Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

Plasmid 50 (2003) 86–93 www.elsevier.com/locate/yplas

Short Communication

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region Brigitta Kurenbach, Christine Bohn, Julia Prabhu,1 Muhtar Abudukerim, Ulrich Szewzyk, and Elisabeth Grohmann* Department for Microbial Ecology, University of Technology Berlin, D-10587 Berlin, Germany Received 16 December 2002, revised 22 April 2003

Abstract The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMb1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G)) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G) Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Conjugative transfer; Intergeneric transfer; Plasmid; Mobilization; Multiple antibiotic resistance; Broad-host-range; Grampositive; Type IV secretion

Conjugative plasmid transfer is the most efficient way of horizontal gene spread. It is considered as one of the major reasons for the emerging *

Corresponding author. Fax: +49-30-314-73460. E-mail address: [email protected] (E. Grohmann). 1 Present address: Actinodrug Pharmaceuticals GmbH, D16761 Hennigsdorf, Germany.

increase of multiple antibiotic-resistant bacteria. The Inc18 plasmids, pIP501, originally isolated from Streptococcus agalactiae (Horodniceanu et al., 1976), and pAMb1 (LeBlanc et al., 1978) confer resistance to the macrolide, lincosamide, streptogramin B (MLS) group of antibiotics, and pIP501 additionally to chloramphenicol. Both plasmids can be transferred to a wide variety of G+ bacteria, including streptococci and lactoba-

0147-619X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-619X(03)00044-1

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cilli (Thompson and Collins, 1988), lactococci (Langella and Chopin, 1989), Listeria spp. (Buuhoi et al., 1984), bacilli (Gibson et al., 1979), clostridia (Oultram and Young, 1985), staphylococci (Engel et al., 1980; Schaberg et al., 1982), and enterococci (Gonz
alez and Kunka, 1983). Enterococci are spread extensively into the environment with the feces and sewage of humans and animals. They are able to multiply in a variety of organic materials, such as fermented food made from meat and milk. Therefore, multiple antibiotic resistance plasmids such as pIP501 may contribute severely to the spread of antibiotic-resistant bacteria via food into the human community. One should be especially concerned about the transmissibility of Inc18 plasmids to pathogens causing life-threatening diseases such as Staphylococcus aureus. Recently another Inc18 plasmid, the 50-kb Rplasmid pRE25 was isolated from an Enterococcus faecalis strain, originating from a dry sausage, and completely sequenced (Schwarz et al., 2001). The plasmid resulted to be very similar to pIP501 (Grohmann et al., 2003; Kurenbach et al., 2002; Schwarz et al., 2001). Inc18 plasmids can mobilize small non-selftransmissible plasmids to a wide range of G+ genera. pRE25 mobilized the 5-kb enterococcal plasmid pESP91 between different E. faecalis strains (Schwarz et al., 2001). van der Lelie et al. (1990) demonstrated that mobilization of pMV158 from E. faecalis JH203 to Lactococcus lactis subsp. lactis IL1403 is dependent on cotransfer of pAMb1 or pIP501. Smith and coworkers showed pMV158 mobilization by pIP501 between different Streptococcus pneumoniae strains (Smith et al., 1980). With a 10-fold recipient excess transfer frequencies (number of transconjugants per donor) were in the range of 10
3 . Non-mobilizable plasmids containing oriTpIP501 were transmitted at high frequencies between L. lactis subsp. lactis strains, if the pIP501 tra functions were provided in trans (Langella et al., 1993). The first report on conjugative transfer from G) E. coli to different Streptomyces species was published in 1989. Mazodier and coworkers efficiently transferred E. coli–Streptomyces shuttle plasmids containing oriTRK2 , oriVpBR322 and the

