Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.     

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.     

Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.     

Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from Listeria monocytogenes BM4293 among Gram-Positive and Gram-Negative Bacteria

We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501.

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

Sequence of the 50-kb Conjugative Multiresistance Plasmid pRE25 from Enterococcus faecalis RE25

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.     

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.     

Identification of a region that influences host range of the streptococcal conjugative plasmid pIP501

pIP501 is a member of a group of conjugative plasmids that are self-transmissible to a wide variety of streptococci as well as to other gram-positive bacteria. Several pIP501 restriction fragment deletion derivatives have been isolated and characterized. In this paper we describe one such derivative (pVA1702) which was conjugally proficient but had a limited host range. The loss of host range ability was seen as decreased conjugal transfer from Enterococcus faecalis to Streptococcus sanguis and was coincident with the deletion of a 4.5-kb DNA fragment. Transformation of pVA1702 into S. sanguis also was dramatically reduced as compared to its progenitor, suggesting the 4.5-kb fragment encoded a factor(s) necessary for stable maintenance in this host but not in E. faecalis. These observations suggest that pIP501 employs specific mechanisms enabling its maintenance in certain gram-positive bacteria.     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp.

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.