03b virology and microbiology

8/13/2019 03B Virology and Microbiology

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Virology

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Table of Contents

Viral modulation of the immune responseAntonio Alcami Pertejo

B 1

Molecular bases of viral pathogenesis and anti-cancer potentialJos M. Almendral del Ro

B 2

Molecular Ecology of Extreme EnvironmentsRicardo Amils Pibernat

B 3

Bacterial Cell Division and Antibiotics ResistanceJuan Alfonso Ayala Serrano

B 4

Biotechnology and Genetics of Extreme thermophilic BacteriaJos Berenguer Carlos

B 5

ASFV: virus models of evasion and protectionngel L. Lpez Carrascosa

B 6

Bacterial Morphogenesis

Miguel ngel de Pedro Montalbn

B 7

Generic variability of RNA virusesEsteban Domingo Solans

B 8

Gene expresin in Streptomycesand yeastsAntonio Jimnez / Mara Fernndez Lobato

B 9

Virus EngineeringMauricio Garca Mateu

B 10

Effects of extrachromosomal elements on behaviour of its host BacillusWilfried J.J. Meijer

B 11

Human immunodefciency virus reverse transcriptase and antiretroviral therapyLus Menndez Arias

B 12

African swine fever virusMaria Luisa Salas Falgueras

B 13

New strategies for prevention and control of viral diseases:foot-and-mouth disease virus as a modelFrancisco Sobrino

B 14

Virology and Microbiology

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CBMSO2009-2010

B1 Viral modulation of the immune response

Research Summary

Staff

Publications

Other Activities

Lnea Investigacin

We are investigating immune evasion mechanisms employed by large DNA

viruses, poxviruses and herpesviruses. Specically, we are characterizing a uniqueimmunomodulatory strategy that consists of the secretion of viral proteins that bind

cytokines and chemokines, important mediators of the inammatory and immuneresponse. We work on two virus systems: (1) Herpesviruses like herpes simplex virusand human cytomegalovirus, which are human pathogens of clinical relevance; and (2)Poxviruses such as vaccinia virus, the smallpox vaccine, and variola virus, the causative

agent of smallpox and the only viral disease eradicated as a result of a global vaccination

campaign. Secreted cytokine decoy receptors are likely to contribute to the pathogenesisof variola virus, which was one of the most virulent viruses known by mankind. The immunemodulatory activity of the viral cytokine receptors and their contribution to pathology are

being addressed in the mousepox model. Mousepox is a smallpox-like disease caused by

ectromelia virus, a natural mouse pathogen, and a classical model of viral pathogenesis.

Viruses have optimized during millions of years of evolution their ability to manipulate thehost immune response. Viruses offer a unique opportunity to develop their immune evasionstrategies as novel therapeutic approaches to block the damage caused by an uncontrolled

inammatory response. In collaboration with Biotech Companies, we are working towardsthe development of viral immunomodulatory proteins as potential medicaments to treat

human allergic and autoimmune diseases caused by immunopathological reactions.

Viruses are the most abundant and diverse biological entities on Earth. We are interested

in the molecular characterization of complex viral communities using new massivesequencing methodologies (Roche-454 Life Sciences). We have described for the rsttime the viral community in an Antarctic lake and are expanding these studies to other

lakes along the Antarctic Peninsula and in the Arctic. We are also interested in using

the new sequencing technologies to identify viruses associated with human pathologies,such as multiple sclerosis.

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Research Summary

Figure 1.Mimicryof cytokines and

cytokine receptors

by herpesviruses

and poxviruses.

Figure 2. Metageno-mic studies of the viralcommunity in Antarctic

lakes.

Staff

Publications

Other Activities

Lnea Investigacin

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CBMSO2009-2010


Research Summary

Publications

Other Activities

Staff

Group Leader:Antonio Alcami Pertejo

Postdoctoral Fellows:Ali Alejo HerbergAbel Viejo Borbolla

Alberto Lpez BuenoDaniel Rubio MuozElena Merino RodrguezSoledad Blanco Chapinal

Imma Montanuy SellartJuan Alonso Lobo

Graduate Students:Marcos Palomo Otero

Sergio Martn PontejoNadia Martnez MartnCarla Mavin

Haleh Heidarieh

Technical Assistance:Roco Martn HernndezCarolina Snchez Fernndez

Visiting Scientists:Joanne Devlin (University ofMelbourne, Australia)Julia Fahel (Trinity College,Dublin, Ireland)

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Publications

Andrs G., Leali, D., Mitola, S., Coltrini, D., Camozzi, M., Corsini, M., Belleri, M., Hirsch, E., Schwendener, R. A., Christofori, Alcami,A. and Presta, M. (2009) A pro-inammatory signature mediates FGF2-induced angiogenesis. J. Cell. Mol. Med. 13, 2083-2108.

Waibler, Z., Anzaghe, M., Frenz, T., Schwantes, A., Phlmann, C., Ludwig, H., Palomo-Otero, M., Alcami, A., Sutter, G. and Kalinke,U. (2009) Vaccinia virus-mediated inhibition of type I interferon responses is a multi-factorial process involving the soluble type Iinterferon receptor B18 and intracellular components. J. Virol.83, 1563-1570.

Carson, C., Antoniou, M., Ruiz-Argello, M. B., Alcami, A., Christodoulou, V., Messaritakis, I., Blackwell, J. M. and Courtenay, O.(2009) A prime/boost DNA/Modied vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safeand immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis. Vaccine27, 1080-1086.

Alcami, A. and Saraiva, M. (2009) Chemokine binding proteins encoded by pathogens.Adv. Exp. Med. Biol.666, 167-179.

