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

Nucleotide sequence of pIP823.Plasmid pIP823 DNA was digested with HindIII, cloned intoHindIII-linearized pUC18, and transformed intoE. coli JM83. The resulting plasmid, pAT459, conferred trimethoprim resistance to this new host. The sequence of the pAT459 insert of 3,712 bp was determined on both strands (Fig.1). Analysis of the sequence revealed the presence of two open reading frames (ORFs) and of a third ORF, which appeared to be disrupted at the HindIII cloning site. In order to confirm this assumption and prove that no segment of pIP823 was lost during cloning, two primers (5′ ATTACTTGTCACGTCTG 3′OH) and (5′ TTTCTTTTGCTCGATCC 3′OH) were designed to be complementary to the pAT459 insert at 261 bp upstream and 245 bp downstream from the HindIII site while allowing amplification of a 514-bp fragment with pIP823 DNA as a template. The sequence of the amplification product corresponded to that predicted for the insert of pAT459 circularized at the HindIII site. The third ORF started 735 bp upstream from theHindIII site in the 3′ part of the insert and ended at 504 bp downstream from this site in the 5′ part of the insert. Thus, the 3,712-bp HindIII insert of pAT459 corresponded to pIP823 linearized at this site.

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Fig. 1.

Schematic representation of the relationship between pIP823 from L. monocytogenes (3,712 bp), pUB110 fromS. aureus (4,525 bp), and pTB913 from Bacillus stearothermophilus (4,525 bp). bleo, gene conferring resistance to bleomycin; dfrD, gene encoding trimethoprim-resistant S2DHFR; dso, double-stranded origin or plus origin of replication; knt, gene conferring resistance to kanamycin; mob, gene encoding the mobilization protein Mob; repU, gene encoding the replication protein RepU; RS_A, palindromic site involved in conjugative mobilization and recombination; ssoU, single-stranded origin or minus origin of replication. The positions of the PCR primers indfrD (D), mob (M), dso (R1), andrepU (R2) are indicated by filled arrowheads.

pIP823 organization and similarities to RC plasmids.The first ORF, from positions 2159 to 2675, corresponds to the dfrDgene, which encodes trimethoprim-resistant dihydrofolate reductase type S2 (S2DHFR) (Fig. 1) (3). The second ORF, from positions 970 to 1975, corresponds to the Rep protein with motifs characteristic of RC replication proteins (Fig. 1). The sequence of Rep was found to be identical to that of RepU of plasmids pUB110 from S. aureus and pTB913 from a thermophilic Bacillus sp. (19, 23), which belong to the pC194/pUB110/pBC16 RC replication plasmid family (12). The degree of amino acid identity of the deduced Rep protein of pIP823 with Rep proteins of the other plasmids of this family ranged from 31.8 to 52.6%. The predicted Rep protein of pIP823 contains a set of three conserved sequence motifs that are typical of initiator Rep proteins of RC replication replicons related to the E. coli bacteriophage φX174, and this Rep is closely related to those of the pC194 family (12). A sequence similar to that of dso of the RC replication plasmids was detected 449 bp upstream from the rep gene. This sequence, from positions 796 to 821, differs by a single nucleotide from the dso of pTB913 (Fig. 1). We also identified in plasmid pIP823, between nucleotides 2775 and 3046 upstream from the mob gene, a conversion signal identical to that of the palU-type sso of pTB913 (8). Therefore, it is likely that both plasmids have the same mechanism of replication initiation and termination. The product of the third ORF, from positions 3206 to 742, corresponds to a Mob protein possessing 97.6% identity with the Pre protein of plasmid pTB913, 90.4% identity with that of pTB53, but only 60.5% identity with the Mob protein of pUB110. Proteins Pre and Mob are involved in site-specific recombination and conjugative mobilization of these plasmids (23). The high level of similarity between the product of the ORF of pIP823 and the Pre and Mob proteins of pTB913 and pUB110 makes it likely that this ORF is responsible for recombination or mobilization of pIP823. A sequence similar to that of the RS_A site of pUB110 was identified between positions 3127 and 3150 upstream from the mob gene. The RS_A site of pUB110 has been shown to be involved in conjugative mobilization, and it has been suggested that the potential palindromic structure of this site might have a function similar to that of the origins of transfer of gram-negative bacteria (24, 28).