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origin of the Streptomyces plasmid pIJ101 from E. coli to various Streptomyces species. Plasmid transfer was dependent on the presence of oriTRK2 in cis and on the RK2 tra functions supplied in trans (Mazodier et al., 1989). Several other reports from different laboratories describe intergeneric conjugative transfer of oriTRK2 -based plasmids (Bierman et al., 1992; Flett et al., 1997; Giebelhaus et al., 1996; Voeykova et al., 1998) or mobilization of RSF1010 from E. coli to various representatives of the order Actinomycetales (Gormley and Davies, 1991). Conjugative plasmid transfer from E. faecalis to E. coli was only obtained by transfer of conjugative shuttle plasmids containing G+ and G) origins of replication, such as oriV of pAMb1 and pBR322, oriTRK2 , the tra functions of pAMb1 and a kanamycin resistance gene expressed in both G+ and G) bacteria. The transfer frequencies (number of transconjugants per donor cell) were low, with values between 3  10
9 and 5  10
9 (DoucetPopulaire et al., 1992; Trieu-Cuot et al., 1988). Charpentier and coworkers report conjugative mobilization of the rolling circle plasmid pIP823 from Listeria monocytogenes among G+ and G) bacteria. Mobilization of pIP823 was obtained by selftransferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis and to E. coli. These data indicate that the gene flux observed in nature from G+ to G) bacteria can occur by conjugative mobilization (Charpentier et al., 1999). The genetic organization of the tra region of the broad-host-range plasmids pIP501 and pRE25 is well conserved with that of the staphylococcal plasmids, pGO1 and pSK41, and the lactococcal plasmid pMRC01. The putative transfer proteins of pRE25 and pIP501 show between 80 and 100%, the tra gene products of pGO1 and pSK41 between 97 and 98% identity (Grohmann et al., 2003). The pMRC01 tra region is the most distantly related with seven unique ORFs, the others exhibit between 25 and 42% identity with the corresponding proteins of pGO1 (Dougherty et al., 1998). The nucleotide sequence of the first

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six genes encoded by the pIP501 tra region (Wang and Macrina, 1995a) was shown to be 100% identical with the respective genes of the enterococcal plasmid pRE25 (Schwarz et al., 2001). The first gene product of the pIP501 tra operon is an IncQ-type DNA relaxase initiating pIP501 transfer by site- and strand-specific cleavage of oriTpIP501 (Kurenbach et al., 2002; Wang and Macrina, 1995b). We determined the nucleotide sequence of orf7– 15 of the pIP501 tra region. pIP501-mediated pMV158-mobilization was shown by intra- and interspecies matings, to E. faecalis and B. subtilis. To our knowledge, we demonstrated for the first time conjugative transfer of a naturally occuring G+ plasmid, pIP501, to S. lividans and to E. coli. The nucleotide sequence of the pIP501 tra genes orf7–15 was determined independently at the same time by Keith ThompsonÕs group and was jointly submitted to GenBank. The sequence is deposited under Accession No. AJ505823. Enterococcus faecalis JH2-2 (Jacob and Hobbs, 1974) harboring pIP501 was grown in brain heart infusion broth (Oxoid, London, England) supplemented with rifampicin (25 lg ml
1 ) and chloramphenicol (20 lg ml
1 ) at 37 °C. pIP501 plasmid DNA was isolated by the procedure of Anderson and McKay (1983). For DNA sequencing, pIP501 DNA was further purified by two consecutive cesiumchloride/EtBr density gradients (Sambrook et al., 1989). CsCl-gradient-purified pIP501 plasmid DNA and five overlapping PCR2 products were applied as templates for sequence determination of the tra region. The nucleotide sequences were determined using an ABI Prism 310 Genetic Analyzer, an ABI373/XL, and an ABI377-Sequencer (Applied Biosystems, Foster City, CA). The sequencing reactions were performed according to manufacturerÕs instructions. Both DNA strands were sequenced with synthetic oligonucleotide primers using a primer walking strategy. Primers were synthesized by MWG AG BIOTECH (Ebersberg, Germany), metabion GmbH (Martinsried, Germany), and TIB MOLBIOL (Berlin, Germany). Computer analysis of the DNA se2 Abbreviations used: PCR, polymerase chain reaction; GST, glutathione S-transferase.

