Characterization of an Active Partition System for theEnterococcus faecalisPheromone-Responding Plasmid pAD1

Abstract

Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. pAD1 is representative of a large and globally disseminated family of conjugative plasmids in Enterococcus faecalis. It is a 60-kb, low-copy-number (one to four copies per chromosome) element that encodes a mating response to the peptide sex pheromone cAD1. It also determines a virulence-related cytolytic (hemolysin/bacteriocin) exotoxin, as well as resistance to UV light. (For recent reviews of pheromone-responding plasmids, see references 6, 7, and 8.) Although aspects of conjugation have been studied extensively, much less is known about the control of pAD1 replication and maintenance. A previous investigation of pAD1 identified a 5-kb region of DNA able to support replication, copy control, and stable inheritance, and it was found that three determinants, designated repA, repB, and repC, were necessary for normal maintenance (46). Data suggested that RepA (336 amino acids) was involved in replication initiation whereas RepB (281 amino acids) and RepC (123 amino acids) were involved in maintaining copy number and stability. As illustrated in Fig. 1, the repA and repB determinants diverge and are separated by a series of iterons, whereas repC is immediately downstream of, and probably transcribed together with, repB. The iterons correspond to two groups of octanucleotide repeats with the sequence TAGTARRR: 13 iterons and 12 iterons are separated by a 75-bp AT-rich (80%) "spacer." There are also three tandemly arranged iterons located downstream of repC.ERecently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were identified and characterized (15). It was found that repA alone, under an artificial promoter, was sufficient for replication when cloned on a plasmid devoid of replication ability. The chimera maintained itself at a high copy number (20 to 30 copies per chromosome), in contrast to the low copy number of intact pAD1. In addition, the replication origin was found to be located within a 170-bp segment of repA, which alone could facilitate replication in cis as long as RepA was provided in trans. RepA was also found to bind specifically to several sites containing inverted-repeat sequences within oriV and bound nonspecifically to singlestranded DNA (15).Whereas RepA alone was able to support replication, the high-copy-number repA chimera was not highly stable in that after 30 generations of unselected growth in broth about 60% of the cells were observed to have lost the plasmid (15); an efficient partitioning system was clearly missing. Good candidates for components of a partition system in the intact pAD1 are...

Modular Evolution of Tn GBS s, a New Family of Integrative and Conjugative Elements Associating Insertion Sequence Transposition, Plasmid Replication, and Conjugation for Their Spreading

“…This sequence represents a putative origin of replication recognized by the TnGBS2 RepA protein. Accessory proteins (RepB and RepC) shown to be involved in plasmid stability (28) are missing in TnGBS2 and in related ICEs. In summary, we identified different replicator proteins and their cognate putative origin of replication in both TnGBS1 and TnGBS2, suggesting that these replication modules are functional.…”

Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative ofBacillus subtilis(natto) Plasmid pLS32

“…The E. coli plasmids, such as F, P1, and R1, carry a cis sequence with which one of the par proteins interacts (4,6,10,15,50). In an attempt to narrow down the cis DNA region necessary for the stable maintenance of pBET131 (Fig.…”

“…Stability determinants involving the active partitioning mechanism have been reported for several plasmids from Gram-positive bacteria. Plasmids from Lactococcus lactis, Streptococcus pyogenes, and Enterococcus faecalis carry proteins with sequence similarities to the type I ATPases (8,9,10,23,36). The partition system encoded by a staphylococcal plasmid, pSK1, is unusual in that the parA gene product, which has no sequence similarity to known type I or type II ATPases, exerts the stabilization function without the involvement of other factors (41).…”

Tales of conjugation and sex pheromones

“…RepC binds cooperatively to the iterons, whereas RepB interacts with the same region in the presence of ATP-but only if RepC is bound. 137 The iteron region probably represents a "centromere-like" structure during partitioning by associating with a host segregation apparatus during cell division. 139 The presence of isolated iteron sequences near the promoters of repA and repB suggests that RepC may contribute to regulating expression of these determinants.…”

“…An operon consisting of repB and repC (see Figs. 3 and 4) encodes a partitioning system 137 with RepB representing a member of the ParA superfamily of ATPases. 138 A series of iterons (12-and 13-octanucleotide diverging repeats [TAGTARRR] separated by an AT-rich, 75-nucleotide "spacer") is located between the diverging repA and repB.…”