Poole, E., Groves, I., Ho, Y., Benedict, C., Alcami, A. and Sinclair, J. (2009) Identication of TRIM23 as a co-factor involved in theregulation of NFkB by the human cytomegalovirus gene product UL144. J. Virol.83, 3581-3590.

Alejo, A., Ruiz-Argello, M. B., Viejo, A., Saraiva, M., Fernndez de Marco, M. M., Salguero, J. and Alcami, A. (2009) Adaptation ofa transient dominant selection method to the generation of ectromelia virus recombinants: in vivo analysis of ectromelia virus CD30deletion mutants. PLoS ONE4(4):e5175.

Alcami, A. and Viejo-Borbolla, A (2009) Identication and characterization of virus-encoded chemokine binding proteins. MethodsEnzymol.460, 173-191.

Shang, L., Thirunarayanan, N., Viejo-Borbolla, A., Martin, A. P., Bogunovic, M., Marchesi, F., Unkeless, J. C., Ho, Y., Furtado, G. C.,Alcami, A., Merad, M., Mayer, L. and Lira, S. A. (2009) Expression of the chemokine binding protein M3 promotes marked changes inthe accumulation of specic leukocytes subsets within the intestine. Gastroenterology137, 1006-1018.

Lpez-Bueno, A., Tamames, J., Velzquez, D., Moya, A., Quesada, A. and Alcami, A. (2009) High diversity of the viral community froman Antarctic lake. Science326, 858-861.

Alejo, A. and Alcami, A. (2010) Chemokine binding proteins as therapeutics. In: Leurs, R., Smit, M. and Lira, S. (eds) ChemokineReceptors as Drug Targets. Wiley-VCH, Weinheim, Germany, pp.359-374.

Rubio, N., Palomo, M. and Alcami, A. (2010) Interferon a/bgenes are upregulated in murine brain astrocytes after Theilers murine

encephalomyelitis virus infection. J. Interf. Cyt. Res.30, 253-262.Viejo-Borbolla, A., Muoz, A., Tabares, E. and Alcami, A. (2010) Glycoprotein G from pseudorabies virus binds to chemokines withhigh afnity and inhibits their function. J. Gen. Virol.91, 23-31.

Fernandez de Marco, M. M., Alejo, A., Hudson, P., Damon, I. K. and Alcami, A. (2010) The highly virulent variola and monkeypoxviruses express secreted inhibitors of type I interferon. FASEB J.24, 1479-1488.

Devlin, J. M., Viejo-Borbolla, A., Browning, G. F., Noormohammadi, A. H., Gilkerson, J. R., Alcami, A. and Hartley, C. A. (2010)Evaluation of immunological responses to a glycoprotein G decient candidate vaccine strain of infectious laryngotracheitis virus.Vaccine28, 1325-1332.

Alcami, A. and Lira, S. A. (2010) Modulation of chemokine activity by viruses. Curr. Opin. Immunol.22, 482-487.

Alcami A. (2010) The interaction of viruses with host immune defenses. Curr. Opin. Microbiol.13, 501-502.

Viejo-Borbolla, A., Martin, A. P., Muniz, L. R., Shang, L., Marchesi, F., Thirunarayanan, N., Harpaz, N., Garcia, R. A., Apostolaki, M.,Furtado, G. C., Mayer, L., Kollias, G., Alcami, A. and Lira, S. A. (2010) Attenuation of TNF-driven murine ileitis by intestinal expressionof the viral immunomodulator CrmD. Mucosal Immunol.3, 633-644.

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Research Summary

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Other Activities

Member of the Editorial Board of Virology

Member of the Editorial Board of Journal of Virology

Advisor to the World Health Organization Advisory Committee on Variola Virus Research

Editor of a special issue of Curr. Opi. Microbiol. on Host-microbe interacions: viruses, vol.13, August 2010.

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CBMSO2009-2010

B2 Molecular bases of viral pathogenesis and anti-cancer potential

Staff

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Doctoral Theses

Research Summary

Research Summary

The research of the laboratory is mainly focused in the molecular mechanisms operatingin viral diseases and in the design of viruses as potential anti-cancer biological tools.

We use as experimental models the parvovirus Minute Virus of Mice (MVM) and thealphavirus Sindbis virus (SV). Among those topics recently addressed, or under currentresearch interest, the following two are within the main ones: (i) The regulation of MVMcapsid assembly in human cancer cells. We have found that VP2, the major MVM capsid

protein, is specically phosphorylated by Raf-1 (Figure 1), a kinase playing key roles inthe development of several common human cancers. The phosphorylation by a mutation-

activated Raf-1 facilitates the nuclear transport of VP2 trimers (Figure 1B), a processessential for the viral maturation. This MVM assembly regulation (Figure 1C) may explainmost aspects of the parvovirus natural tropism toward human cancer cells, and it doesidentify a relevant target for possible oncolytic virotherapies. (ii) Translation control of viralmRNA. Our working hypothesis is that translation of viral mRNA may be directing keyaspects of virus cycle such as host range and evolution, tropism for cells and tissues, and

pathogenesis. Our main goal is to understand how the PKR cellular kinase modulatesreplication of SV (Figure 2) and MVM through the phosphorylation of translation initiationfactor 2 (eIF2). By means of systems biology, we are also characterizing the molecularmechanism used by SV to overcome the antiviral effect of PKR, which involves specic

structures in viral mRNA and the use of alternative initiation factors supporting viraltranslation.

The group of Begoa Aguado (*) is focused on Alternative Splicing as a factor contributingto complexity and diversity among primates and other mammals, through the generation

of different transcripts, including chimeric and non-coding ones. We are also investigating

alternative splicing alterations which can cause disease, such as Rheumatoid Arthritis andMyotonic Dystrophy. We are using new technologies such as nano Q-PCRq, microarraysand Massive Sequencing.

