In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system.
TL;DR: In vivo studies reveal a short halflife of the antitoxin and a long lifetime of the ζ toxin and when transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.
Abstract: Streptococcus pyogenes pSM19035-encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid-deprived cells and epsilon as an antitoxin that neutralises the activity of zeta. The epsilon and zeta proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co-crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half-life of the epsilon antitoxin (-18 min) and a long lifetime of the zeta toxin (>60 min). When transcription-translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the background.
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TL;DR: Evidence now indicates that toxin–antitoxin loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.
Abstract: Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.
991 citations
TL;DR: The dissection of the interaction of the toxins with intracellular targets and the elucidation of the tertiary structures of toxin-antitoxin complexes have provided exciting insights into toxin-antsitoxin behavior.
Abstract: Antibiotic resistance, virulence, and other plasmids in bacteria use toxin-antitoxin gene pairs to ensure their persistence during host replication. The toxin-antitoxin system eliminates plasmid-free cells that emerge as a result of segregation or replication defects and contributes to intra- and interspecies plasmid dissemination. Chromosomal homologs of toxin-antitoxin genes are widely distributed in pathogenic and other bacteria and induce reversible cell cycle arrest or programmed cell death in response to starvation or other adverse conditions. The dissection of the interaction of the toxins with intracellular targets and the elucidation of the tertiary structures of toxin-antitoxin complexes have provided exciting insights into toxin-antitoxin behavior.
572 citations
TL;DR: Toxin–antitoxin systems are small genetic elements composed of a toxin gene and its cognate antitoxin and their potential to combat viral infection may aid in controlling infectious diseases.
Abstract: Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin. The toxins of all known TA systems are proteins while the antitoxins are either proteins or non-coding RNAs. Based on the molecular nature of the antitoxin and its mode of interaction with the toxin the TA modules are currently grouped into five classes. In general, the toxin is more stable than the antitoxin but the latter is expressed to a higher level. If supply of the antitoxin stops, for instance under special growth conditions or by plasmid loss in case of plasmid encoded TA systems, the antitoxin is rapidly degraded and can no longer counteract the toxin. Consequently, the toxin becomes activated and can act on its cellular targets. Typically, TA toxins act on crucial cellular processes including translation, replication, cytoskeleton formation, membrane integrity, and cell wall biosynthesis. TA systems and their components are also versatile tools for a multitude of purposes in basic research and biotechnology. Currently, TA systems are frequently used for selection in cloning and for single protein expression in living bacterial cells. Since several TA toxins exhibit activity in yeast and mammalian cells they may be useful for applications in eukaryotic systems. TA modules are also considered as promising targets for the development of antibacterial drugs and their potential to combat viral infection may aid in controlling infectious diseases.
275 citations
Cites background from "In vitro and in vivo stability of t..."
...Instead, the activity of promoter P ω is regulated by dimeric ω 2 , a global regulator of transcription.(39,42) In addition, basal levels of ε and ζ are expressed from the constitutive but very weak promoter Pε....
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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 "In vitro and in vivo stability of t..."
...The plasmid-encoded ζ toxin is induced by artificial inhibition of transcription or translation which leads to more rapid decay of the ε antitoxin by Lon protease (Camacho et al., 2002)....
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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 from "In vitro and in vivo stability of t..."
.... The epsilon/zeta system is encoded from a bicistronic operon, which is regulated by the repressor protein omega...
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References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: An analysis of the solvent content of 116 different crystal forms of globular proteins found that in many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.
Abstract: An analysis has been made, from the data which are currently available, of the solvent content of 116 different crystal forms of globular proteins. The fraction of the crystal volume occupied by solvent is most commonly near 43 %, but has been observed to have values from about 27 to 65%. In many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.
7,857 citations
"In vitro and in vivo stability of t..." refers background or methods in this paper
...Assuming one ε2ζ2 complex to occupy the asymmetric unit, the Matthews coefficient (Matthews, 1968) calculates to 2....
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...Assuming one ε2ζ2 complex to occupy the asymmetric unit, the Matthews coefficient (Matthews, 1968) calculates to 2.63 Å3/Da with 53% solvent content in the crystals....
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...The stoichiometry of the ε and ζ proteins in the crystals was determined by the Matthews coefficient (Matthews, 1968) and by densitometric scanning and N-terminal amino acid sequencing of the CB stained, SDS-PAGE separated ε and ζ bands of the complexes obtained from dissolved crystals....
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TL;DR: In this article, a method for calculating accurate molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition, was presented, and the method was calibrated against 18 "normal" globular proteins.
Abstract: Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.
5,077 citations
TL;DR: This chapter describes three prediction methods that use evolutionary information as input to neural network systems to predict secondary structure (PHDsec), relative solvent accessibility, and transmembrane helices (P HDhtm).
Abstract: Publisher Summary The first step in a PHD prediction is generating a multiple sequence alignment. The second step involves feeding the alignment into a neural network system. Correctness of the multiple sequence alignment is as crucial for prediction accuracy as is the fact that the alignment contains a broad spectrum of homologous sequences. This chapter describes three prediction methods that use evolutionary information as input to neural network systems to predict secondary structure (PHDsec), relative solvent accessibility (PHDacc), and transmembrane helices (PHDhtm). It illustrates the possibilities and limitations in practical applications of these methods with results from careful cross-validation experiments on large sets of unique protein structures. All predictions are made available by an automatic Email prediction service. The baseline conclusion after some 30,000 requests to the service is that 1-D predictions have become accurate enough to be used as a starting point for the expert-driven modeling of protein structure.
1,316 citations
"In vitro and in vivo stability of t..." refers methods in this paper
...A secondary structure prediction from the amino acid sequences of the ε and ζ proteins was performed with the program PHDsec (Rost and Sander, 1993; Rost, 1996)....
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897 citations