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Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S. cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.

International audience; One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication. This article compares these two cases within a single whole-cell model framework. This model relies upon a membrane embedding a chemical reaction network (CRN) synthesizing all the cellular constituents, including the membrane, by feeding from an external nutrient. Assuming the osmolarity is kept constant, the system dynamics are governed by a set of nonlinear differential equations coupling the chemical concentrations and the surface-area-to-volume ratio. The resulting asymptotic trajectories are used to determine the cellular shape by minimizing the membrane bending energy (within an approximate predefined family of shapes). While the stationary case can be handled quite generally, the oscillatory one is investigated using a simple oscillating CRN example, which is used to identify features that are expected to hold for any network. It is found that cellular self-replication can be reached with or without chemical oscillations, and that a requirement common to both stationary and oscillatory cases is that a minimum spontaneous curvature of the membrane is required for the cell to divide once its area and volume are both doubled. The oscillatory case can result in a greater variety of cellular shape trajectories but raises additional constraints for cellular division and self-replication: (i) the ratio of doubling time to oscillation period should be an integer, and (ii) if the oscillation amplitude is sufficiently high, then the spontaneous curvature must be below a maximum value to avoid early division before the end of the cycle. Because of these additional stringent constraints, it is likely that early protocells did not rely upon chemical oscillations. Biochemical oscillations typical of modern evolved cells may have emerged later through evolution for other reasons (e.g., metabolic advantage) and must have required additional feedback mechanisms for such a self-replicating system to be robust against even slight environmental variations (e.g., temperature fluctuations).

Every day, almost one billion children around the world experience violent punishment. Eliminating all violence against children is a key target of the United Nations’ 2030 Agenda for Sustainable Development. This is a monumental challenge due to the diversity of cultural, economic and social contexts in which children live. Violence-prevention programs developed in wealthy countries cannot be assumed to be transferable to low- and middle-income countries. We assessed the relevance of Positive Discipline in Everyday Parenting (PDEP) to 525 parents living in countries with high ( n = 201), medium ( n = 166), or low ( n = 158) Inequality-Adjusted Human Development Indices. The outcome measures were parents’ satisfaction with the program and their perceptions of its impact on their parenting. Across IHDI categories, almost all parents were “mostly” or “very satisfied” with the overall program (98.4%), the PDEP parent book (97.9%), and the program activities (97.8%). Parent satisfaction scores were higher in the Low IHDI category than in the High IHDI category. Across IHDI categories, large majorities of parents perceived PDEP as having positive impacts on their parenting. While parents in the Medium IHDI category had the strongest perceptions of PDEP’s positive impact, more than 90% of parents in the Low IHDI category believed that the program will help them to understand their children’s development and feelings, communicate better with their children, control their anger, and build stronger relationships with their children. PDEP is a promising tool for preventing punitive violence against children across human development contexts.

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1–Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro. However, this nonspecific integration activity is rare in vivo. Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1–Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1–Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

Physical and emotional punishment of children is highly prevalent in the Asia-Pacific region. These actions predict a range of physical and emotional harms, prompting a worldwide effort to eliminate them. A key strategy in this effort is to change parental beliefs regarding the acceptability of physical and emotional punishment. The Positive Discipline in Everyday Parenting (PDEP) program was designed to change those beliefs by teaching parents about child development and strengthening their problem-solving skills. A sample of 377 parents in the Asia-Pacific region completed the program: 329 mothers and 47 fathers of children ranging in age from infancy to adolescence. The parents lived in Australia ( n = 135), Japan ( n = 172) or the Philippines ( n = 70). In all three countries, parents’ approval of punishment in general, and physical punishment specifically, declined and they became less likely to attribute typical child behavior to intentional misbehavior. By the end of the program, at least 75% of parents in each country felt better prepared to respond nonviolently to conflict with their children.