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CBMSO2009-2010

B2

Research Summary

Molecular bases of viral pathogenesis and anti-cancer potential

Staff

Publications

Doctoral Theses

Research Summary

Figure 1. The Raf-1 kinase regulatesMVM assembly. (A)T w o - d i m e n s i o n a lphosphopeptide map ofMVM capsid subunitsin vitro phosphorylatedby Raf-1. (B) Trimers ofthe MVM capsid proteinisolated from humanand insect cells differ intheir nuclear transport

competence. (C) The roleof Raf-1 in the nucleartranslocation of MVMassembly intermediates.

Figure 2. The shut-offphenomenon in mouse brain

tissues infected with Sindbisvirus. Translation of cellular,but not of viral mRNAs,was inhibited due to eIF2phosphorylation.

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CBMSO2009-2010


Research Summary

Publications

Doctoral Theses

Staff

Group Leader:Jos M. Almendral del Ro

Scientic Staff:

Begoa Aguado Orea (*)Antonio Talavera DezIvn Ventoso Bande

Postdoctoral Fellows:Elena Domingo Gil

Esther Grueso HierroFrancisco Hernndez-Torres (*)

Graduate Student:Noelia Blanco MenndezMaria Elizalde ArbillaRen Toribio Lpez

Olatz Villate Bejarano (*)Alberto Rastrojo Lastras (*)Raquel Lpez-Dez (*)

Undergraduate Students:Marian Fernandez EstevezJulia Wienke (*)

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Publications

Sanz MA, Castell A, Ventoso I, Berlanga JJ, Carrasco L. (2009). Dual mechanism for the translation ofsubgenomic mRNA from Sindbis virus in infected and uninfected cells. PLoS One. 4(3):4772.

Blanco, AM, L Rausell, B Aguado, M Perez-Alonso, R Artero. (2009). A FRET-based assay for characterizationof alternative splicing events using peptide nucleic acid uorescence in situ hybridization. Nucleic AcidsResearch37e116.

Riolobos, L., Valle, N., Hernando, E., Maroto, B., Kann, M., and J. M. Almendral. (2010). Viral oncolysis thattargets Raf-1 signaling control of nuclear transport. J. Virol.,84, 2090-2099.

Ventoso, I., Berlanga, J.J., and J. M. Almendral. (2010). Translational control by the Protein Kinase Rrestricts Minute Virus of Mice infection: role in parvovirus oncolysis. J. Virol.,84, 5043-5051.

Toribio R, Ventoso I. Inhibition of host translation by virus infection in vivo. (2010). Proc Natl Acad SciUSA. 107(21):9837-42.

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Doctoral Theses

Olatz Villate.Splicing en el MHC de clase III: caracterizacin y expresin de las iso-formas del gen NFkBIL1. Estudio de su relacin con artritis reumatoide. Directora,Begoa Aguado.

Ren Toribio Lpez.Cambios traduccionales que regula la interaccin virus hospeda-dor. Implicacin en el desarrollo de virus oncolticos. Director, Ivn Ventoso

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CBMSO2009-2010

B3 Molecular Ecology of Extreme Environments

Staff

Publications

Patents

Doctoral Theses

Research Summary

Research SummaryTwo main lines of research are currently in progress in our group:Molecular Ecology of Extreme Environments:This area of research has the followingobjectives:- Acidophiles: conventional microbial ecology (enrichment cultures, isolation, physiology),molecular ecology (DGGE, FISH, CARD-FISH, cloning), molecular biology (genomics,metagenomics, proteomics, differential gene expression using DNA arrays) andbiotechnology (control of bioleaching, specic metal sequestering and phytoremediation)of extreme acidic environments (Ro Tinto basin, different acidic lakes of the Iberian PyriticBelt, ro Agrio (Argentina), volcanic areas of Island, Antartica),

- Geomicrobiological characterization of extreme environments as habitability models ofastrobiological interest: Tinto basin (Mars geochemical analogue), sulde deposits fromAntartica (Mars analogue), Tirez hypersaline lagoon (Europa analogue, in collaborationwith I. Marn, associate professor of the Department of Molecular Biology), Uyuni salt lake(Europa analogue, in collaboration with I. Marn), volcanic areas of Island, permafrostareas of Alaska (Mars analogue).- Geomicrobiology of the Iberian Pyritic Belt (IPB) subsurface: different techniques arebeing implemented in the characterization of the subsurface bioreactor responsible of theextreme acidic conditions of Ro Tinto. This work is done in collaboration with the Centrode Astrobiologa (Origin Project IPBSL)- The line of microbial ecology of anaerobic environments directed by professor J.L. Sanz(UAM) is being developed in the new facilities that the Department of Molecular Biology

has in the Biology Building. This collaborative work is centred in the anaerobic activitiesdetected in the different model systems studied by our group (Tinto basin, IPB, Tirezlagoon). Micology, This area of research directed by Dr. Aldo Gonzlez has the followingobjectives:- Molecular genetics and microbiology of Basidiomicetes (Pleurotus ostreatusas modelsystem).- Use as lamentous fungi as a source of secondary metabolites, lignolytic enzymes andspecic sequestering of toxic metals.- Control and elimination of fungi from air-indoor.

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Research Summary Figura 1.Specic Cr(III) sequestering acidophilic fungi.

Figura 2.Acidophilic photosynthetic biolm.

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Publications

Patents

Doctoral Theses

Staff

Group Leader:Ricardo Amils Pibernat

Scientic Staff:

Aldo Gonzlez Becerra

Posdoctorales:Moustafa Malki

Monika Oggerin de Orube

Lourdes Rufo NietoJavier Ruiz Prez

Graduate Students:Patxi San Martn UrizEnrique Marn PalmaCarlotta VizioliIrene Snchez Andrea

Technical Assistance:

Nuria Rodrguez GonzlezCatalina del Moral Juarez

Visiting Scientists:Linda Amaral (MBL, Woods Hole, USA)Jim Field (Arizona State University, USA)Alberto Gonzlez Fiaren (NASA-Ames, USA)Eric Zettler (MBL, Woods Hole, USA)

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Publications

Publications

Fairen, A.G., Dvila, A.F., Duport, L.G., Amils, R., McKay, C.P. (2009) Nature, 459: 398-400.

Fairen, A.G., Schulze-Makuch, D., Rodrguez, A.P., Fink, W., Dvila, A., Uceda, E.R., Furfaro, R., Amils, R.,McKay, C.P. (2009)Planet.Space Sci., 57: 276-287.

Daz-Garretas, B., Asensi, A., Rufo, L., Rodrguez, N., Snchez-Mata, D., Amils, R., de la Fuente, V. (2009)Northeastearn Naturalist,16: 56-64.

Carnicero, D., Daz, E., Escolano, O., Rubinos, D., Ballesteros, O., Barral, M.T., Amils, R., Garca Frutos,F.J. (2009) Adv. Materials Research, 71-73: 677-680.

Eugenio, M.E., Carbajo, J.M., Martn, J.A., Gonzlez, and A.E. Villar, J.C. (2009) J. Basic Microbiol.49(5): 433-440.

Gonzlez-Toril, E., Aguilera, A., Souza-Egipsy, V., Diez, M., Lpez-Pamo, E., Snchez-Espaa, J., Amils,R. (2009) Adv. Materials Research, 71-73: 113-116.

Amils, R., Gonzlez-Toril, E., Aguilera, A., Rodrguez, N., Fernndez-Remolar, D., Daz, E., Garca-Moyano, A., Sanz, J.L. (2009) Adv.Materials Research, 71-73: 13-19.

Garca-Moyano, A., Gonzlez-Toril, E., Amils, R. (2009) Adv. Materials Research, 71-73: 109-112.

Gonzlez-Toril, E; Amils, R; Delmas, RJ, et al. 2009. Biogeosciences, 6(1): 33-44.

de la Fuente, V., Rufo, L., Rodrguez, N., Amils, R., Zuluaga, J. (2009) Biol Trace Elem Res, DOI10.1007/s12011-009-8471-1.

Aguilera, A., Souza-Egipsy, V., Gonzlez-Toril, E., Rendueles, O., Amils, R. (2010). Internat. Microbiol., 13:29-40. DOI: 10.2436/20,1501.01.109.

Gonzlez-Toril, E., Aguilera, A., Rodrguez, N., Fernndez-Remolar, D., Gmez, F., Daz, E., Garca-Moyano, A., Sanz, J.L., Amils, R. (2010) Hydrometallurgy, 104: 329-333.

Cid, C., Garcia-Descalzo, L., Casado-Lafuente, V., Amils, R., Aguilera, A. (2010) Proteomics, 10: 2026-2036 DOI 10.1002/pmic.200900592.

Souza-Egipsy, V., Aguilera, A., Mateo-Mart, E, Martn-Gago, J.A., Amils, R. (2010). Geomicrobiol. J., 27:692-706.

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Lalueza, J., Rius, A., Puig, R., Mart, E., Mart, J.F., Rodrguez, N., Amils, R. (2010). Journal of AmericanLeather Chemists Association, 105: 210-221.

Amaral -Zett ler, L ., Zettler, E.R., Theroux, S.M., Palacios, C., Agu ilera, A., Ami ls, R. (20010) ISME J.doi:10.1038/ismej.2010.101.

Arana-Cuenca, A., Tllez Jurado A., Yage, S., Fermian, E., Carbajo, J.M., Gonzlez, T., Domnguez, A.,Villar, J.C., and Gonzlez, A.(2010) Forest Systems19(2): 234-240.

Eugenio, M.E., Santos, S.M., Carbajo, J.M., Martn, J.A., Martn-Sampedro, R., Gonzlez, A.E., and Villar,J.C. (2010) Bioresources Technology101(6): 1866-1870.

Fairn, A.G., Chevier, V., Abramov, O., Marzo, G.A., Gavin, P., Davila, A.F., Tornabene, L.L. , Bishop, J.L.,Roush, T.L., Gross, C., Kneissl, T., Uceda, E.R., Dohm, J.M., Schulze-Makuch, D., Rodrguez, J.A.P., Amils,R., McKay, C.P. (2010) PNAS (USA), doi/10.1073/pnas.1002889107.

Fairen, A. G., Dvila, A.F., Lim, D., Bramall, N., Bonaccorsi, R., Zavaleta, J., Uceda, E.R., Stoker, C.,,Wierzchos, J., Amils, R., Dohm, J.M., Andersen, D., McKay, C. (2010)Astrobiology, 821- 843. DOI:10.1089/ast. 2009.0440.

Fernndez-Remolar, D., Snchez-Romn, M., Amils, R. (2010) Sustainability, 2: 2541-2554,doi:10.3390/su2082541.

Gmez; F., Mateo-Mart, E., Prieto.Ballesteros, O., Martn-Gago, J., Amils, R. (2010) Icarus, doi:10.1016/j.icarus.2010.05.027.

Carbajosa, S., Malki, M., Caillard, R., Lpez, M.F., Palomares, F.J., Martn-Gago, J.A., Rodrguez, N.,Amils, R., Fernndez, V.M., De Lacey, A.L. (2010) Biosensors and Bioelectronics, 26: 877-880.

Fernndez-Remolar, D., Prieto-Ballesteros, O., Gmez-Ortiz, D., Fernndez-Sampedro, M., Sarrazin, P.,Gailhanou, M., Amils, R. (2010) Icarus, 211: 114-138.

Menor-Salvn, C., Tornos, F., Fernndez-Remolar, D., Amils, R. (2010) Earth Planet. Sci. Lett., doi:10.1016/j.epsl.2010.09.020.

Captulos de libros:Cinco captulos de libro.Libros:Amils, R., Segura, J. (2010) Ro Tinto viaje a Marte, ediciones Alfar (Sevilla).

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Patents

Amils, R. Malki, M., Fernndez, V., de Lacey, A.L.Ttulo: Electrodo bacteriano aerbico para nodo de una pila de combustible sin mediadoresredox ni membrana intercambiadora de protones. N de solicitud: 200701534. Fecha deconcesin: 23/12/2009

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Doctoral Theses

Eric Zettler, enero 2009, The relationship between environment and the microbialcommunity in a patchwork of geochemical islands, Ricardo Amils, Universidad Autnomade Madrid.

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CBMSO2009-2010

B4

Research Summary

Bacterial Cell Division and Antibiotics Resistance

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Research Summary

The objectives of our group are the analysis of the bacterial growth and cell division underdiverse molecular approaches, and to study the mechanisms of resistance to the -lactamantibiotics, that have been developed by pathogens of clinical origin. Under this generalscheme, we have developed two main aspects. One is the molecular characterization ofthe CTX-M -lactamases family, their mobilization mechanisms and the ancestral originof the CTX-M-1 subfamily. Also the identication of PBPs and their role on the resistancemechanisms of anaerobic strains and Gram-negative enteric bacteria was analyzed. A newfamily of PBPs, belonging to the COG1680, with high homology with class C -lactamasesand been involved in morphogenesis, are going to be analyzed in a new project.

Based on analysis of peptidoglycan structure of AeromonasPBP4 mutants, we haveproposed a model for induction of the expression of -lactamase mediated by a twocomponent regulatory system. (See joint gure)

During cell division, assembly of proteins at a division ring has the effect of constricting

the membrane and producing a cell wall septum. The synthesis of a rigid peptidoglycanseptum involves a set of dedicated enzymes, as penicillin-binding proteins. We haveachieved the purication of several of these enzymes (PBP1B, PBP3, and inactive

variants) together with a good number of substrates (including sacculi, as the largestavailable structure, fragmented peptidoglycan and smaller-sized precursors as lipid II andUDP-muramyl pentapeptide) to assist in the set up of in vitroscreening assays.

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CBMSO2009-2010

B4 Bacterial Cell Division and Antibiotics Resistance

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Research Summary

Figure 1. Model for beta-lactamase induction inAeromonas spp. and the role of the blr regulon.Inhibition of PBPs by b-lactams causes an increasein the concentration of the BlrB activatory ligand. Thisligand then interacts with BlrB (dotted arrow), causingit to auto-phosphorylate (P in the diagram). Thisphosphate is transferred to BlrA, which binds to the cre/blr tag sequence found upstream of all blr regulon genepromoters. The effect of this is to recruit RNA polymeraseand activate blr regulon transcription. Known blr regulongenes encode three blactamases, Amp, Cep and Imi,which directly hydrolyse the b-lactam, helping to prevent

further peptidoglycan damage. The blr regulon is alsoknown to include non-b-lactamase-encoding genes, e.g.blrD, and it is proposed that their products have a role in

protecting the cell from b-lactam challenge through anancillary mechanism.

Figure 2.DD-endopeptidase activity of AmpH identiedby HPLC. Peaks correspond to M4.- NAcGlc-NAcMur-tetrapeptide , M5.- NAcGlc-NAcMur-pentapeptide, D45.-Disacharide-tetra-pentapeptide, and C.- cefmetazol.

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Research Summary

Publications

Other Activities

Staff

Group Leader:Juan Alfonso Ayala Serrano

Postdoctoral Fellow:Silvia Marina Gonzlez Leiza

Graduate Students:Cristian Gustavo Aguilera RossiAlaa Ropy Mahmoud Sayed

Undergraduate Students:Godofrey Cherry

Yasemin Ezgi ErtrkAndrs Caballero

Laura VuoloJose Roberto Angeles VzquezNicolas Cordeiro

Ana Fernndez GonzlezSorelis Urdaneta FernndezLucia LozanoPaula Andrea Espinal

Mara Margarita Bernal

Visiting Scientists:Ayelen Patricia Porto (Universidad de Bue-nos Aires, Buenos Aires, Argentina)Ana Catarina Souza Lopes (Universidade

Federal de Pernambuco, Brasil)Bartolome Moya (Universidad Islas Balea-res, Mallorca)Judith J. Velasco (Universidad de los An-des, Mrida, Venezuela)-Jzsef Ski University of Szeged,-Elizabeth Nagy University of Szeged,-Gabriella Terhes(University of Szeged, Szeged, Hungary)Sergei Borchsenius (Institute of Cytology,

RAS, Saint-Petersburg, Russia)

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Publications

D. Korsak, Z. Markiewicz, G. O. Gutkind, and J. A. Ayala. (2010) Identication of the full set of Listeriamonocytogenespenicillin-binding proteins and characterization of PBPD2 (Lmo2812). BMC Microbiology,10:239-251

Ins Bado, Nicols F. Cordeiro, Luciana Robino, Virginia Garca-Fulgueiras, Vernica Seija, Cristina Bazet,Gabriel Gutkind, Juan A. Ayala, and Rafael Vignoli. (2010). Detection of class 1 and 2 integrons, extended-spectrum -lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive CareUnit inpatients. Intern. J. Antimicrob. Agents,36(5):453-458

A.C.S. Lopes, D. Leal Veras, A.M.S. Lima, R.C. Andrade Melo, and J.A. Ayala. (2010). bla(CTX-M-2) and

bla(CTX-M-28) extended-spectrum beta-lactamase genes and class 1 integrons in clinical isolates ofKlebsiella pneumoniaefrom Brazil.. MIOC. 105(2):163-167.