Host range, stability, and copy number of pIP823.Plasmid pIP823 was introduced by transformation into B. subtilis 168, E. coli HB101, E. faecalis JH2-2, L. monocytogenes LO17RF,S. aureus RN4220, and E. coli DH5α. The plasmid conferred high-level resistance to trimethoprim (MICs from 1,028 to 2,056 μg/ml) and was stably maintained in the various hosts, with 100% of the cells retaining the plasmid after growth for ca. 100 generations in the absence of trimethoprim. The copy number of pIP823 was estimated to vary from 2 to 20 copies per chromosome equivalent, depending on the host.

Identification of ssDNA pIP823 replication intermediates.Numerous RC replication plasmids from gram-positive bacteria accumulate ssDNA molecules (32, 33). We screened for the presence of pIP823 ssDNA intermediates in lysates of L. monocytogenes BM4293, E. faecalis JH2-2 (pIP823), S. aureus RN4220 (pIP823),B. subtilis 168 (pIP823), and E. coliHB101 (pIP823). As shown in Fig. 2 (lane 2), in the absence of rifampin and erythromycin L. monocytogenes BM4293 accumulated ssDNA molecules, suggesting thatsso of pIP823 is not efficient in this host. In the presence of erythromycin (lane 3), ssDNA was converted to dsDNA. In the presence of erythromycin and rifampin, ssDNA molecules accumulated inL. monocytogenes BM4293 (lane 4) but also inB. subtilis (pIP823) (lane 7), S. aureus (pIP823) (lane 6), and E. coli HB101 (pIP823) (lane 8), confirming that conversion of ssDNA to dsDNA is dependent on RNA polymerase activity in these hosts. Under similar culture conditions, multimer ssDNA intermediates were detected inE. faecalis JH2-2 (pIP823) lysates (lane 5). In contrast to pUB110, which does not replicate in E. coli, pIP823 replicated and accumulated ssDNA in this species. Thesso of pIP823 was RNA polymerase dependent (Fig. 2 and data not shown). Usually sso shows activity only in a limited number of hosts (11, 12). The amount of ssDNA molecules accumulated during RC replication is plasmid and host dependent since the conversion rate of ssDNA to dsDNA depends on the efficiency of the host machinery to recognize a given plasmid’s sso(36).

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Fig. 2.

Detection of single-stranded pIP823 DNA. Bacterial cultures were grown to mid-logarithmic phase and treated for 2 h with (i) no addition, (ii) erythromycin (100 μg/ml), or (iii) erythromycin plus rifampin (100 μg/ml each). Total DNA was prepared, and equivalent amounts of samples were run on a 0.9% agarose gel, transferred to a Nytran membrane, and hybridized to an in vitro³²P-labeled pIP823 probe. Lanes: 1, supercoiled-DNA ladder; 2 to 4, L. monocytogenes BM4293: 2, no addition; 3, erythromycin added; 4, erythromycin and rifampin added; 5 to 8, erythromycin and rifampin added: 5, E. faecalis JH2-2 (pIP823); 6, S. aureus RN4220 (pIP823); 7,B. subtilis 168 (pIP823); 8, E. coliHB101 (pIP823).

pIP823 was not self-transferable by conjugation.In a previous study, we proposed that transferable plasmid-mediated multiple-antibiotic resistance and transposon-borne tetracycline resistance in L. monocytogenes result from acquisition of these genetic elements from enterococci (4). Based on the identity of dfrD of L. monocytogenes BM4293 with the corresponding gene in Staphylococcus haemolyticus, we also suggested that trimethoprim resistance in Listeriamay have originated in Staphylococcus (3). The data obtained since then show that trimethoprim resistance in strain BM4293 is associated with an RC replicon, a plasmid type common in staphylococci. It is thus possible that trimethoprim resistance is due to plasmid transfer, and we therefore studied the transferability of pIP823. As expected, attempts to transfer pIP823 by conjugation between L. monocytogenes, E. faecalis,S. aureus, B. subtilis, andE. coli were unsuccessful, suggesting that pIP823 was not self-transferable (data not shown). The ability of pIP823 to be mobilized by various conjugative plasmids and transposons between these gram-positive and gram-negative species in which it replicates was then studied (Table 2).

Conjugative mobilization of pIP823 by self-transferable plasmids from gram-positive bacteria.The broad-host-range enterococcal plasmid pAMβ1 was introduced by conjugation fromE. faecalis BM4110 (pAMβ1) into L. monocytogenes BM4293 and S. aureus RN4220 (pIP823) at mobilization frequencies of 3.7 × 10⁻² and 9.3 × 10⁻⁶, respectively. Transfer by conjugative mobilization of plasmid pIP823 was obtained from the L. monocytogenes BM4293 (pAMβ1) donor to L. monocytogenes LO17RF andE. faecalis JH2-2 recipients. From transconjugantL. monocytogenes LO17RF (pAMβ1 plus pIP823), pIP823 could be mobilized to L. monocytogenesEGDSmR and E. faecalis BM4110, and from transconjugantE. faecalis JH2-2 (pAMβ1 plus pIP823), it could be mobilized to L. monocytogenes EGDSmR. The resistance phenotypes of 100 transconjugants from each mobilization experiment indicated that pAMβ1 was cotransferred in all cases. Mobilization of pIP823 by pAMβ1 was not obtained with (i) L. monocytogenes BM4293 (pAMβ1) as a donor and the other gram-positive bacteria or E. coli as recipients or (ii) an S. aureus donor and either L. monocytogenes or S. aureus recipients. Nevertheless, pAMβ1 could be conjugated from L. monocytogenes BM4293 to S. aureus 80CR5RF and fromS. aureus RN4220 to L. monocytogenesLO17RF at frequencies of 10⁻⁵ and 2 × 10⁻⁹, respectively. No mobilization of pIP823 by pAMβ1 could be detected from L. monocytogenes toS. aureus, B. subtilis, andE. coli (Table 2). The gram-negative and gram-positive shuttle plasmid pAT191 contains the origin of replication and the transfer functions of pAMβ1, the replication origin of pBR322, and the transfer origin of IncP plasmid RK2 from gram-negative bacteria (33). This bireplicon, which conjugates from E. faecalis to E. coli, was introduced by conjugation from E. faecalis BM4110 (pAT191) to L. monocytogenes BM4293 and E. faecalis JH2-2 (pIP823) with frequencies of 1.8 × 10⁻³ and 1.7 × 10⁻², respectively. Conjugative mobilization of pIP823 by pAT191 was obtained from L. monocytogenes BM4293 and E. faecalis JH2-2 (pIP823) donors to E. faecalis BM4110 and E. coli HB101 recipients. Plasmid pIP823 could not be mobilized by conjugation by pAT191 from E. coli HB101 toE. coli K802N, L. monocytogenes LO17RF, or E. faecalis JH2-2.

Conjugative mobilization of pIP823 by the streptococcal conjugative transposon Tn1545.Plasmid pIP823 was introduced by electrotransformation into E. faecalisJH2-2::Tn1545 and L. monocytogenesLO17RF::Tn1545. The plasmid was successfully mobilized by conjugative transposon Tn1545 fromL. monocytogenes LO17RF::Tn1545(pIP823) to L. monocytogenes EGDSmR and E. coli HB101 and from E. faecalisJH2-2::Tn1545 (pIP823) to L. monocytogenes EGDSmR (Table 2). None of the transconjugants contained Tn1545. No conjugative mobilization of pIP823 by Tn1545 from L. monocytogenesLO17RF::Tn1545 (pIP823) to E. faecalis BM4110 and E. coli HB101 and fromE. faecalis JH2-2::Tn1545 (pIP823) to E. faecalis BM4110 was obtained.

Conjugative mobilization of pIP823 by self-transferable plasmids from gram-negative bacteria.Plasmids RP4, R64, and pIP55-1 were introduced by conjugation into E. coliHB101 (pIP823) with frequencies of 1.4 × 10⁻⁴, 2.2 × 10⁻⁴, and 1.4 × 10⁻⁴, respectively. Plasmid pIP823 could be mobilized from E. coli HB101 to E. coli K802N by the three plasmids (Table 2). In the experiment with RP4 as a helper plasmid, of the 100 transconjugants analyzed, all had acquired both pIP823 and RP4 whereas R64 was cotransferred to only 32% of the 100 transconjugants studied. Plasmid pIP823 was mobilizable from E. coli K802N (RP4 plus pIP823) to L. monocytogenes EGDSmR but not to E. faecalis BM4110 or from E. coli K802N (RP64 plus pIP823) or K802N (pIP55-1 plus pIP823) toL. monocytogenes EGDSmR and E. faecalisBM4110.