International audience; Selenium (Se) is an essential trace element for mammals. It is involved in redox functions as the amino acid selenocysteine, translationally inserted in the active site of a few proteins. However, at high doses it is toxic and the mechanisms underlying this toxicity are poorly understood. Because of the high level of conservation of its genes and pathways with those of higher organisms and the powerful genetic techniques that it offers, Saccharomyces cerevisiae is an attractive model organism to study the molecular basis of Se toxicity. High-throughput technologies developed in this yeast include genome-wide screening of bar-coded systematic deletion sets, as well as whole-transcriptome, -proteome, and -metabolome analysis.This chapter focuses on the contribution of S. cerevisiae to the understanding of the mechanisms of selenocompound toxicity, combining results from classical biochemistry with genome-wide analyses and more detailed gene-by-gene approaches. Experimental data demonstrate that toxicity is compound specific. Inorganic Se induces DNA damage whereas selenoamino acids cause proteotoxicity.

International audience; Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3΄-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus.

Selenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress. However, it has become apparent that the mode of action of seleno-compounds varies, depending on its chemical form and speciation. Recent studies in various eukaryotic systems, in particular the model organism Saccharomyces cerevisiae, provide new insights on the cytotoxic mechanisms of selenomethionine and selenocysteine. This review first summarizes current knowledge on reactive oxygen species (ROS)-induced genotoxicity of inorganic selenium species. Then, we discuss recent advances on our understanding of the molecular mechanisms of selenocysteine and selenomethionine cytotoxicity. We present evidences indicating that both oxidative stress and ROS-independent mechanisms contribute to selenoamino acids cytotoxicity. These latter mechanisms include disruption of protein homeostasis by selenocysteine misincorporation in proteins and/or reaction of selenols with protein thiols.

It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution. Here, we analyze the non-electrostatic component of the free-energy difference between B-DNA and Z-DNA. We also compute the composition of the counterion sheath in a wide range of mixed-salt solutions and of temperatures: in contrast with the large difference of composition between the B-DNA and Z-DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed-salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small-Angle X-ray Scattering and complemented by (31) P NMR. With due consideration for the kinetics of the B-Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5'-d[T-(m(5) C-G)12 -T] 26-mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson-Boltzmann equation is described in detail. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 369-384, 2016.

The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATPdependent transport of glutathione–metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol 36, 679–687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated. Using secretory vesicles isolated from a sec6-4 mutant transformed either with the plasmid harbouring YCF1 or the control plasmid, we establish that the glutathione-conjugate selenodigluthatione is a highaffinity substrate of this ATP-binding cassette transporter and that oxidized glutathione is also efficiently transported. Finally, we show that the presence of Ycf1p impairs the glutathione ⁄ oxidized glutathione ratio of cells subjected to a selenite stress. Possible mechanisms by which Ycf1p-mediated vacuolar uptake of selenodiglutathione and oxidized glutathione enhances selenite toxicity are discussed.

International audience; Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand. Proteins 2008. © 2007 Wiley-Liss, Inc.

International audience; GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active d-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous d-Tyr- tRNA(Tyr) deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by d-amino acids are considered.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

International audience; Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity.

International audience; The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.

The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five β-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys → Asp mutation inserted into the β-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.

Cell growth inhibition by severald-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules.Escherichia coli and yeast cells express an enzyme,d-Tyr-tRNATyr deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA andd-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimeric E. coli d-Tyr-tRNATyr deacylase at 1.55 A resolution is reported. The structure corresponds to a β-barrel closed on one side by a β-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the β-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.

