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.     

RNase/anti-RNase activities of the bacterial parD toxin-antitoxin system

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.     

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection.     

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.     

Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity

Kid and Kis are, respectively, the toxin and antitoxin encoded by the parD operon of plasmid R1. The recently solved crystal structure of Kid has revealed that this protein closely resembles the CcdB toxin of plasmid F. In CcdB, the residues involved in toxicity are located at the carboxy-terminal end of the protein. However, an analogous information on the Kid toxin was not available. Here, we have characterized a collection of non-toxic mutants of the Kid protein and identified the residues that affected the toxicity but not the co-regulatory activity of Kid. These are located in two discrete regions of the protein, at the amino and carboxy-terminal ends. Particularly, residues E18 and R85, that are conserved in the Escherichia coli ChpAK and RelE toxins, are affected by amino-acid changes that alter neither the overall structure of the protein nor its state of association, as shown by CD and sedimentation equilibrium analyses. However, thermal denaturation and intrinsic tryptophan fluorescence emission data point to subtle local changes at the N-terminal end of the protein. The implications of these results in the current model on the structure and function of Kid-related bacterial toxins are discussed.     

An ABC transporter is essential for resistance to the antitumor agent mithramycin in the producerStreptomyces argillaceus

The organization of the phosphate-specific transport (pst) operon inPseudomonas aeruginosa has been determined. The gene order of thepst operon ispstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the knownEscherichia coli pst operon is the lack of thepstS gene encoding a periplasmic phosphate (P_i)-binding protein. Even though the threepst genes were absolutely required for P_i-specific transport, expression of thepst operon at high levels did not increase P_i uptake inP. aeruginosa. DNA sequences for thepstB andphoU genes have been determined previously. The newly identifiedpstC andpstA genes encode possible integral membrane proteins of 677 amino acids (M _r 73 844) and 513 amino acids (M _r 56 394), respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in theirE. coli counterparts. A chromosomal deletion of the entirepst operon renderedP. aeruginosa unable to repress P_i taxis under conditions of P_i excess. ThephoU andpstB genes are essential for repressing P_i taxis. However, mutants lacking either PstC or PstA alone were able to repress P_i taxis under conditions of P_i excess.     

SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3′ end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3′ end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3′ end of the histone mRNA with the 5′ end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.     

Identification of two new genes,mukE andmukF,involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.