quences was performed with the ABI software, applying the programs Factura, Seq-Edit, and Seq-Align, the Wisconsin package Version 10.2 (Genetics Computer Group, Madison, WI) and BLASTP (Altschul et al., 1997). The genetic organization of the pIP501 tra region is depicted in Fig. 1. orf1–15 are highly similar to the respective genes of E. faecalis pRE25 (Table 1). ORF1–6, ORF8–9, and ORF14 are 100% identical with the corresponding pRE25 gene products. In pIP501 one large ORF (ORF11, 306 amino acids) comprises the regions of the corresponding ORF34 and ORF35 in pRE25 (Grohmann et al., 2003). ORF13 (321 amino acids) is significantly larger than the corresponding ORF37 (231 amino acids) encoded by pRE25. Recently we showed that a glutathione Stransferase (GST)-ORF1 fusion protein mediates cleavage of supercoiled oriTpIP501 DNA in vitro (Kurenbach et al., 2002). Hence, the ORF1 protein was termed TraA relaxase. ORF5 contains the Walker motif, typical for NTP-binding proteins, with a well-conserved Walker A motif and a weak Walker B motif. It is tempting to speculate that by ATP-hydrolysis the ORF5 protein would deliver energy required for substrate export during the conjugative transfer process. The putative ORF7 gene product contains the SLT transglycosylase domain (http://www.ncbi. nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid ¼ pfam 01464&version ¼ v1.54) of lytic transglycosylases. Bacterial lytic transglycosylases ( ¼ muramidases) degrade murein via cleavage of the b-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. For ORF7 membrane location was predicted with the PSORT program (http://psort.nibb.ac.jp). This would be in agreement with its putative role in local opening of the peptidoglycan in the cell envelope in order to facilitate substrate export. ORF10 shows the domains conserved in TraG/ TraD/VirD4-like proteins (pfam02534 family). These so-called coupling proteins contain a P-loop (Gly-rich motif in the nucleotide-binding region) and a Walker B-motif. They are thought to link the DNA transfer intermediate to, and perhaps lead it through, the mating channel. The putative ORF10

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Fig. 1. Genetic organization of the pIP501 tra region. Homologues and (putative) functions of the gene products are shown. Ptra , promoter of the tra operon, and the putative transcription terminator are indicated.

Table 1 Amino acid similarity of putative proteins encoded by the pIP501 tra region to sequences in public databases ORF

Nucleotide position on tra-region

Highest similarity/(putative) function

Putative RBS

aac identity (%)

ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7 ORF8 ORF9 ORF10

1394–3379a 3403–3735a 3754–4137a 4211–4783a 4794–6755a 6789–8121a 43–1152b 1165–1716b 1721–2152b 2145–3800b

AGAGG-8-TTG AGAGG-8-ATG AGAAG-10-ATG AGAAG-8-TTG AGGAG-7-ATG AGAAG-10-ATG GGAAG-17-ATG AGGAG-8-ATG AGGAG-5-ATG AGGAG-8-ATG

100 100 100 100 100 100 96 100 100 99

ORF11 ORF12 ORF13 ORF14 ORF15

3818–4741b 4741–5673b 5690–6658b 6687–7055b 7115–7963b

ORF24 of pRE25/DNA relaxase ORF25 of pRE25 ORF26 of pRE25 ORF27 of pRE25 ORF28 of pRE25/putative ATPase ORF29 of pRE25 ORF30 of pRE25/putative lytic transglycosylase ORF31 of pRE25 ORF32 of pRE25 ORF33 of pRE25/putative TraG-/VirD4-like Coupling protein ORF34 of pRE25 ORF36 of pRE25/putative TrsL-like protein ORFeta of pSM19035 ORFtheta of pSM19035 and ORF38 of pRE25 CopF of pAMb1

GGAGG-7-ATG GGAGG-9-ATG GAAGA-13-ATG AAGAG-12-ATG AGGAG-8-ATG

96 93 97 100 96

a

Nucleotide position refers to Accession No.: L39769. Nucleotide position refers to Accession No.: AJ505823. c Amino acid. b

gene product could fulfill such a role by linking the relaxosome, probably consisting only of oriT and tightly bound TraA, with the G+ mating channel. ORF12 shows 22% identity with the putative transfer complex proteins TrsL encoded by the staphylococcal plasmids pSK41 (Berg et al., 1998; AF051917) and pGO1 (Morton et al., 1993; L11998), and with TraL encoded by the lactococcal plasmid pMRC01 (Dougherty et al., 1998; AE001272). Membrane location was predicted for ORF12, TrsL, and TraL. However, their function in the conjugative transfer process remains to be elucidated. ORF13 and ORF14 show high similarity with the putative pSM19035 proteins, ORFeta and ORFtheta, respectively. ORFtheta is identical