Amy E. Tayler, Juan A. Ayala, Pannika Niumsup, Katrin Westphal, Timothy R. Walsh, Bernd Wiedemann,Peter M. Bennett, and Matthew B. Avison. (2010). Induction of beta-lactamase production inAeromonashydrophila is responsive to beta-lactam-mediated changes in peptidoglycan composition. Microbiology.

156(Pt 8):2327-35.

Porto, Ayeln; Ayala, Juan; Gutkind, Gabriel; and Di Conza, Jos. (2010). A novel OXA-10-like -lactamaseis present in different Enterobacteriaceae. Diagnostic Microbiology and Infectious Disease.66:228-229.

M. Macedo-Vias, N. F. Cordeiro, I. Bado, S. Herrera-Leon, M. Vola, L. Robino, R. Gonzalez-Sanz, S.Mateos, F. Schelotto, G. Algorta, J. A. Ayala, A. Echeita and R. Vignoli. (2009). Surveillance of antibioticresistance evolution and detection of class 1 and 2 integrons in human isolates of multiple resistant

Salmonella Typhimurium obtained in Uruguay from 1976 to 2000. International Journal of Infectious

Diseases, 13:342-348.

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Profesor Ad Honorem, Facultad de Medicina, Universidad de la Republica, UruguayJulio, 2009.

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CBMSO2009-2010

B5 Biotechnology and Genetics of Extreme thermophilic Bacteria

Research Summary

Staff

Publications

Patents

Research Summary

We use the extreme thermophilic bacteria Thermus thermophilus (Tth) as our main

laboratory model. Tthgrows quite fast at 70 C, forms colonies on plates, and shows naturalcompetence. As other thermophiles, its enzymes show a greater ability to crystallize thantheir mesophilic homologues, thus making it an excellent model for Structural Biology. Itsancient phylogenetic origin also adds biological and evolutionary interest to this model.

We pursue both biotechnological and basic-science objectives with this model. Inbiotechnology: i) We overproduce and use thermostable enzymes (thermozymes) for

biotransformations in collaboration with external groups specialized in biocatalysis; ii)We evolve enzymes and proteins towards either increasing their thermostability throughin vivo selection, or their specic activities at low temperatures; and iii) We develop newgenetic tools for its use in thermophiles. In the last two years we have overproducedmore than 50 thermozymes (dehydrogenases, estherases, and glycosidases) and carriedout selection procedures for the isolation of thermostable mutants of three proteins.

In basic-science we study the energy metabolism of Tth, specically the respiration ofnitrogen oxides denitrication-, its genetic control, and its lateral gene transfer (LGT).Through massive sequencing we have shown that the process is catalyzed by threereductases encoded within a denitrication island (DI) that is the subject of frequent LGT

events. This DI concentrates information on new type of enzymes that replace the aerobicrespiratory complexes I and III, by more simple ones or by bifunctional enzymes acting asreductases-electrons transporter. The DI also encodes new sensory-signal transductionsystem that allows the organism to select the components of its respiratory chainsaccording to environmental signals. For the next years we will continue these studiesand we start new projects on unconventional mechanisms that prevent the LGT betweenbacterial species.

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B5

Research Summary

Biotechnology and Genetics of Extreme thermophilic Bacteria

Staff

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Patents

Research Summary

Figure 1. The denitricationapparatus of Thermus

thermophilus. An scheme of

the localization and role of theNADH dehydrogenase (Nrc),and the nitrate (Nar), nitrite(Nir), and nitric oxide (Nor)reductases is shown. Notethe role of Nar as electron

transporter replacing the

complex III. Electrons pathwayis shown as red arrows.

Figure 2. Evolving an

esterase. (R)-1-phenyl-2-propyl acetate docked

in the active site of a

quadruple mutant (mutantQ) of the Pseudomonasfuorescens esteraseI.

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CBMSO2009-2010


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Staff

Group Leader:Jos Berenguer Carlos

Scientic Staff:

Aurelio Hidalgo Huertas

Postdoctoral Fellows:Daniel VegaLeticia Torres

Graduate Students:Federico AcostaEloy Ferreras PuenteZahra ChahlaMarcos Almendros GimenezLaura lvarez MuozCarlos Bricio Garber

No Rigoberto RiveraYamal Al-Ramahi GonzlezMartin Hesseler

Undergraduate Students:Alba M Sanchez Nio,Akbar Espaillat Fernndezngel Cantero CamachoDaan Swarts

Technical Assistance:Esther Snchez FreireMaria Jos de Soto Lpez

Visiting Scientists:Estariette van HeerdenKoos AlbertinDerek LitthauerGodfrey TlouPhilip Armand Bester

Mariana Erasmus

Novalanda Betty MabizelaSusana Alarico

Hernn Costa

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Patents

Publications

Larsen,M., Zielinska, D.F., Martinelle, M., Hidalgo, A., Jensen, L., Bornscheuer, U.T. and Hult, K. (2010)Suppression of water as a nucleophile in Candida antarcticalipase B catalysis. ChemBioChem11,796-801.

Cava, F, Hidalgo, A., and Berenguer, J. (2009) Thermus thermophilusas biological model. Extremophiles,13, 213-231.

Schliemann, A., Hidalgo, A., Berenguer, J. and Bornscheuer, U.T (2009). Increased Enantioselectivity byEngineering Bottleneck Mutants in an Esterase from Pseudomonas fuorescens. ChemBioChem 10,2920-2923 (cover feature).

