV158 by purified MobM protein required a supercoiled or single-stranded substrate, indicating that the nic site should be exposed in a single-stranded DNA configuration for MobM-dependent cleavage (Grohmann et al., 1999
).
In spite of the above in vitro findings, a direct definition of the in vivo functionality of pMV158-oriT has not been tested. This functionality has been addressed here by uncoupling the mobM gene from the oriT region and showing that transfer did occur in these conditions. To this end we have cloned pMV158-oriT into an Escherichia coli plasmid vector, whereas the mobM gene was cloned into a compatible plasmid, and placed under the control of an inducible promoter. Upon induction, transfer of the pMV158-oriT-containing vector was observed at a moderately high frequency when the IncP plasmid RP4 provided the conjugative machinery (Pansegrau et al., 1994 ). In addition, RP4 or the IncW plasmid R388 were able to mobilize pMV158 at a frequency similar to that found when pMV158 was mobilized in S. pneumoniae (Priebe & Lacks, 1989
) or in Lactococcus lactis (Farías et al., 1999
) by plasmids pIP501 or pAMß1 (Wang & Macrina, 1995
and references therein). Plasmids harbouring genes involved in the RP4 transfer systems were able to mobilize pMV158, except when the RP4-traG or the traF genes were absent, indicating that these gene products participate in the mobilization of pMV158.
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METHODS |
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Filter matings.
Cells harbouring plasmids (donors) were grown to 3x108 c.f.u. ml-1 and 200 µl of the cultures was mixed with 2 ml of the recipient E. coli HMS174, grown to the same cell density. In the case of strain BL21(DE3) harbouring the three plasmids (RP4, pNLF4 and pLGM2), when the cultures reached the above density, they were induced with various concentrations of IPTG for several time periods, and the same strain HMS174 was used as recipient. In all cases, and to mimic the mating assays that we routinely perform among Gram-positive bacteria, the cell mixtures were filtered onto sterile 13 mm diameter nitrocellulose filters (0·22 µm, Millipore), placed cell-side up on TY-agar plates and incubated at 37 °C for 4 h. Cells were removed by washing the filters with 10 mM MgCl2, 2 µg bovine serum albumin ml-1 and 10% (v/v) glycerol, and plated on the appropriate solid media containing the selective antibiotics. Transconjugants were allowed to grow for 18 h. All experiments were performed in the presence of DNase I (100 µg ml-1). Frequency of conjugation was expressed as the ratio between the number of transconjugants per donor cell and the values are the mean of three independent experiments.
Cloning of the pMV158-oriT.
The EcoRV restriction site of plasmid pACYC184 was used to clone the pMV158 region encompassing the in vitro defined oriT. This region was obtained by PCR amplification with the following primers: 5'-GTCCCTTGCTGATTTTTAAACGAG-3' (coordinates 34083431 of pMV158) and 5'-CAACCATGTAACTCATAGATTTCC-3' (coordinates 37203743).
The former primer contains a DraI restriction site (underlined), whereas the second includes the first codons of gene mobM (see Fig. 1.) The resulting amplified DNA harbours a Tth111I restriction site, so that it was digested with DraI and Tth111I. The protruding Tth111I-generated ends were filled in with PolIK enzyme, and the resulting 247 bp DNA fragment was ligated to DNA from plasmid pACYC184 linearized at its single EcoRV site. The ligation mixture was used to electroporate E. coli BL21(DE3), and clones harbouring plasmids of the expected size were selected for further characterization. Plasmid DNA from one clone harbouring the desired recombinant plasmid (termed pNLF4), was isolated and the integrity of the cloned region was verified by determination of its nucleotide sequence on an automated sequencer (ABI 373A) and the dyedeoxynucleotide termination procedure (Applied Biosystems). Finally, DNA from pNLF4 was used to electroporate E. coli BL21(DE3) harbouring plasmids pLGM2 and RP4.
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RESULTS AND DISCUSSION |
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A number of plasmids and transposons were shown to be transferable by conjugation between Gram-positive and Gram-negative bacteria by means of the functions supplied by plasmids of the IncP family (Bertram et al., 1991 ; Smith et al., 1980
; Trieu-Cuot et al., 1987
). To test whether conjugal transfer of pMV158 occurred in E. coli, cells harbouring plasmid RP4 were transformed with pMV158 DNA. As controls, we used the donor strain harbouring only RP4 or pMV158. In addition, the pMV158-derivative plasmid pLS1, which lacks the mobilization functions, was also used (see Fig. 1a
). Mating experiments showed that pMV158 could be transferred at a moderate efficiency when plasmid RP4 was employed as the auxiliary plasmid (Table 2
.) Transfer of pMV158 was about 1000-fold less efficient than that of RP4. Analysis of the DNA content of six transconjugants showed the presence of pMV158 in all of them (not shown). No transconjugants were detected when only pMV158 was tested and, as expected, the
mobM derivative plasmid pLS1 was not transferred (Table 2
). We conclude that, in addition to plasmids of the Inc18 family, plasmid RP4 can provide the functions needed for conjugative mobilization of the streptococcal plasmid pMV158. At a similar low frequency, the IncW plasmid R388 (Datta & Hedges, 1972
) was also able to mobilize pMV158 (Table 2
). However, under the same experimental conditions, pMV158 could not be mobilized by F (not shown). Transfer mediated by RP4 was also observed from E. coli to the Gram-positive host L. lactis, although at very low frequency (2x10-7), indicating that the RP4 transfer machinery enables promiscuous plasmids, like pMV158, to surpass the so-called barrier between these two types of bacteria. The above finding may provide new tools for transferring DNA cloned into pMV158 among a variety of species in which this plasmid replicates (del Solar et al., 1998
), although the efficiency of transfer should be improved.
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Uncoupling of pMV158-oriT from the mobM gene: mobilization of pNLF4
To assay the functionality of the cloned pMV158-oriT, the E. coli BL21(DE3)/pLGM2 strain was transformed with DNA from plasmids RP4 and pNLF4. The resulting strain contains, in addition to the cloned pMV158-oriT (pNLF4) and RP4 as the auxiliary plasmid, the pMV158-mobM gene placed under the control of the 10 promoter of phage T7 (plasmid pLGM2). Thus, synthesis of MobM is inducible by IPTG (Guzmán & Espinosa, 1997
), so that the intracellular levels of MobM should depend upon the amount of IPTG added to the cultures. The results of the mating experiments (Table 4
) showed that the number of transconjugants increased with the IPTG concentration used, the optimum being 0·5 mM. At 1 mM IPTG, a 100-fold decrease in the frequency of conjugation was found, probably because overproduction of MobM could be toxic to the host harbouring pLGM2. Time of induction was also observed to be an important parameter for the transfer efficiency, the optimal period being 10 min. The integrity of the mobilized plasmid pNLF4 was checked by analysis of the plasmid content of some of the transconjugants (Fig. 2
.) Most of the vector pACYC184, and part of plasmid pLGM2, appeared as dimers (Fig. 2
), as deduced from restriction analysis of the plasmid DNA. In all cases analysed, simultaneous transfer of RP4 was observed (Fig. 2
), probably because of the high frequency of RP4 conjugation (Table 2
). Our results allow us to conclude that: i) the functional pMV158-oriT is encompassed between the 247 bp DraITth111I DNA fragment, a region that contains the in vitro defined site where MobM protein introduces its nick on supercoiled pMV158 DNA (Guzmán & Espinosa, 1997
); and ii) MobM is not only acting in cis, but also behaves like a trans-acting protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 February 2000;
revised 5 June 2000;
accepted 20 June 2000.