Analysis of the Conjugal Transfer System of the Pheromone- Independent Highly Transferable Enterococcus Plasmid pMG1: Identification of a tra Gene ( traA ) Up-Regulated during Conjugation

Abstract

ABSTRACT pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance. pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis . To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation. RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes. One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating. The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture. The increase was not observed in cultures of the donor cells or recipient cells. These findings suggested that the increase was a conjugation-specific event. The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci. The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 × 10 −7 and 1.1 × 10 −3 per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 × 10 −3 and 8.5 × 10 −2 per donor cell, respectively, in filter mating experiments. This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating. 71ORF2, which is designated traA , is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating.