Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 07 Dec 2018 11:08:05 Microbiology (2002), 148, 2489–2495 Printed in Great Britain Differential effects of Kid toxin on two modes of replication of lambdoid plasmids suggest that this toxin acts before, but not after, the assembly of the replication complex Katarzyna Potrykus, 1 Sandra Santos, 2 Marc Lemonnier, 2 Ramon Diaz-Orejas 2 and Grzegorz We grzyn 1,3 Author for correspondence : Grzegorz We grzyn. Tel : 48 58 346 3014. Fax: 48 58 301 0072. e-mail : wegrzynbiotech.univ.gda.pl 1 Department of Molecular Biology, University of Gdan  sk, Kladki 24, 80-822 Gdan  sk, Poland 2 Centro de Investigaciones Biolo  gicas, C.S.I.C., Vela  zquez 144, 28006 Madrid, Spain 3 Institute of Oceanology, Polish Academy of Sciences, S  w. Wojciecha 5, 81-347 Gdynia, Poland Kid is a small protein that is encoded by plasmid R1. It is a toxin that belongs to a killer system that ensures the stability of the plasmid in host cells. The results of previous studies have suggested that Kid is an inhibitor of DNA replication, possibly acting at the onset of initiation. Here, the authors tested the effects of Kid on oriλ-intitiated and oriJ-initiated replication, which may be driven by both the newly assembled replication complex and the heritable complex. It was found that Kid inhibits only replication that is driven by the newly assembled replication complex. The authors also report that Kid inhibits ColE1-like plasmid replication in vivo, in agreement with the previously reported inhibition of ColE1 during in vitro replication. It is proposed that the Kid toxin acts at the level of replication either by preventing de novo assembly of the replication complex or by impairing the functional interactions of the replication complex at the initiation stage. Keywords : plasmid R1, λ plasmids, oriJ-based plasmid, inheritance of the replication complex INTRODUCTION Plasmid R1 contains the parD region, which is re- sponsible for the stabilization of the plasmid in host cells. This stabilization is based on the mechanism involved in the killing of plasmid-free cells. Two proteins are encoded by the parD system, Kid and Kis. Kid is a toxin that is responsible for killing cells and Kis neutralizes the lethal activity of Kid (Bravo et al., 1987, 1988). Previous studies have revealed that Kid inhibits the in vitro replication of a DnaB-dependent replicon, ColE1, at an early stage and that this toxin also prevents the induction of the λ prophage. Moreover, the overpro- duction of the DnaB protein (a helicase) results in a considerable increase in the viability of cells expressing the kid gene (Ruiz-Echevarria et al., 1995). These results led to the original proposal that Kid could target DnaB, thus poisoning the replication complex at the initiation stage (Ruiz-Echevarria et al., 1995). Alternatively, Kid could interact with another component of the replication complex in a manner that could be disrupted by an excess of DnaB in the cell. However, it remains unknown whether this toxin acts before or after the assembly of the replication complex. To test at which step Kid inhibits the replication process, we used in vivo systems based on two phage-derived replicons, λ and oriJ. There are two ‘pathways’ of replication for both of these replicons. After a round of replication, the replication complex is inherited by one of two daughter plasmid copies and it may function in future rounds of replication ; a new replication complex has to be assembled in the second plasmid copy (We  grzyn & Taylor, 1992 ; Potrykus et al., 2000). The heritable replication complex contains λ-encoded O and P replication proteins and two bacterial proteins : DnaB and DnaK (a molecular chaperone) (We  grzyn & We  grzyn, 1995, 2001 ; We  grzyn et al., 1995a, b, 1996; Z  ylicz et al., 1998 ; Potrykus et al., 2002). It is possible to restrict the replication of plasmids λ and oriJ solely to the pathway that is based on the heritable replication complex (Fig. 1). Amino-acid starvation prevents the synthesis of new replication proteins, thus 0002-5710  2002 SGM 2489