Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen

Promotion of protocell self-assembly from mixed amphiphiles at the origin of life

Vesicles formed from single-chain amphiphiles (SCAs) such as fatty acids probably played an important role in the origin of life. A major criticism of the hypothesis that life arose in an early ocean hydrothermal environment is that hot temperatures, large pH gradients, high salinity and abundant divalent cations should preclude vesicle formation. However, these arguments are based on model vesicles using 1–3 SCAs, even though Fischer–Tropsch-type synthesis under hydrothermal conditions produces a wide array of fatty acids and 1-alkanols, including abundant C10–C15 compounds. Here, we show that mixtures of these C10–C15 SCAs form vesicles in aqueous solutions between pH ~6.5 and >12 at modern seawater concentrations of NaCl, Mg2+ and Ca2+. Adding C10 isoprenoids improves vesicle stability even further. Vesicles form most readily at temperatures of ~70 °C and require salinity and strongly alkaline conditions to self-assemble. Thus, alkaline hydrothermal conditions not only permit protocell formation at the origin of life but actively favour it

Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence

Recuits de la résistivité électrique produite dans le cuivre par trempe et par irradiation - I. comparaison des trempes sous atmosphère d'hélium et sous atmosphère d'hydrogène.

A specimen of copper has been in turn quenched in a hydrogen atmosphère and in a helium atmosphère. After each of thèse quenches, isothermal or isochronal anneals were performed in order to compare the mobilities of the point defects produced. Near room tempe-rature, the activation energies for motion have been found equal to 0.77 ± 0 10 eV after helium quenches and to 0.80 ± 0.06 eV after hydrogen quenches. Between — 30 °C and + 50 °C, the isochronal curves (in percent of annealed resistivity) are identical. We conclude that it is not possible to distinguish a difference in the mobility of the point defects produced in either case.Un même échantillon de cuivre a subi successivement des trempes en atmosphère d'hydrogène et des trempes en atmosphère d'hélium. Chacune de ces trempes était suivie de recuits isothermes ou isochrones, en vue de comparer les mobilités des défauts ponctuels créés par la trempe. Aux environs de la température ordinaire les énergies d'activation pour le mouvement ont été trouvées égales à 0,77 ± 0,10 eV après trempes dans l'hélium et à 0,80 ± 0,06 eV après trempes dans l'hydrogène. De plus, entre — 30 °C et + 50 °C, les isochrones (exprimées en pour cent de résistivité disparue) sont confondues. On en conclut qu'il n'est pas possible de distinguer la mobilité produits dans l'un ou l'autre cas

Recuits de la résistivité électrique produite dans le cuivre par trempe et par irradiation - II. Étude du cuivre irradié aux électrons. Comparaison avec du cuivre trempé

Using the same specimen, we have carried out alternately irradiation with 1.5 MeV electrons at -175 °C and quencheing in a hydrogen atmosphere. After each quench or eachirradiation, isothermal or isochronal anneals were performed in order to compare the mobilities of the point defects produced in each case. Activation energy determinations are not conclusive. Isochronal anneals show that after irradiation the specimen anneals in stage III at lower temperature than after a quench.Nous avons effectué alternativement sur un même échantillon de cuivre, des irradiations par des électrons de 1,5 MeV à—175 °C et des trempes en atmosphère d'hydrogène. Après chaque irradiation ou trempe, des recuits étaient faits en vue de comparer les mobilités des défauts ponctuels qui recuisent au voisinage de la température ordinaire. Les déterminations d'énergie d'activation ne permettent pas de conclure à une différence. Les recuits isochrones montrent qu'il existe toujours un décalage qui peut atteindre 30 °C entre les températures moyennes des recuits dans le cuivre irradié (étape III) et trempé, le métal irradié recuisant à plus basse température

Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts