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- Results and discussion
- Experimental procedures
pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid-free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone-responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone-binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface-exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917–lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.
- Top of page
- Results and discussion
- Experimental procedures
Enterococcus faecalis and Enterococcus faecium are normally harmless commensals that inhabit the human intestine, but they can act as opportunistic pathogens causing urinary tract infections, bacteraemia and infective endocarditis (Murray, 1990; Moellering, 1992; Jett et al., 1994). Enterococci are among the three most common types of bacteria involved in nosocomial infections in the USA (Lewis and Zervos, 1990). Multiple antibiotic resistance, including resistance to the ‘last resort’ antibiotic vancomycin, is widespread among the enterococci and represents a serious and growing clinical problem; endocarditis infections involving such organisms are associated with high mortality rates (Edmond et al., 1996; Huycke et al., 1998). The phenomenon of conjugative DNA transfer is particularly common among the enterococci and frequently involves highly transmissible plasmids and/or conjugative transposons, and antibiotic resistance determinants commonly reside on such elements (Clewell, 1990).
A group of highly conjugative plasmids found commonly in enterococci, especially in E. faecalis, are those which encode a mating response to small peptide sex pheromones secreted by plasmid-free (recipient) strains (for recent reviews, see Dunny and Leonard, 1997; Maqueda et al., 1997; Clewell, 1999). The response by donor bacteria involves synthesis of a plasmid-encoded protein surface adhesin referred to as ‘aggregation substance’ which facilitates the initial contact with recipient cells. Aggregation substance coats the donor cell surface and adheres to ‘enterococcal-binding substance’ (EBS) on the recipient surface. There is evidence that EBS corresponds at least in part to lipoteichoic acid (Ehrenfeld et al., 1986; Bensing and Dunny, 1993). As EBS is also located on the donor surface, donors exposed to a culture filtrate of recipients undergo a clumping response, a phenomenon that serves as a convenient assay in the quantification of pheromone activity (Dunny et al., 1979).
Once a copy of the plasmid is acquired by the recipient, production of the related pheromone in the transconjugants is shut down or masked; however, unrelated pheromones that induce a mating response by donors bearing different families of plasmids continue to be secreted. Each plasmid encodes a peptide that acts as a competitive inhibitor of the corresponding pheromone and is believed to help prevent self-induction by endogenous pheromone that may be produced in plasmid-containing cells. Some of the best studied pheromone-responding plasmids are pAD1, pCF10 and pPD1, which confer responses to the octa- or heptapetides cAD1, cCF10 and cPD1 respectively; their corresponding inhibitor peptides are designated iAD1, iCF10 and iPD1. The chromosome-encoded pheromone determinants have recently been identified in E. faecalis genome databases and show that the peptides represent components of the signal sequences of lipoprotein precursors (Clewell et al., 2000).
pAM373 is a relatively small (36.7 kb) pheromone-responding plasmid that was identified as one of at least five plasmids present in the E. faecalis clinical isolate RC73 (Clewell et al., 1985). It was of interest at that time because, unlike the case for other plasmids, the related pheromone activity (cAM373) detectable in culture filtrates of plasmid-free E. faecalis was also produced by Staphylococcus aureus but not by coagulase-negative staphylococci. Activity was also produced by Streptococcus gordonii (formerly Streptococcus sanguis) (strains Challis and G9B) and Enterococcus hirae (strain 9790); however, no activity was detected in culture filtrates of a number of other species tested. Filter matings between E. faecalis cells carrying pAM373::Tn917 (encodes erythromycin resistance) and recipient S. aureus or S. gordonii strains did not give rise to transconjugants, implying that the plasmid was unable to establish in these hosts; however, transfer to E. hirae was observed.
The cAM373 peptides from both E. faecalis and S. aureus have been characterized and found to differ only in their carboxyl-terminal amino acids; the structures were found to be AIFILAS and AIFILAA respectively (Mori et al., 1986; Nakayama et al., 1996). The inhibitor peptide encoded by pAM373 was determined to be SIFTLVA (Nakayama et al., 1995a).
pAM373 is also interesting in that although most other pheromone-responding plasmids have determinants for aggregation substance that are strongly homologous with each other based on hybridization data the determinant on pAM373 appears to be significantly different (Galli and Wirth, 1991; Hirt et al., 1996). This suggests possible differences in the mechanism of adherence to recipient cells. Indeed, Muscholl-Silberhorn (1999) has very recently reported that the pAM373 aggregation substance is not only structurally different but also binds to cells defective in EBS.
The initial report of pAM373 15 years ago (Clewell et al., 1985) preceded the rapid appearance of vancomycin resistance in enterococci that has occurred during the past decade. Now several classes of highly evolved vancomycin resistance gene clusters (e.g. vanA, vanB, vanC, etc.) can be found associated with clinical isolates of enterococci (Evers et al., 1996). Some are found on conjugative plasmids and reside on transposons such as Tn1546 (Arthur et al., 1993; Jensen et al., 1999; Willems et al., 1999). Thus far, only a few vancomycin-resistant clinical isolates of S. aureus have been reported (Hiramatsu et al., 1997; Turco et al., 1998); these are relatively low levels of resistance and are unrelated to the resistances that have been appearing in the enterococci. Considering the ability of pAM373 to respond to a pheromone that is produced by S. aureus, it would seem that this plasmid, or closely related plasmids, could play a significant role in facilitating movement of vancomycin resistance determinants from enterococci into this important human pathogen. Although pAM373 itself may not be capable of replicating in S. aureus, an involvement in the mobilization or delivery of other resistance-bearing elements is a real possibility. For this reason and because, when compared with other pheromone-responding plasmids, pAM373 has been reported to have significantly different structural properties based on hybridization, further analyses of this interesting element were initiated. The complete sequence of the plasmid as well as genetic analyses which include identification of determinants relating to regulation of the pheromone response are presented.