Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria

Abstract

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.

FIG. 1

Schematic representation of the relationship between pIP823 from L. monocytogenes (3,712 bp), pUB110 from S. 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 in dfrD (D), mob (M), dso (R1), and repU (R2) are indicated by filled arrowheads.

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 vitro ³²P-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. coli HB101 (pIP823).