Rocha-Martin, J., Vega D., Cabrera Z, Bolivar JM. Fernandez-Lafuente R., Berenguer J. and Guisan, J.M.(2009) Purication, immobilization and stabilization of a highly enantioselective alcohol dehydrogenasefrom Thermus thermophilusHB27 cloned in E. coli.Proccess in Biochemistry, 44,1004-1012.

Bolivar J.M., Rocha-Martin J., Mateo C., Cava F., Berenguer, J, Vega D. , Fernandez-Lafuente R., GuisanJ.M. (2009) Purication and stabilization of a glutamate dehygrogenase from Thermus thermophilusviaoriented multisubunit plus multipoint covalent immobilization. Journal of Molecular Catalysis B: Enzymatic58, 158-163.

Almendros M., Sinisterra JV, and Berenguer J. (2009) Thermus thermophilusStrains Active in PurineNucleoside Synthesis. Molecules2009, 14, 1279-1287.

Bolivar J.M, Rocha-Martin J. Mateo C., Cava F., Berenguer J., Fernandez-Lafuente R., Guisan J. M.(2009) Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternarystructure of multimeric enzymes. Biomacromolecules10, 742-747

Bolivar J., Mateo, C., Rocha-Martin, Cava F., Berenguer J., Fernandez-Lafuente R., Guisan J. M. 2009.The adsortion of multimeric enzymes on very lowly activated supports involves more enzyme subunits:stabilization of glutamate dehydrogenase from Thermus thermophilusby immobilization on heterofunctionalsupports. Enzyme Microb. Technol.44, 139-144.

Cava, F., Chahla, Z., Alavare, L., and Berenguer, J. (2009) Respiracin y desnitricacin en Thermusthermophilus.In: Bonete, M. J., and Martnez-Espinosa R. M. (ed) Avances en el metabolismo del nitrgeno.Editorial Club Universitario, Alicante. pp. 173-186.

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Patents

Procedimiento de inmovilizacin de una glutamato deshidrogenasa.PCT/ES2008/070166.

Nuevo marcador termoestable para la seleccin gentica de Thermus sppP200603279.(Concesin 28-05-2010).

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CBMSO2009-2010

B6

Research Summary

ASFV: virus models of evasion and protection

Staff

Publications

Research Summary

During the last years we have been searching for established cell lines sensitive to theAfrican Swine Fever Virus (ASFV), for the production and titration of virus isolates bothfrom the eld or laboratory, as well as for the generation of virus recombinants with speci-c genes deleted. These deletion mutants facilitate the study of the role of several ASFVgenes involved in the evasion of the antiviral response of the cell during virus infection,

and may serve as attenuated virus models in the generation of possible vaccines to in-

duce protective immunity against ASF. So, we have conrmed the role of the ASFV lectin

(EP153R gene) in the modulation of the expression of MHC-I antigens in the membraneof the infected cell, and we are also inactivating several viral genes in a partially-attenua-ted ASFV isolate (NHV), able to induce protection against the virulent L60 isolate, but stillretaining a residual virulence unacceptably high to be used as a vaccine.

The porcine cell lines sensitive to ASFV infection are also being used for the determinationof the level of induction of selected cytokines by ASFV isolates with various degreesof virulence, searching for a possible virulence/attenuation prole, which might resultin a remarkable reduction of the in vivo infections required to determine the degree ofvirulence of the virus isolates.

Another objective of our Group is the optimization of the use of non-conventional antiviralsas the lauryl gallate (LG) in the prevention and/or treatment of viral diseases of clinicaland veterinarian importance. We have demonstrated its low cytotoxicity and its efciencyin the inhibition of the virus production in several models (ASFV, inuenza, herpes,...) incells infected in the laboratory, and we pretend to extend the study of the protective levelprovided by the LG in in vivoinfections.

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CBMSO2009-2010

B6

Research Summary

ASFV: virus models of evasion and protection

Figure 1.Interaction EP153R - SLA-I. Residues involved

Staff

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CBMSO2009-2010

B6 ASFV: virus models of evasion and protection

Research Summary

Publications

Staff

Group Leader:ngel L. Lpez Carrascosa

Postdoctoral:Patricia de Len Valds

Undergraduated Student:Alba Martnez Flrez

Technical Assistance:Maria Jos Bustos Snchez

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CBMSO2009-2010

B6 ASFV: virus models of evasion and protection

Publications

Research Summary

Staff

PublicationsPublications

Hurtado, C., Bustos, M. J. and Carrascosa, A. L. (2010). The use of COS-1 cells for studies of eld and la-boratory African swine fever virus samples. J. Virol. Methods164, 131-134.

Hurtado, C., Bustos, M. J., Granja, A. G., de Len, P., Sabina, P., Lopez-Vias, E., Gomez-Puertas, P., Re-villa, Y., and Carrascosa, A. L. (2010). The African swine fever virus lectin EP153R modulates the surfacemembrane expression of MHC class I antigens.Arch. Virol.DOI: 10.1007/s00705-010-0846-2.

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CBMSO2009-2010

B7 Bacterial morphogenesis

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Research Summary

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Publications

Research Summary

Our main interest is the investigation, at the molecular and structural levels, of the bacterial

cell wall (sacculus or peptidoglycan layer) as the primary morphogenetic element ofthe prokaryotic cell. We are presently working on cell development, adaptation anddifferentiation of appendages (prostheca, agella, pili and hold-fast) in alfa-proteobacteria,mainly inAsticcacaulis biprosthecium, and other bacterial groups of complex morphology.

