The ribonucleolytic activity of the ribotoxin α-sarcin is not essential for in vitro protein biosynthesis inhibition.

Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.

Kis antitoxin couples plasmid R1 replication and parD (kis,kid) maintenance modules.

The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

Structure and function of bacterial kid-kis and related toxin-antitoxin systems.

Toxin-antitoxin systems were discovered as plasmid auxiliary maintenance cassettes. In recent years, an increasing amount of structural and functional information has become available about the proteins involved, allowing the understanding of bacterial cell growth inhibition by the toxins on a molecular level. A well-studied TA system is formed by the proteins Kid and Kis, encoded by the parD operon of the Escherichia coli plasmid R1. The toxicity of Kid has been related to its endoribonuclease activity, which is counteracted by binding of the antitoxin Kis at the proposed active site. In this review, the structural studies on the Kid-Kis system are compared to those of three related toxin-antitoxin systems: MazF-MazE, CcdB-CcdA and RelE-RelB.

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.

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

parD toxin-antitoxin system of plasmid R1--basic contributions, biotechnological applications and relationships with closely-related toxin-antitoxin systems.

Toxin-antitoxin systems, as found in bacterial plasmids and their host chromosomes, play a role in the maintenance of genetic information, as well as in the response to stress. We describe the basic biology of the parD/kiskid toxin-antitoxin system of Escherichia coli plasmid R1, with an emphasis on regulation, toxin activity, potential applications in biotechnology and its relationships with related toxin-antitoxin systems. Special reference is given to the ccd toxin-antitoxin system of plasmid F because its toxin shares structural homology with the toxin of the parD system. Inter-relations with related toxin-antitoxin systems present in the E. coli chromosome, such as the parD homologues chpA/mazEF and chpB and the relBE system, are also reviewed. The combined structural and functional information that is now available on all these systems, as well as the ongoing controversy regarding the role of the chromosomal toxin-antitoxin loci, have made this review especially timely.