Identification and modeling of a novel chloramphenicol resistance protein detected by functional metagenomics in a wetland of Lerma, Mexico

The exploration of novel antibiotic resistance determinants in a particular environment may be limited because of the presence of uncultured microorganisms. In this work, a culture independent approach based on functional metagenomics was applied to search for chloramphenicol resistance genes in agro-industrial wastewater in Lerma de Villada, Mexico. To this end, a metagenomic library was generated in Escherichia coli DH10B containing DNA isolated from environmental samples of the residual arsenic-enriched (10 mg/ml) effl uent. One resistant clone was detected in this library and further analyzed. An open reading frame similar to a multidrug resistance protein from Aeromonas salmonicida and responsible for chloramphenicol resistance was identifi ed, sequenced, and found to encode a member of the major facilitator superfamily (MFS). Our results also showed that the expression of this gene restored streptomycin sensitivity in E. coli DH10B cells. To gain further insight into the phenotype of this MFS family member, we developed a model of the membrane protein multiporter that, in addition, may serve as a template for developing new antibiotics. [Int Microbiol 2013; 16(2):103-111]Keywords: Escherichia coli; chloramphenicol; functional metagenomics; major facilitator superfamily; homology models; membrane proteins; arseni

Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment. © 2015 Mirete, Mora-Ruiz, Lamprecht-Grandío, de Figueras, Rosselló-Móra and González-Pastor.This work was funded by the Spanish Ministry of Science and Innovation (CGL2012-39627-C03/02 and 03); the latter also supported with European Regional Development Fund (FEDER). MM-R Ph.D. is supported by fellowship CVU 265934 of the National Council of Science and Technology (CONACyT), MexicoPeer Reviewe

The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal

<p>Abstract</p> <p>Background</p> <p><it>Bacillus subtilis </it>3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, <it>B. subtilis </it>encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in <it>B. subtilis </it>3610.</p> <p>Results</p> <p>We show that <it>B. subtilis </it>3610 produces intracellular NO <it>via </it>NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the <it>nos </it>gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the <it>nos </it>knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal.</p> <p>Conclusion</p> <p>The results suggest that biofilm formation and swarming motility in <it>B. subtilis </it>represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in <it>B. subtilis </it>might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of <it>B. subtilis </it>from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.</p

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.Fil: Lamprecht Grandío, María. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Cortesão, Marta. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Mirete, Salvador. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Benguigui de la Cámara, Macarena. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: de Figueras, Carolina G.. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Pérez Pantoja, Danilo. Universidad Tecnológica Metropolitana; EspañaFil: White, Joseph John. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Rosselló Móra, Ramon. Instituto Mediterráneo Estudios Avanzados; EspañaFil: González Pastor, José Eduardo. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; Españ

Search for WZ resonances in the fully leptonic channel using pp collisions at s=8 TeV with the ATLAS detector

A search for resonant WZ production in the ℓνℓ'ℓ' (ℓ,ℓ'=e,μ) decay channel using 20.3 fb-1 of s=8TeV pp collision data collected by the ATLAS experiment at LHC is presented. No significant deviation from the Standard Model prediction is observed and upper limits on the production cross sections of WZ resonances from an extended gauge model W' and from a simplified model of heavy vector triplets are derived. A corresponding observed (expected) lower mass limit of 1.52 (1.49) TeV is derived for the W' at the 95% confidence level.Peer Reviewe

Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √s = 8 TeV pp collisions with the ATLAS detector

Aad, G. et al.A search for the direct production of charginos and neutralinos in final states with three leptons and missing transverse momentum is presented. The analysis is based on 20.3 fb−1 of s√ = 8 TeV proton-proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with the Standard Model expectations and limits are set in R-parity-conserving phenomenological Minimal Supersymmetric Standard Models and in simplified supersymmetric models, significantly extending previous results. For simplified supersymmetric models of direct chargino (χ˜±1) and next-to-lightest neutralino (χ˜02) production with decays to lightest neutralino (χ˜01) via either all three generations of sleptons, staus only, gauge bosons, or Higgs bosons, (χ˜±1) and (χ˜02) masses are excluded up to 700 GeV, 380 GeV, 345 GeV, or 148 GeV respectively, for a massless (χ˜01).We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWF and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF, DNSRC and Lundbeck Foundation, Denmark; EPLANET, ERC and NSRF, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, DFG, HGF, MPG and AvH Foundation, Germany; GSRT and NSRF, Greece; ISF, MINERVA, GIF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; BRF and RCN, Norway; MNiSW and NCN, Poland; GRICES and FCT, Portugal; MNE/IFA, Romania; MES of Russia and ROSATOM, Russian Federation; JINR; MSTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey.Peer reviewe

Measurement of the production cross section of prompt J/ψ mesons in association with a W ± boson in pp collisions at √s = 7 TeV with the ATLAS detector