Plasmid pOX38-Km, an F-factor derivative, was introduced by conjugation into E. coli HB101 (pIP823) and E. coliDH5α (pIP823) at frequencies of 1.2 × 10⁻⁴and 1.1 × 10⁻¹, respectively. Plasmid pIP823 was mobilized from E. coli DH5α (pOX38-Km plus pIP823) to E. coli HB101 but not from E. coli HB101 (pOX38-Km plus pIP823) to E. coliDH5α and L. monocytogenes LO17RF (Table 2).E. coli SM10, which contains the IncP group transfer functions of RK2 integrated into the chromosome, was electrotransformed with pIP823 DNA. Conjugative mobilization was obtained from E. coli SM10 (pIP823) toE. coli K802N and L. monocytogenesEGDSmR but not to E. faecalis BM4110. This result indicates that mobilization of pIP823 by RK2 occurs in the absence of cointegration.

The presence of pIP823 in transconjugants from all of the mobilization experiments described above was examined by amplification of total DNA with oligonucleotides specific for rep, dfrD, andmob followed by hybridization of the PCR products obtained with pIP823 DNA as a probe (data not shown). For each strain, fragments of the expected size which hybridized with the pIP823 probe were obtained. Small nonconjugative plasmids have been shown to be mobilized by conjugative plasmids among gram-positive bacteria. The S. aureus plasmid pC221 is mobilized by pG01 (25), whereas pLS20 can mediate transfer of pBC16 and pUB110 (15). The nonconjugative streptococcal plasmid pMV158 can be mobilized by both pAMβ1 and pIP501 (24). The mechanism of mobilization of relaxable plasmids in S. aureus appears to be analogous to that of mobilization by donation in gram-negative bacteria (25). Transfer by mobilization of pC194 and pUB110 by the conjugative transposons Tn925 and Tn916between Bacillus species has been described previously (20, 29). In contrast to pIP823, pTB913 was reported to be nonmobilizable (22). This is surprising in view of the similarities between the Mob proteins and the RS_A sites of the two plasmids. It would be interesting to examine transfer of pTB913 in the context developed here.

In conclusion, we have characterized the natural RC plasmid pIP823 fromL. monocytogenes BM4293, which bears the gene that encodes trimethoprim-resistant S2DHFR. This plasmid belongs to the pC194/pUB110 family of RC replication plasmids. The similarities with pUB110 from Staphylococcus and pTB913 fromBacillus include all the features essential for this replication mode: (i) a dso sequence for initiation of plus-strand synthesis, (ii) a rep lication protein, and (iii) ansso sequence for initiation of minus-strand synthesis. Rep of pIP823 was identical to those of pTB913 and pUB110. Plasmid pIP823 bears the gene that encodes a Mob protein showing a high degree of identity with Mob of pTB913 and possesses a potential RS_A site closely related to those of members of the pC194/pUB110 RC replication plasmid family. Plasmid pIP823 has an exceptionally broad host range of replication since it is maintained stably in L. monocytogenes, E. faecalis, S. aureus, B. subtilis, and E. coli, in which plasmid ssDNA intermediates accumulated. One of the main features of RC plasmids is their promiscuity, which is attributed to replication functions that appear to be active in a large variety of hosts. Other functions such as transferability by mobilization, cassette exchange, and recombination may also contribute to dissemination of these plasmids. Thebleo and knt genes of pTB913 and pUB110 confer resistance to bleomycin and kanamycin, respectively. Plasmid pIP823 may have diverged from pTB913 by replacement of the bleomycin and kanamycin resistance cassettes by the trimethoprim resistance determinant at the recombination-specific site RS_A (Fig. 1); the reverse filiation is as likely. However, it is noteworthy that pTB913 has been detected only in an integrated state as part of plasmid pTB19 ofBacillus. The high degree of similarity between the RC plasmids indicates easy horizontal transfer of these replicons between gram-positive cocci and bacilli, including Listeria. In addition, we have demonstrated conjugative mobilization of pIP823 by plasmids and a transposon between L. monocytogenes,E. faecalis, and E. coli. These findings raise, once more, the question of the so-called genetic barrier between gram-positive and gram-negative bacteria (6). Based on its properties, pIP823 might be attractive for the development of new broad-host-range cloning and expression gram-positive–gram-negative shuttle vectors. Characterization of pIP823 is also of interest in view of the possible dissemination of trimethoprim resistance in Listeria.