The aminoacyl‐tRNA synthetases (aaRSs) are a family of enzymes well known for their role in protein synthesis. More recent investigations have discovered that this classic family of enzymes is actually capable of a broad repertoire of functions that not only impact protein synthesis, but also extend to a number of other critical cellular activities (Figure 1; Martinis et al ., 1999). Specific aaRSs play roles in cellular fidelity, tRNA processing, RNA splicing, RNA trafficking, apoptosis and transcriptional and translational regulation. A recent EMBO workshop entitled Structure and Function of Aminoacyl‐tRNA Synthetases (Mittelwihr, France, October 10–15, 1998) highlighted the diversity of the aaRSs' role within the cell. These novel activities, as well as significant advances in delineating mechanisms of substrate specificity and the aminoacylation reaction, affirm the family of aaRSs as pharmaceutical targets. Herein we report major advances in the field during the short period since the last aaRS meeting (Francklyn et al ., 1997). Figure 1. Cellular roles and functions of aaRS. Particular aaRSs involved in each of the represented activities are indicated by the three letter code of the amino acid that they activate. ### aaRSs perform dual and essential roles in the cell Eukaryotic pre‐tRNAs are synthesized in the nucleus and require extensive processing to yield functional tRNAs. An elegant series of experiments clearly show that mature tRNAs are charged in the nucleus prior to export to the cytoplasm (Lund and Dahlberg, 1998). Blockage of the tRNA export machinery results in accumulation of nuclear charged tRNAs. Introduction of specific aaRS inhibitors decreases export of the relevant uncharged tRNA. This nuclear aaRS‐dependent aminoacylation reaction was proposed to serve as a stringent quality control mechanism prior to export of mature tRNA to the cytoplasm where it is required for protein synthesis. Nuclear aminoacylation of tRNAs (Arts et al ., 1998; Lund and Dahlberg, 1998) presumably requires import of aaRSs from the cytoplasm. …

The aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes well known for their role in protein synthesis. More recent investigations have discovered that this classic family of enzymes is actually capable of a broad repertoire of functions which not only impact protein synthesis, but extend to a number of other critical cellular activities. Specific aaRSs play roles in cellular fidelity, tRNA processing, RNA splicing, RNA trafficking, apoptosis, transcriptional and translational regulation. A recent EMBO workshop entitled `Structure and Function of Aminoacyl-tRNA Synthetases' (Mittelwihr, France, October 10–15, 1998), highlighted the diversity of the aaRSs'role within the cell. These novel activities as well as significant advances in delineating mechanisms of substrate specificity and the aminoacylation reaction affirm the family of aaRSs as pharmaceutical targets.

Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal of the 5'-phosphate. The tRNA 5'-phosphate plays therefore the role of a relay between the enzyme and the status of the 1-72 base pair. In this study, the receptor site for the 5'-phosphate of elongator peptidyl-tRNAs and the position at the surface of PTH of the 3'-end of complexed peptidyl-tRNA are identified by site-directed mutagenesis experiments. The former site comprehends two cationic side chains (K105 and R133) which are likely to clamp the phosphate. The second corresponds to a four asparagine cluster (N10, N21, N68, and N114). By using these two positional constraints, the acceptor arm of elongation factor Tu-bound Phe-tRNAPhe could be docked to PTH. Contacts involve the acceptor and TPsiC stems. By comparing the obtained 3D model to that of EF-Tu:Phe-tRNAPhe crystalline complex in which the 5'-phosphate of the ligand also lies between a K and an R side chain, we propose that, in both systems, the capacity of the 5'-phosphate of a tRNA to reach or not a receptor site is the main identity element governing generic selection of elongator tRNAs. On the other hand, while the 1-72 mismatch acts as an antideterminant for PTH or EF-Tu recognition, it behaves as a positive determinant for the formylation of initiator Met-tRNAfMet.

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P 1 ,P 3 -triphosphate (Ap 3 A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap 3 A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap 3 A-hydrolyzing activity and a 30-fold-elevated Ap 3 N concentration.

International audience; Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator. In parallel, the abilities of these mixtures to produce colloids were assessed. Studied metal cations could be classified in three groups: (i) metal ions that protect cells against selenium toxicity and form insoluble colloids with selenide (Ag+, Cd2+, Cu2+, Hg2+, Pb2+ and Zn2+), (ii) metal ions which protect cells by producing insoluble metal-selenide complexes and by catalyzing hydrogen selenide oxidation in the presence of dioxygen (Co2+ and Ni2+) and, finally, (iii) metal ions which do not afford protection and do not interact (Ca2+, Mg2+, Mn2+) or weakly interact (Fe2+) with selenide under the assayed conditions. When occurring, the insoluble complexes formed from divalent metal ions and selenide contained equimolar amounts of metal and selenium atoms. With the monovalent silver ion, the complex contained two silver atoms per selenium atom. Next, because selenides are compounds prone to oxidation, the stabilities of the above colloids were evaluated under oxidizing conditions. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the reduction of which can be optically followed, was used to promote selenide oxidation. Complexes with cadmium, copper, lead, mercury or silver resisted dissolution by DTNB treatment over several hours. With nickel and cobalt, partial oxidation by DTNB occurred. On the other hand, when starting from ZnSe or FeSe complexes, full decompositions were obtained within a few tens of minutes. The above properties possibly explain why ZnSe and FeSe nanoparticles were not detected in animals exposed to selenocompounds.

In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU. The two corresponding enzymes could be purified at homogeneity from a delta lysU and a delta lysS strain, respectively. Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PPi exchange as well as in the tRNALys aminoacylation reaction. Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex. In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase. This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium. Remarkably, these conditions also induce lysU expression. Homogeneous LysU and LysS were also compared in Ap4A synthesis. LysU is only 2-fold more active than LysS in the production of this dinucleotide. This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of Ap4A inside stressed Escherichia coli cells. This conclusion could be strengthened by determining the concentrations of Ap4N (N = A, C, G, or U) in a delta lysU as well as in a lysU+ strain, before and after a 1-h temperature shift at 48 degrees C. The measured concentration values were the same in both strains.

The constitutive lysyl-tRNA synthetase (LysRS) of the Escherichia coli strain OEL134 differs from the wild-type enzyme by the single substitution of threonine 208 with methionine. In vitro study of the isotopic [32P]PPi-ATP exchange reaction catalyzed by purified T208M LysRS revealed specific features that are not observed with the wild-type LysRS: (i) The steady state of the reaction was reached after a approximately 1-min lag when the addition of the enzyme was used to initiate the reaction. This lag disappeared upon preincubation of the enzyme with lysine and ATP. (ii) The variation of the steady state rate as a function of the lysine concentration in the assay was sigmoidal (Hill coefficient of 1.65), suggesting cooperativity of lysine binding to this dimeric enzyme. The allosteric behavior of the mutant enzyme was further established by showing that, at low concentrations of lysine, low amounts of cadaverine stimulated T208M LysRS activity. T208A LysRS, in which threonine 208 had been changed into alanine by site-directed mutagenesis, displayed the same properties as T208M LysRS. Remarkably, Thr 208 makes part of the first signature motif of class II aminoacyl-tRNA synthetases, a motif likely to be involved in the dimerization of the enzyme subunits. Therefore, the behavior of the Thr 208 mutants of LysRS supports the idea that the dimerization of class II aminoacyl-tRNA synthetases is important for an efficient structuration of their active site.

International audience; Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (*)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.

In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive regulatory protein (Lrp). To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region. The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo. In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner. This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association. These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn. Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp.

A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetase, was cloned from the extreme thermophile prokaryote Thermus thermophilus VK-1 and sequenced. The analysis indicated an open reading frame encoding a protein of 492 amino acids. This putative protein has significant homologies to previously sequenced lysyl-tRNA synthetases and displays the three motifs characteristic of class II aminoacyl-tRNA synthetases. The T. thermophilus lysS gene was overexpressed in Escherichia coli by placing it downstream of the E. coli beta-galactosidase gene promoter on plasmid pBluescript and by changing the ribosome-binding site. The overproduced protein was purified by heat treatment of the crude extract followed by a single anion-exchange chromatography step. The protein obtained is remarkably thermostable, retaining nearly 60% of its initial tRNA aminoacylation activity after 5 h of incubation at 93 degrees C. Finally, lethal disruption of the lysRS genes of E. coli could not be compensated for by the addition in trans of the T. thermophilus lysS gene despite the fact that this gene was overexpressed and that its product specifically aminoacylates E. coli tRNA(Lys) in vitro.