with ORFB of pAMb1 and ORF38 of pRE25. The function of these proteins is not known. ORF15 has 96% identity with CopF, the copy number repressor encoded by pAMb1 (Le Chatelier et al., 1994), and 85% identity with ORFiota of pSM19035 (Ceglowski and Alonso, 1994). CopF shares up to 95% identity with the copy number repressor CopR of pIP501 and CopS, the corresponding protein of plasmid pSM19035. The high similarity of ORF15 with these copy number repressors makes a role as DNA-binding regulatory protein likely. orf16 encodes CopR (Accession No. X72021). In order to investigate the pIP501 host range we performed solid surface matings with multicellular

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G+ and with G) recipients. Transconjugants were analysed by specific PCR (Fig. 2) and plasmid DNA isolation (Fig. 3). Plasmid DNA from G+ bacteria was isolated by the procedure of Anderson and McKay (1983). The presence of the tra region was confirmed by PCR specific for orf1/2, orf4, and orf13/14 of the pIP501 tra region. orf1/2 was amplified with oligonucleotide primers annealing to the 30 region of orf1 (fw: 50 -GATCAA TCCCAAGAATTGGATA-30 , position 3185– 3203 on the pIP501 tra region; Accession No. L39769) and to the 50 region of orf2 (rev: 50 -CTT CGCACAAGCAATCCCACC-30 , position 3571– 3591 in L39769) with an annealing temperature of 60 °C; orf4 by using oligonucleotide primers specific for orf4 (fw: 50 -AAAGAGAGGTGATCAA ATTGTTTGA-30 , position 4137–4161 in L39769) and (rev: 50 -CGTCCTGATTGTCCTTGTC-30 , position 4681–4699 in L39769) with an annealing temperature of 50 °C. orf13/14 was synthesized with primers binding to orf13 (fw: 50 -AAGGT AGATATATCCGATTGG-30 , position 6437– 6457 in AJ505823) and to orf14 (rev: 50 -TCTTTCC TAACAACTGGCTT-30 , position 6893–6912 in AJ505823) with an annealing temperature of 56 °C. First, we applied E. faecalis JH2-2 (pIP501) as donor and S. lividans TK23 (pGM11) as recipient. S. lividans TK23 (Hopwood et al., 1983) harboring plasmid pGM11 (Wohlleben and Muth, 1993) was cultivated at 30 °C in LB medium with 200 lg ml
1 kanamycin. pGM11 (5501 bp) contains a ts replicon and the aphII gene of Tn5. The mating procedure described by Kieser et al. (2000) was slightly modified: exponentially growing E. faecalis JH2-2 (pIP501) cells were mixed with 107 to 108 spores of S. lividans TK23 (pGM11) (10 excess of recipients) and incubated on LB agar at 30 °C overnight. Transfer frequencies of 3  10
5 transconjugants per donor cell were obtained (Table 2). One of the 11 transconjugants was further analysed by PCR. The PCR product with the correct size of 406 bp for orf1/2 is shown in Fig. 2 (lane 2). Plasmid DNA isolation showed that pIP501 is maintained at very low copy number in S. lividans (data not shown). With the parental strain S. lividans TK23 (pGM11) no PCR products for orf1/2, orf4, and orf13/14 were obtained (data not shown).

Fig. 2. PCR analysis of transconjugants. The orf1/2-specific 406-bp and the 142-bp oriTpMV158 product were loaded onto a 2.5% agarose gel and subjected to electrophoresis in 0.5 TAE buffer. Lanes 2–5, orf1/2-specific products. Lanes 1, 6, and 13, 100-bp DNA ladder (MBI Fermentas, St. Leon-Rot, Germany); lane 2, S. lividans TK23 (pGM11, pIP501); lane 3, E. faecalis V583 (pIP501); lane 4, E. coli XL1-Blue (pIP501); lane 5, E. faecalis JH2-2 (pIP501). Lanes 7–12, orf1/2 and oriTpMV158 -specific PCR for B. subtilis and E. faecalis transconjugants. Lanes 7, 9, and 11 contain orf1/2-specific PCRs and lanes 8, 10, and 12 oriTpMV158 -specific PCR products. Lanes 7 and 8, B. subtilis MB46 SL601 (pIP501, pMV158); lanes 9 and 10, E. faecalis V583 (pIP501, pMV158), and lanes 11 and 12, E. faecalis JH2-2 (pIP501, pMV158).

Fig. 3. Plasmid DNA isolated from B. subtilis and E. faecalis transconjugants was loaded onto a 0.7% agarose gel and separated by electrophoresis. Lane 1, B. subtilis MB46 SL601; lane 2, B. subtilis MB46 SL601 (pIP501, pMV158); lane 3, E. faecalis V583; lane 4, E. faecalis V583 (pIP501, pMV158); lane 5, E. faecalis JH2-2; lane 6, E. faecalis JH2-2 (pIP501); lane 7, a mixture of the BAC-Tracker supercoiled DNA ladder (Epicentre, Madison, WI; 38, 55, 95, and 120 kb DNA bands; the broad band of DNA that appears below the 38 kb band is nonmarker DNA) and the supercoiled DNA ladder (Invitrogen, Groningen, The Netherlands; 5.012, 6.030, 7.045, 8.066, 10.102, 12.138, 14.174, and 16.210 kb DNA bands). The DNA bands for plasmids pIP501 and pMV158, and for the chromosomal DNA (chrom.) are marked by arrows. pIP501 seems to occur predominantly in multimeric form in B. subtilis as well as in E. faecalis.

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Table 2 pIP501-transfer and pIP501-mediated pMV158 mobilization Donor

Recipient

Donor:recipient ratio

Cotransfer/transferratea

E. faecalis JH2-2 (pIP501, pMV158) E. faecalis OG1X (pIP501, pMV158) E. faecalis V583 (pIP501, pMV158) E. faecalis JH2-2 (pIP501) E. faecalis JH2-2 (pIP501) S. lividans TK23 (pGM11, pIP501) E. coli XL1-Blue (pIP501) B. subtilis MB46 SL601 (pIP501, pMV158)

E. faecalis V583 E. faecalis V583 B. subtilis MB46 SL601 E. faecalis JH2-2

1:2 1:10 1:2 1:10 1:10

2.0  10
6 3.3  10
5 2.6  10
6 4.7  10
6 1.1  10
2

S. lividans TK23 (pGM11) E. coli XL1-Blue

1:10

3.0  10
5b

1:10

0.9  10
5

E. faecalis V583

1:10

0.9  10
7

E. faecalis JH2-2

1:10

1.9  10
3

E. faecalis OG1X

1:10

>4  10
2

a Transfer and cotransfer frequencies are given as number of CmR (EmR ), and CmR TcR transconjugants/recipient, respectively. The given values are means of three independent measurements (except for the last mating). b The pIP501 transfer rate for the mating E. faecalis JH2-2 (pIP501)  S. lividans TK23 (pGM11) is expressed as number of transconjugants/donor cell. Antibiotic concentrations for selection of transconjugants were, for E. faecalis V583 (pIP501, pMV158), 20 lg ml
1 Cm, 4 lg ml
1 Tc, and 1 mg ml
1 Kan; for E. faecalis JH2-2 (pIP501), 20 lg ml
1 Cm and 50 lg ml
1 Rif; for E. faecalis JH22 (pIP501, pMV158), 20 lg ml
1 Cm, 4 lg ml
1 Tc, and 50 lg ml
1 Rif; for E. faecalis OG1X (pIP501, pMV158), 20 lg ml
1 Cm, 4 lg ml
1 Tc, and 1 mg ml
1 Sm; for S. lividans (pIP501, pGM11), 25 lg ml
1 Em and 200 lg ml
1 Kan; for E. coli XL1-Blue (pIP501), 20 lg ml
1 Cm and 20 lg ml
1 Tc; for E. faecalis V583 (pIP501), 20 lg ml
1 Cm and 1 mg ml
1 Kan, and for B. subtilis MB46 SL601 (pIP501, pMV158), 20 lg ml
1 Cm, 4 lg ml
1 Tc, and 50 lg ml
1 Rif. Cm, chloramphenicol; Em, erythromycin; Kan, kanamycin; Rif, rifampicin; Sm, streptomycin; and Tc, tetracycline.

