Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family

Abstract

Multidrug-resistant staphylococci often harbour plasmids that carry genes conferring resistance to several antimicrobial compounds. Many of these multiresistance plasmids appear to utilize a related theta-type replication system for which multiresistance plasmid pSK1 serves as a prototype. Essential pSK1 replication elements were identified by cloning segments of the replication region and testing the resulting plasmids for replication proficiency. An iterated region within rep and a DNA segment of up to 68 bp upstream of the rep promoter were both found to be essential for origin activity. The Rep protein was overexpressed as a 6¾His-tagged C-terminal fusion protein and was shown to bind in vitro to four Rep boxes located within the rep coding region. Inactivation of a divergently oriented promoter upstream of rep, designated P rnaI , resulted in an elevated plasmid copy number. Comparative analyses suggest that the replication systems of many staphylococcal multiresistance plasmids share a similar genetic organization and utilize an antisense-RNA-mediated regulatory mechanism for copy number control.

Genetic requirements for replication initiation of the staphylococcal multiresistance plasmid pSK41

“…No evidence was found to suggest that any of the four Rep boxes are involved in negative regulation of pSK41 replication, since all Rep box deletions reduced plasmid copy number. Given that replication of staphylococcal multiresistance plasmids such as pSK41 and pSK1 is controlled by an antisense RNA (Kwong et al, 2006(Kwong et al, , 2008, a role in the negative regulation of plasmid replication may not be required for the oriV iterons of these plasmids.…”

“…The RepA_N family includes a group of conjugative pheromone response plasmids from Enterococcus faecalis (De Boever et al, 2000;Francia et al, 2004;Hedberg et al, 1996), the vast majority of currently identified multiresistance plasmids from staphylococci Gering et al, 1996;Gill et al, 2005;Kwong et al, 2004Kwong et al, , 2008Zhang et al, 2003), as well as a number of plasmids from Lactococcus (Hagen et al, 2010), Lactobacillus (Thompson et al, 1999) and Bacillus (Tanaka et al, 2005). Several Rep proteins from this family have been shown to bind to repeated sequences that are located centrally within the rep gene, and these repeats have been shown to form part of the oriV (Francia et al, 2004;Kwong et al, 2004Kwong et al, , 2008Tanaka et al, 2005).…”

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

“…Hence, looping within a single origin would require that RepA proteins bind to nonconsecutive iterons (e.g., iterons 1-3, 1-4, or 2-4). However, DNase I protection studies on RepA-origin interactions clearly revealed that when RepA binds, every box is protected, which would not be consistent with nonconsecutive looping (28)(29)(30). Moreover, the pattern of protection in the footprints is strikingly periodic, which indicates essentially identical modes of RepA binding to each iteron, in particular for boxes 2-4 in the pSK41 origin (Fig.…”

“…genus-specific clusters, suggesting that this domain may perform a host-specific role (28)(29)(30)(31)(32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that reside within the plasmid origin (30).…”

“…The multiresistance RepA proteins are arguably among the most abundant of plasmid Rep proteins, yet how they function is not known. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long and variable linker region (∼30-50 residues), and a C-terminal domain (CTD) of ∼120 residues (28)(29)(30)(31). The NTD and CTD are both essential for replication.…”