Efficient transfer of the pheromone-independent Enterococcus faecium plasmid pMG1 (Gmr) (65.1 kilobases) to Enterococcus strains during broth mating

Abstract

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10(-4) per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMbeta1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.

FIG. 1

Physical map of pMG1. Fragments produced by restriction endonuclease restriction of pMG1 DNA are denoted by letters.

FIG. 2

Kinetics of transfer for pheromone-independent plasmid pMG1 and pheromone-dependent plasmid pAM714. Matings were carried out with mixtures that each consisted initially of 0.05 ml of the donor, 0.45 ml of the recipient, and 4.5 ml of fresh N2GT broth. The mixtures were incubated at 37°C with gentle shaking. At the indicated time points, 0.1-ml samples were removed, diluted appropriately, and plated on THB agar plates containing gentamicin or erythromycin for the selection of pMG1 or pAM714 transconjugants, respectively, plus drugs for selection of the recipient strains. Symbols: ○, transfer from E. faecalis FA2-2(pMG1) to E. faecalis JH2SS; ▵, transfer from E. faecium KTSS(pMG1) to E. faecium KTRF; □, transfer from E. faecalis JH2SS(pAM714) to E. faecalis FA2-2.

FIG. 3

Microscopic observation. The mating mixture (A) and pheromone-exposed donor cells (B) were observed by microscopy (Nikon Optiphoto 2 at ×1,000 magnification) after 2 h of mating between E. faecalis FA2-2(pMG1) donor cells and E. faecalis JH2SS recipient cells, as shown in Fig. 2 (A), and after 2 h of exposure of E. faecalis FA2-2(pMG1) to FA2-2 culture filtrate (pheromone), as shown in Table 3 (B).

FIG. 4

Agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs and hybridization with plasmid pMG326, which contains a part (11.5 kb) of EcoRI fragment A, or the pMG1 probe. (A) Agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs. Plasmid pIP501 was digested with KpnI, and the other plasmids were digested with EcoRI. Lanes: 1, pAMβ1; 2, pAM373; 3, pIP501; 4, pMG1; 5, pAD1; 6, pPD1; 7, HindIII-digested lambda DNA. Duplicate gels were Southern blotted and hybridized to pMG326 DNA (B) or pMG1 DNA (C).