Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Abstract

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.PemK | mRNA interferase | parD | plasmid stability | RNase

Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

“…Improving copy number or partitioning at cell division has been pointed out to explain the unexpected high stability of low-copy plasmids [ 28 ]. It has been pointed recently that Kid can inhibit cell division and that this toxin achieves plasmid retention by uncoupling plasmid replication and cell division [ 21 ]; in addition, the same study proposes that the hok-sok antitoxin-toxin system of this plasmid could eliminate plasmid free cells that could arise due to failures in this process.…”

“…Similarly, in plasmid R1, coordination of the basic replicon functions and the kis-kid system activates the Kid toxin to rescue replication of cells with very low copy number [ 16 ]. Activated Kid toxin inhibit cell division, thus effectively achieving plasmid R1 retention by increasing plasmid replication and timing this replication rescue before cell division can occur [ 21 ]. Our report adds to this that the kis-kid system plays a more relevant role in control of plasmid copy number than previously suspected.…”

“…Two findings were key to explaining, in molecular terms, this new and intriguing signature of the coupling: (i) the identification of the RNase activity of the Kid toxin [ 13 , 14 , 15 ] and (ii) the finding that copB-repA mRNA contains two sites in the intergenic region of copB-repA mRNA that are efficiently cleaved by Kid [ 20 ]; this cleavage reduces the CopB levels, activates the repA promoter and increases plasmid replication efficiency. It has been recently reported that Kid cleaves mRNA of key cell division proteins; in this instance, Kid replication rescue occurs before cell division and effectively enforces plasmid retention by uncoupling plasmid replication and cell division [ 21 ]. We reported recently that increasing in trans the Kis antitoxin levels suppressed the “interference” phenotype; this suggested that Kis antitoxin levels could act as the switch connecting the replication and kis-kid toxin-antitoxin maintenance functions [ 22 ].…”

The Importance of the Expendable: Toxin–Antitoxin Genes in Plasmids and Chromosomes

“…Plasmid maintenance linked to the coordination of TAs with plasmid replication was initially reported by the Diaz-Orejas laboratory on the kis-kid TA encoded by plasmid R1 ( Ruiz-Echevarría et al, 1995a , b ), and further analyzed ( Pimentel et al, 2005 ; López-Villarejo et al, 2012 , 2015 ). Additional work revealed a further coordination of the kis-kid TA with cell cycle functions ( Pimentel et al, 2014 ). On the whole the above work supports that, failures in plasmid R1 replication reduces the levels of the Kis antitoxin and increases the activity of the Kid toxin.…”

Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host

“…The toxin component, Kid, is activated in cells which are under threat of loosing the plasmid through copy number depression ( Ruiz-Echevarría et al., 1995 ). It has been shown recently that Kid activation results in inhibition of cell division and stimulation of DNA replication, preventing plasmid loss and increasing its copy number ( Pimentel et al., 2014 ). The mechanism of cell division inhibition differs from the Rcd-indole system and Kid directs cleavage of mRNA of two proteins, FtsZ and ZapA, involved in cell division.…”