pIP501-transfer from the S. lividans TK23 (pGM11, pIP501) transconjugant to the kanamycin resistant E. faecalis V583 strain (P. Courvalin, Institut Pasteur, Paris, France) occurred at a transfer rate of 0.9  10
7 indicating that the pIP501 transfer factors were maintained and fully functional in S. lividans. pIP501 could also be transferred from E. faecalis JH2-2 (pIP501) to E. coli XL1-Blue (Stratagene, La Jolla, USA). pIP501 was stably maintained, albeit at very low copy number, without selection over at least 50 generations in the G) host (data not shown). An average transfer rate of 0.9  10
5 transconjugants/recipient was obtained for overnight matings with a 10-fold excess of recipients. Functionality of the pIP501 tra genes in E. coli was proved by conjugative transfer of pIP501 from the E. coli transconjugant to E. faecalis JH2-2 (Table 2). pIP501 mobilized the tetracycline resistance plasmid pMV158 in intra- (among different E.

faecalis strains) and interspecies matings from E. faecalis to B. subtilis MB46 SL601 (M. Espinosa, CSIC, Madrid, Spain). For E. faecalis V583 recipients a cotransfer frequency of 3.3  10
5 transconjugants/recipient was obtained, while for E. faecalis JH2-2 recipients under optimum conditions (exponentially growing cells, donor:recipient ratio of 1:10) frequencies of up to 4.1  10
2 were reached. The low transfer efficiency observed with E. faecalis V583 recipients could probably be explained by incompatibility events. A BLAST search of the recently finished E. faecalis V583 genome at http://tigrblast.tigr.org/ ufmg with pIP501 revealed that the pIP501 replication gene, repR, is 96% identical with a region termed repS on the E. faecalis V583 genome. The coding region of a putative CopS protein on the E. faecalis V583 genome shows 100% identity to the first third (97 of 279 nucleotides) of the pIP501 copy number repressor copR. CopR amino acids 18–37 form the DNA-binding helix-turn-helix-

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motif (Steinmetzer et al., 2000). repS and copS are located in close vicinity to each other on plasmid-1 (plasmid-1, 66,320 bp; plasmid-2, 17,963 bp; plasmid-3, 57,660 bp) encountered in E. faecalis V583. Cotransfer of pIP501 and pMV158 was also observed in interspecies matings between E. faecalis OG1X (strain collection of M. Espinosa, CSIC, Madrid) harboring pIP501 and pMV158 and B. subtilis MB46 SL601 recipients. pIP501 could be further transferred from the B. subtilis MB46 SL601 transconjugants to E. faecalis OG1X indicating that the pIP501 tra region is fully functional in B. subtilis. The presence of pIP501 and pMV158 in the transconjugants was verified by tra region specific PCR for pIP501, oriT-specific PCR for pMV158 and plasmid DNA isolation. oriTpMV158 was synthesized with primers binding to the oriT region (fw: 50 -CTACCTGTCCCTTGCTGAT-30 , position 3407–3425 in X15669) and (rev: 50 -GC AGTGCCGACCAAAACCA-30 , position 3531– 3549 in X15669) with an annealing temperature of 55 °C. All transfer data are summarized in Table 2. The pIP501 tra region revealed an extremely broad-host-range including transfer to multicellular G+ bacteria and to G) E. coli. Functionality of the pIP501 tra functions in the new hosts was proved by use of Streptomyces and E. coli transconjugants as donors in further matings. We determined the nucleotide sequence of the pIP501 tra genes, orf7–15. The high similarity of the tra regions of enterococcal (pRE25), staphylococcal (pSK41, pGO1), and lactococcal plasmids (pMRC01) with that of pIP501 makes pIP501—the plasmid with the broadest host range of these—a very good model system to study conjugative transfer in G+ bacteria. Studies on the cellular location of the pIP501-encoded putative type IV components and on protein–protein interactions of the Tra components shall help throw some light on the conjugative transfer mechanisms in G+ bacteria.

Acknowledgments We thank D. Grothe and M. Meixner for sequence determination of orf7 and orf8–15, re-

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