RNase/Anti-RNase Activities of the BacterialparDToxin-Antitoxin System

Abstract

The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single-and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

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

“…Kid had been reported to cleave mRNA at UAA, UAC (26), and UAU sites (collectively, UAH sites) (25). However, we observed cleavage at UUACU sites but not at UAA and UAU sequences close to them, and our results also seemed incompatible with cleavage simply at UAC sites (ref.…”

“…In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25,26), a view that has prevailed in the literature (14,16,(27)(28)(29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30,31).…”

“…These observations explained why activation of Kid in our mR1KK samples was not paralleled by a decrease in OD 600 relative to control samples at early time points. Moreover, although Kid cleaves mRNA and inhibits protein synthesis (24)(25)(26), the cell enlargement shown in Fig. 2A suggested that the toxin does not halt protein production completely in E. coli.…”

Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

“…16 In cell extracts, Kid, MazF and ChpBK have been shown to be able to inhibit protein synthesis and these proteins can act both in vitro and in vivo as endoribonucleases able to cleave RNA in the absence of ribosomes. [17][18][19][20][21][22][23] The Kid toxin cleaves RNA preferentially at the 5 0 side of the A residue in the nucleotide sequence 5 0 -UA(A/C)-3 0 of singlestranded regions, although cleavage in doublestranded regions, 17 and at the 3 0 side of the adenosine base, has been observed as well. 17,20 The presence of the antitoxins Kis and MazE prevents RNA cleavage by Kid and MazF, respectively.…”

“…[17][18][19][20][21][22][23] The Kid toxin cleaves RNA preferentially at the 5 0 side of the A residue in the nucleotide sequence 5 0 -UA(A/C)-3 0 of singlestranded regions, although cleavage in doublestranded regions, 17 and at the 3 0 side of the adenosine base, has been observed as well. 17,20 The presence of the antitoxins Kis and MazE prevents RNA cleavage by Kid and MazF, respectively. 17,18,20,21,23 The mechanism of RNA binding and cleavage by the Kid toxin is not known.…”