Genetic and physiological analysis of conjugation in Streptococcus faecalis

Abstract

In an effort to elucidate the mechanisms of conjugal plasmid transfer in Streptococcus faecalis, a genetic analysis of the sex pheromone-dependent tetracycline resistance plasmid pCF-10 was initiated. Rare transconjugants obtained from short matings with wild-type donors not exposed to sex pheromones were screened for increased donor potential in a subsequent mating. From this screening, a mutant plasmid, designated pCF-11, whose transfer functions are expressed in the absence of pheromone induction was isolated. Cells carrying pCF-11 spontaneously clump when grown in broth culture but do not excrete sex pheromones active against wild-type donors. In the course of initial experiments, it was observed that physiological conditions could affect plasmid transfer frequency. Therefore, a set of standardized optimal mating conditions was defined. The experiments carried out to determine these conditions revealed that a transient increase in transfer frequency of about 2 orders of magnitude occurred in early-exponential-phase donor cells. This peak of activity is independent of sex pheromone response, since it was observed with induced or uninduced donor cells carrying either pCF-11 or pCF-10.In Streptococcusfaecalis, donor cell aggregation and transfer functions of certain conjugative plasmids are induced by sex pheromones, or clumping-inducing agents (CIA), produced by recipient cells (2)(3)(4). Most of the experimental work on sex pheromone-dependent plasmid transfer has been carried out with hemolysin and bacteriocin plasmids (24). We recently reported the identification of a CIA-dependent R factor carrying tetracycline resistance (Tetr) (5).This plasmid, designated pCF-10, is a useful model for a genetic study of conjugation since it carries a selectable marker on a plasmid whose transfer genes can be turned on and off by the addition or removal of CIA. We have begun to isolate a series of mutant derivatives of this plasmid and to analyze the effects of the mutations on the donor phenotype. In the course of these initial genetic studies, occasional variations in plasmid transfer efficiencies which appeared to be related to physiological conditions of the cells in the mating mixtures were observed. In light of these observations, we felt that it would be important to define optimal mating conditions for the demonstration of differences in mating behavior between wild-type and mutant donors. In this paper, we describe the isolation of a mutant plasmid which exhibits a high donor potential in the absence of CIA. We t Present address: Department of Microbiology, University of Michigan, Ann Arbor, MI 48109.also discuss experiments in which we employed the wild-type and mutant plasmids to determine the effects of growth conditions and cell density on plasmid transfer and CIA response. MATERIALS AND METHODSBacteria and growth medium. The strains used in this study are listed in Table 1. BYGT medium (5) was employed in all experiments.Mating experiments. For the initial experiments described in this paper, donor and recipi...

Enterococcus faecalisstrains with compromised CRISPR-Cas defense emerge under antibiotic selection for a CRISPR-targeted plasmid

“…Due to the critical catalytic function of the RuvC domain, we hypothesize that the Ala749Thr substitution confers a loss of Cas9 function. We tested the ability of the BHI- and erythromycin-passaged WT4 populations to interfere with plasmid transfer using derivatives of the PRP, pCF10 (45), that are targeted by CRISPR3 spacers 1, 6, and 7. These experiments revealed that the Ala749 mutation indeed renders these populations deficient for CRISPR-Cas activity (Fig 2).…”

Control of genes for conjugative transfer of plasmids and other mobile elements

“…A number of Enterococcal plasmids (which are all bigger than 25 kb) are transferred very efficiently in liquid cultures – as high as 0.5 per donor [144]. The most extensively studied plasmids from this group are: pAD1 (60 kb), encoding hemolysin‐bacteriocin production and UV resistance, pCF10 (54 kb), encoding tetracycline resistance, and pPD1 (59 kb), a bacteriocin producer.…”

Control of genes for conjugative transfer of plasmids and other mobile elements

“…A number of Enterococcal plasmids (which are all bigger than 25 kb) are transferred very e¤ciently in liquid cultures^as high as 0.5 per donor [144]. The most extensively studied plasmids from this group are: pAD1 (60 kb), encoding hemolysin-bacteriocin production and UV resistance, pCF10 (54 kb), encoding tetracycline resistance, and pPD1 (59 kb), a bacteriocin producer.…”