The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

El sistema toxina-antitoxina, [épsilon] [dseda], como inhibidor de la proliferación celular e inductor de tolerancia

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 08-03-201

Distinct Activities of Bacterial Condensins for Chromosome Management in Pseudomonas aeruginosa

International audienceThree types of structurally related structural maintenance of chromosomes (SMC) complexes, referred to as condensins, have been identified in bacteria. Smc-ScpAB is present in most bacteria, whereas MukBEF is found in enterobacteria and MksBEF is scattered over the phylogenic tree. The contributions of these condensins to chromosome management were characterized in Pseudomonas aeruginosa, which carries both Smc-ScpAB and MksBEF. In this bacterium, SMC-ScpAB controls chromosome disposition by juxtaposing chromosome arms. In contrast, MksBEF is critical for chromosome segregation in the absence of the main segregation system, and it affects the higher-order architecture of the chromosome by promoting DNA contacts in the megabase range. Strikingly, our results reveal a prevalence of Smc-ScpAB over MksBEF involving a coordination of their activities with chromosome replication. They also show that E. coli MukBEF can substitute for MksBEF in P. aeruginosa while prevailing over Smc-ScpAB. Our results reveal a hierarchy between activities of bacterial condensins on the same chromosome

Toxin zeta Triggers a Survival Response to Cope with Stress and Persistence

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation.Spanish Ministerio de Economía y Competividad and the European Union (MINECO-FEDER) BFU2015-67065-P4.019 JCR (2017) Q1, 31/125 MicrobiologyUE

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host.

International audienceIn Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution-ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness

A reforma do pensamento: por uma educação que descubra as relações ocultas do saber

Reforming education involves reforming thought, and a reform of thought implies a paradigm shift in the current knowledge organization model. Complex thinking, interdisciplinary and transdisciplinary research methods and recognition of the relationship between knowledge, the knower and the surrounding medium are elements that can contribute to the construction of educational models that address the problems faced by the world nowadays. A holistic view in education contributes and commits the educator and the learner with society and its problems.Reformar la educación implica reformar el pensamiento y una reforma del pensamiento implica un cambio de paradigma en el modelo actual de organización del conocimiento. El pensamiento complejo, los métodos de investigación inter y transdisciplinar y el reconocimiento de la relación entre el saber, el sujeto cognoscente y el medio que lo rodea son elementos que pueden contribuir a la construcción de modelos educativos que respondan a los problemas que enfrenta el mundo actual. Una visión holística dentro de la educación contribuye y compromete al educador y al educando con la sociedad y sus problemáticas.Reformar a educação implica reformar o pensamento e uma reforma do pensamento implica uma mudança de paradigma no modelo atual de organização do conhecimento. O pensamento complexo, os métodos de pesquisa inter e transdisciplinar e o reconhecimento da relação entre o saber, o sujeito cognoscente e o meio que o rodeia são elementos que podem contribuir para a construção de modelos educativos que respondam aos problemas enfrentados pelo mundo atual. Uma visão holística dentro da educação contribui e compromete o educador e o educando com a sociedade e suas problemáticas

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins

International audienceAs in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization

RelA is required for ζY83C toxin enhanced efficacy to different antimicrobials.

<p>A) BG1145 cells (Δ<i>relA</i>) were grown to ∼5×10⁷ cells/ml. Then 0.5% Xyl and/or an AM were added and the cultures were incubated for 120 min. B) BG1145 cells were pre-treated with limiting relacin (1 mM) concentrations (+ Rel) or not (− Rel), and then 0.5% Xyl, Amp or both were added and the cultures were incubated for 120 min. The symbols, the plating conditions, and the antimicrobial concentrations were those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086615#pone-0086615-g001" target="_blank">Figure 1</a>. Error bars show 95% confidence intervals of more than three independent experiments.</p

Effect of a chromosomal-encoded TA locus on the formation of antimicrobial persisters.

<p>BG689 (ζY83C <i>mazF</i>⁺) and BG1243 (ζY83C Δ<i>mazF</i>) cells were grown in MMS7 to ∼5×10⁷ cell ml⁻¹. Then 0.5% Xyl^a or 2x-MIC Amp^b or both was added and the culture was incubated for 120 min.</p>c<p>Induction or not of the ζY83C toxin is indicated by + and − superscript symbols, respectively.</p>d<p>The CFUs were measured after 120 min of toxin induction and/or antimicrobial addition by plating appropriate dilutions on LB plates. The results are the average of at least three independent experiments and are within a 10% standard error.</p

Expression of the ε₂ antitoxin reverses the effect of ζ toxin, but does not increase MDT.

<p>(A and B) BG1125-borne ζ gene was induced by the addition of 1 mM IPTG, and pCB799-borne ε gene was induced by addition of Xyl. BG1125 cells were grown to ∼5×10⁷ cells/ml in MMS7 containing traces of Xyl (0.005%). Then, expression of ζ was induced and/or cells treated with an antimicrobial, and the cultures incubated for 120 min (A) or 240 min (B). At those times, samples were taken and plated on LB agar or on LB-0.5% Xyl plates to induce the expression of the ε₂ antitoxin. Expression of the ε₂ antitoxin reverses the effect of ζ toxin in non-growing cells. (C and D) BG1125 cells were grown in MMS7, containing traces of Xyl (0.005%), up to early stationary phase, and diluted to ∼1×10⁹ cells/ml. Then, expression of <b>ζ</b> was induced and/or cells treated with an antimicrobial, and the cultures incubated for 120 min (C) or 240 min (D) with agitation at 37°C. At these times samples were taken and plated on LB agar or LB-0.5% Xyl to induce the expression of the ε₂ antitoxin. The symbols, the plating conditions, and the antimicrobial concentrations were those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086615#pone-0086615-g001" target="_blank">Figure 1</a>. Error bars show 95% confidence intervals of more than three independent experiments.</p