Aad, G. et al.The process pp → W ± J/ψ provides a powerful probe of the production mechanism of charmonium in hadronic collisions, and is also sensitive to multiple parton interactions in the colliding protons. Using the 2011 ATLAS dataset of 4.5 fb−1 of s√ = 7 TeV pp collisions at the LHC, the first observation is made of the production of W ± + prompt J/ψ events in hadronic collisions, using W ± → μν μ and J/ψ → μ + μ −. A yield of 27.4+7.5−6.5 W ± + prompt J/ψ events is observed, with a statistical significance of 5.1σ. The production rate as a ratio to the inclusive W ± boson production rate is measured, and the double parton scattering contribution to the cross section is estimated.We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWF and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF, DNSRC and Lundbeck Foundation, Denmark; EPLANET, ERC and NSRF, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, DFG, HGF, MPG and AvH Foundation, Germany; GSRT and NSRF, Greece; ISF, MINERVA, GIF, DIP and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; BRF and RCN, Norway; MNiSW and NCN, Poland; GRICES and FCT, Portugal; MNE/IFA, Romania; MES of Russia and ROSATOM, Russian Federation; JINR; MSTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey; STFC, the Royal Society and Leverhulme Trust, United Kingdom; DOE and NSF, United States of America.Peer reviewe

Charged-particle multiplicities in pp interactions at root s=900 GeV measured with the ATLAS detector at the LHC

22 páginas, 4 figuras, 1 tabla.-- et al.(ATLAS Collaboration).-- arXiv:1003.3124v2The first measurements from proton-proton collisions recorded with the ATLAS detector at the LHC are presented. Data were collected in December 2009 using a minimum-bias trigger during collisions at a centre-of-mass energy of 900 GeV. The charged-particle multiplicity, its dependence on transverse momentum and pseudorapidity. and the relationship between mean transverse momentum and charged-particle multiplicity are measured for events with at least one charged particle in the kinematic range vertical bar eta vertical bar 500 MeV. The measurements are compared to Monte Carlo models of proton-proton collisions and to results from other experiments at the same centre-of-mass energy. The charged-particle multiplicity per event and unit of pseudorapidity eta = 0 is measured to be 1.333 +/- 0.003(stat.) +/- 0.040(syst.), which is 5-15% higher than the Monte Carlo models predict.We are greatly indebted to all CERN’s departments and to the LHC project for their immense efforts not only in building the LHC, but also for their direct contributions to the construction and installation of the ATLAS detector and its infrastructure. All our congratulations go to the LHC operation team for the superb performance during this initial data-taking period. We acknowledge equally warmly all our technical colleagues in the collaborating Institutions without whom the ATLAS detector could not have been built. Furthermore we are grateful to all the funding agencies which supported generously the construction and the commissioning of the ATLAS detector and also provided the computing infrastructure. The ATLAS detector design and construction has taken about fifteen years, and our thoughts are with all our colleagues who sadly could not see its final realisation. We acknowledge the support of ANPCyT, Argentina; Yerevan Physics Institute, Armenia; ARC and DEST, Australia; Bundesministerium für Wissenschaft und Forschung, Austria; National Academy of Sciences of Azerbaijan; State Committee on Science & Technologies of the Republic of Belarus; CNPq and FINEP, Brazil; NSERC, NRC, and CFI, Canada; CERN; CONICYT, Chile; NSFC, China; COLCIENCIAS, Colombia; Ministry of Education, Youth and Sports of the Czech Republic, Ministry of Industry and Trade of the Czech Republic, and Committee for Collaboration of the Czech Republic with CERN; Danish Natural Science Research Council and the Lundbeck Foundation; European Commission, through the ARTEMIS Research Training Network; IN2P3-CNRS and Dapnia-CEA, France; Georgian Academy of Sciences; BMBF, HGF, DFG and MPG, Germany; Ministry of Education and Religion, through the EPEAEK program PYTHAGORAS II and GSRT, Greece; ISF, MINERVA, GIF, DIP, and Benoziyo Center, Israel; INFN, Italy; MEXT, Japan; CNRST, Morocco; FOM and NWO, Netherlands; The Research Council of Norway; Ministry of Science and Higher Education, Poland; GRICES and FCT, Portugal; Ministry of Education and Research, Romania; Ministry of Education and Science of the Russian Federation and State Atomic Energy Corporation “Rosatom”; JINR; Ministry of Science, Serbia; Department of International Science and Technology Cooperation, Ministry of Education of the Slovak Republic; Slovenian Research Agency, Ministry of Higher Education, Science and Technology, Slovenia; Ministerio de Educación y Ciencia, Spain; The Swedish Research Council, The Knut and Alice Wallenberg Foundation, Sweden; State Secretariat for Education and Science, Swiss National Science Foundation, and Cantons of Bern and Geneva, Switzerland; National Science Council, Taiwan; TAEK, Turkey; The Science and Technology Facilities Council and The Leverhulme Trust, United Kingdom; DOE and NSF, United States of America.Peer reviewe

ATLAS search for a heavy gauge boson decaying to a charged lepton and a neutrino in pp collisions at root s=7 TeV

The ATLAS detector at the LHC is used to search for high-mass states, such as heavy charged gauge bosons (W'), decaying to a charged lepton (electron or muon) and a neutrino. Results are presented based on the analysis of pp collisions at a center-of-mass energy of 7 TeV corresponding to an integrated luminosity of 4.7 fb(-1). No excess beyond Standard Model expectations is observed. A W' with Sequential Standard Model couplings is excluded at the 95 % credibility level for masses up to 2.55 TeV. Excited chiral bosons (W*) with equivalent coupling strength are excluded for masses up to 2.42 TeV