Molecular and structural characterization of the PezAT chromosomal toxin-antitoxin system of the human pathogen Streptococcus pneumoniae
06 Jul 2007-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology [etc.])-Vol. 282, Iss: 27, pp 19606-19618
TL;DR: Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site and in the PezA2PezT2 complex, Pez
Abstract: The chromosomal pezT gene of the Gram-positive pathogen Streptococcus pneumoniae encodes a protein that is homologous to the zeta toxin of the Streptococcus pyogenes plasmid pSM19035-encoded epsilon-zeta toxin-antitoxin system. Overexpression of pezT in Escherichia coli led to severe growth inhibition from which the bacteria recovered ∼3 h after induction of expression. The toxicity of PezT was counteracted by PezA, which is encoded immediately upstream of pezT and shares weak sequence similarities in the C-terminal region with the epsilon antitoxin. The pezAT genes form a bicistronic operon that is co-transcribed from a σ70-like promoter upstream of pezA and is negatively autoregulated with PezA functioning as a transcriptional repressor and PezT as a co-repressor. Both PezA and the non-toxic PezA2PezT2 protein complex bind to a palindrome sequence that overlaps the promoter. This differs from the epsilon-zeta system in which epsilon functions solely as the antitoxin and transcriptional regulation is carried out by another protein designated omega. Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site. In the PezA2PezT2 complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance.
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TL;DR: Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems.
Abstract: Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems. The cellular targets of these toxins are quite diverse and include DNA replication, mRNA stability, protein synthesis, cell-wall biosynthesis, and ATP synthesis. These toxins are co-expressed and neutralized with their cognate antitoxins from a TA (toxin-antitoxin) operon in normally growing cells. Antitoxins are more labile than toxins and are readily degraded under stress conditions, allowing the toxins to exert their toxic effect. Presence of at least 33 TA systems in Escherichia coli and more than 60 TA systems in Mycobacterium tuberculosis suggests that the TA systems are involved not only in normal bacterial physiology but also in pathogenicity of bacteria. The elucidation of their cellular function and regulation is thus crucial for our understanding of bacterial physiology under various stress conditions.
587 citations
Cites background from "Molecular and structural characteri..."
...This binding is mediated by the DNA-binding domain of the antitoxin, which has a specific structure, ribbon-helix-helix (RHH) or helix-turnhelix (HTH), for binding to double-stranded DNA (11, 58, 68, 73, 77, 79, 86)....
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TL;DR: This work has suggested that regulated cell death and lysis in bacteria play an important role in both developmental processes, such as competence and biofilm development, and the elimination of damaged cells,such as those irreversibly injured by environmental or antibiotic stress.
Abstract: Summary: Although the phenomenon of bacterial cell death and lysis has been studied for over 100 years, the contribution of these important processes to bacterial physiology and development has only recently been recognized. Contemporary study of cell death and lysis in a number of different bacteria has revealed that these processes, once thought of as being passive and unregulated, are actually governed by highly complex regulatory systems. An emerging paradigm in this field suggests that, analogous to programmed cell death in eukaryotes, regulated cell death and lysis in bacteria play an important role in both developmental processes, such as competence and biofilm development, and the elimination of damaged cells, such as those irreversibly injured by environmental or antibiotic stress. Further study in this exciting field of bacterial research may provide new insight into the potential evolutionary link between control of cell death in bacteria and programmed cell death (apoptosis) in eukaryotes.
347 citations
Additional excerpts
...pneumoniae (123, 178) have recently been studied....
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TL;DR: TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.
Abstract: Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.
232 citations
Cites background from "Molecular and structural characteri..."
...Binding of antitoxins to regulatory sites can be achieved either by HTH motifs (Khoo et al., 2007; Brown et al., 2009; Schumacher et al., 2009; Arbing et al., 2010) or by RHH folds (Takagi et al., 2005; Madl et al., 2006; Mattison et al., 2006; Oberer et al., 2007; Li et al., 2008; Dalton &…...
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...Homologs of ε and ζ that are sometimes denoted PezA and PezT, respectively, are encoded by certain Gram-positive species (Khoo et al., 2007)....
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...Binding of antitoxins to regulatory sites can be achieved either by HTH motifs (Khoo et al., 2007; Brown et al., 2009; Schumacher et al., 2009; Arbing et al., 2010) or by RHH folds (Takagi et al....
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TL;DR: It is demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin, the first crystal structure of a zeta toxin bound to its substrate.
Abstract: Most genomes of bacteria contain toxin–antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics.
207 citations
Cites background or methods from "Molecular and structural characteri..."
...(F) E. coli BL21 (DE3) cells expressing nontoxic PezTDC242 (D66T) show rRNA degradation only upon treatment with ampicillin (upper panel), but not after treatment with the translation inhibitor tetracycline (lower panel)....
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...Strikingly, we could provoke almost identical characteristics of cell death in E. coli expressing nontoxic PezT (D66T) by additional treatment of the culture with ampicillin (Figures 1C and S1F)....
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...structurally similar to poly- and mononucleotide kinases [17,18]....
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...To exclude the possibility that the observed phenotype is due to general overexpression, we performed control experiments with cells bearing the expression plasmid of the nontoxic variant PezT (D66T) [17]....
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...Structures of zeta toxins as well as mutational studies suggested that their toxicity is connected to an ATP-dependent phosphorylation event [17,18]....
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TL;DR: An overview of the literature for 2005 is attempted to highlight works of interest and novelty and draw attention to those works which it is felt have provided a route to better analysis and increased the ability to understand the meaning of thermodynamic change on binding.
Abstract: Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naive, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding.
191 citations
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