International audience; In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strains VK.-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 Å resolution (space group P212121, a = 61.4 Å, b = 156.1 Å, c = 177.3 Å) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

International audience; Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P(i) concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P(i) transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P(i) medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P(i) transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.

International audience; 2D images of label-free biochips exploiting resonant waveguide grating (RWG) are presented. They indicate sensitivities on the order of 1 pg/mm2 for proteins in air, and hence 10 pg/mm2 in water can be safely expected. A 320×256 pixels Aluminum-Gallium-Nitride-based sensor array is used, with an intrinsic narrow spectral window centered at 280 nm. The additional role of characteristic biological layer absorption at this wavelength is calculated, and regimes revealing its impact are discussed. Experimentally, the resonance of a chip coated with protein is revealed and the sensitivity evaluated through angular spectroscopy and imaging. In addition to a sensitivity similar to surface plasmon resonance (SPR), the RWGs resonance can be flexibly tailored to gain spatial, biochemical, or spectral sensitivity.

International audience; In the frame of biological threat, security systems require label free biochips for rapid detection. Biosensors enable to detect biological interactions, between probes localized at the surface of a chip, and targets present in the sample solution. Here, we present an optical transduction, enabling 2D imaging, and consequently parallel detection of several reactions. It is based on the absorption of biological molecules in the UV domain. Thus, it is based on an intrinsic property of biological molecules and does not require any labelling of the biological molecules. DNA and proteins absorb UV light at 260 and 280 nm respectively. Sensitivity is a major requirement of biosensing devices. Configurations leading to enhancement of the interaction between light and biological molecules are of interest. For a better sensitivity, resonant grating structures are then studied. They enable to confine the electric field close to the biological layer. Imaging of resonant grating is not largely studied, even for visible wavelengths, but it results in good sensitivity. The protein used in this study is the methionyl-tRNA synthetase. Its absorption is representative of protein absorption, and it can then serve as a model for immunological detection. The best experimental contrast due to a monolayer of proteins is 40%. With data processing currently employed for biochip imaging: average on several acquisitions and on all the pixels imaging the biological spots, the device is able to detect a surface density of proteins in the 10 pg/mm range. © (2010) COPYRIGHT SPIE--The International Society for Optical Engineering

Three recent improvements in solvent-signal suppression are described. First, PJR (“power-adapted jump and return”) replaces JR (jump and return). It goes beyond the “infinitely short π 2 pulse” approximation and can therefore be used even when the available RF power is low. The phase shift is small, and the amplitude correction providing a flat response is simple and power-independent. With a postexcitation delay, PJR generates an echo. Second, “modified binomials,” i.e., binomials sandwiched between two π pulses are studied. This simple modification eliminates the linear phase shift, which is the main problem of binomial sequences. It has a clear theoretical basis. These two sequences are applied to the observation of a test sample of thymidine, following a proposed standard procedure for the evaluation of methods of solvent-signal suppression. Third, it is pointed out that the elimination of radiation damping would avoid the resulting rotation of the magnetic moment during the RF-free intervals within solvent-signal-suppression excitation sequences. It may be achieved by incorporating the coil in an open, series-tuned, matching circuit.

International audience; Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).

International audience; This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260nm and 280nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates. We analyze them through models and experiments and validate the possibility of measuring absorptions of the order of 10(-3). These multilayer structures display a high reflectivity, and maximize the interaction of the electric field with the biological element at the chip surface. Optimization of the experimental absorption, which includes effects such as roughness of the biochip, spectral and angular resolution of the optics, illumination, etc., is carried out with an inorganic ultraviolet absorber (titanium dioxide) deposit. We obtained an induced absorption contrast enhanced by a factor of 4.0, conferring enough sensitivity to detect monolayers of DNA or proteins. Experimental results on an Escherichia coli histidine-tagged methionyl-tRNA synthetase protein before and after complexation with an anti-polyHis specific antibody validate our biosensing technique. This label-free optical method may be helpful in controlling biochip coatings, and subsequent biological coupling at the surface of a biochip.

Escherichia coli strains with abnormally high concentrations of bis(5'-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.