We want to extend our experience in simple cell-cycle organisms as Escherichia coli,

to more complex bacteria as A.biprosthecium. This species presents a dimorphic cellcycle with a mobile agellated form (swarmer cell) which eventually differentiates into asessile form displaying two laterally opposed prostheca. Only the sessile form is divisionprocient, and generates a sessile and a swarmer cell upon division. At present we areinvestigating how the metabolism and structure of the cell wall are modied in accordancewith the cell cycle differentiation processes, and in relation with the generation of cellappendages (prostheca, agella, and hold-fast) at precise places and times in the cellcycle. Recently we started a new line of work centred on the discovery of a novel regulatorymechanism of cell wall metabolism in Vibrio choleraeand other bacteria. The mechanismis mediated by the production and release of specic D-amino acids, is part of the general

stress response mechanism, and has the potential to act as an inter and intra specicsignalling system. We are also involved in cooperative projects concerning structural and

biochemical aspects of cell wall metabolism in other organisms, Listeria monocitogenesor Leptospira bifexa, associated to the development of symbiotic or pathologic relations

and to peculiar morphologies.

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Figure 1.Asticcacaul is biprosthecium cellsvisualized by confocal microscopy.

Figure 2. Actively growing (A) and resting (B)cells of Vibrio cholera as visualized by ESM.

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Publications

StaffStaff

Group Leader:Miguel Angel de Pedro Montalbn

Graduate Students:Said Taimani

Undergraduate Students:

Marisela Domnguez DomnguezMara Hidalgo GarcaIrene Cartas

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Publications

Lam, H, Oh, D.C., Cava, F., Takacs, C.N., Clardy, J., de Pedro, M.A. and Waldor, (2009) D-amino acidsgovern stationary phase cell wall remodeling in bacteria. Science325:1552-1555.

de Pedro, M.A. (2009). Peptidoglycan (Murein), In Encyclopedia of Microbiology. (Moselio Schaechter,Ed.), pp. 453-469, Oxford: Elsevier.

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CBMSO2009-2010

B8

Research Summary

Genetic variability of RNA viruses

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Doctoral Theses

Research Summary

The main interest of our group is to understand the molecular bases of RNA virusextinction by lethal mutagenesis, and to explore antiviral protocols using mutagenic

agents and antiviral inhibitors. We have documented a double mutagenic and inhibitory

activity of 5-uorouracil on foot-and-mouth disease virus (FMDV), and have characterizedFMDV mutants resistant to the mutagenic purine analogue ribavirin. Specically, wehave described an amino acid substitution in the FMDV polymerase which avoids thedeleterious activity of ribavirin by means of a modulation of the mutation types produced

by the drug. Virus survival through modulation of mutation types, without an alterationof the general copying delity of the polymerase, constitutes a new mechanism ofresistance to a mutagenic agent. Despite the presence of mutagen-resistance mutations,

virus extinction can be achieved with alternative mutagenic treatments. Contrary to whathas been established for classic antiviral treatments, a sequential administration of aninhibitor rst, and then of a mutagenic agent can be more effective for virus extinctionthan the administration of the two drugs simultaneously.

With regard to the understanding of the biological implications of quasispecies behavior, wehave characterized a competition-colonization dynamics among FMDV subpopulation thatarose in cell culture through diversication of a biological clone of the virus. Our laboratory

has participated in collaborations with various teams on theoretical and experimentalvirology, on structural studies with the FMDV polymerase, and biological implications ofquasispecies, including new vaccine designs. Such collaborations can be identied by thenames of the authors of the publications included in the present summary.

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Research Summary

Genetic variability of RNA viruses

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Figure 1.Antiviral strategy based on the extinction of viruses by increasing their mutationrates during replication.

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CBMSO2009-2010

B8 Genetic variability of RNA viruses

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Publications

Patents

Doctoral Theses

Staff

Group Leader:Esteban Domingo Solans

Postdoctoral Fellows:Celia Perales Viejo

Vernica Martn GarcaArmando Arias Esteban

Julie Sheldon

Graduate Students:Rubn Agudo TorresMarta Sanz-Ramos RojoSamuel Ojosnegros Martos

Hctor Moreno BorregoHctor Tejero FrancoIgnacio de la Higuera

Ana M Ortega Prieto

Technical Assistance:Ana Isabel de vila LucasEva Garca CuetoIsabel Gallego JimnezM Eugenia Soria Benito

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CBMSO2009-2010

B8 Genetic variability of RNA virusesPublications

Research Summary

Staff

Patents

Doctoral Theses

Publications

Agudo, R. Ar ias, A . a nd Domingo, E. (2009). 5-Fluorourac il in l ethal mutagenesis of f oot-and-mout h dis ease virus. Future MedicinalChemistry,1(3), 529-539.Domingo, E. and Wain-Hobson, S. (2009). The 30th anniversary of quasispecies. Meeting on quasispecies: past, present and future.EMBO Reports, 10, 444-448.Emonet, S.F., de la Torre, J.C., Domingo, E. and Sevilla, N. (2009). Phylogeny/molecular taxonomy and evolution or Arenaviruses. Infection Geneticsand Evolution, 9, 417-429.Perales, C., Agudo, R. and Domingo, E. (2009). Counteracting quasispecies adaptability: extinction of a ribavirin resistant virus mutant by analternative mutagenic treatment. PLoS One,4(5): e5554.doi: 10.1371/journal.pone.0005554.Escarms, C., Perales, C. and Domingo, E. (2009). Biological effect of Mullers ratchet. Distant capsid site can affect picornavirusprotein processing. J. Virol. 83, 6748-6756.Domingo, E. (2009). Nueva gripe humana de origen porcino. Cunto de nuevo? Investigacin y Ciencia.393, 10-12.Ferrer-Orta, C., Agudo, R., Domingo, E. and Verdaguer, N. (2009). Structural insights into replication and elongation processes by the FMDVRNA-dependent RNA polymerase. Curr. Op. in Structural Biol.19(6), 752-8.Perales, C., Agudo, R., Tejero, H., Manrubia, S.C. and Domingo, E. (2009). Potential benets of sequential inhibitor-mutagen treatments of RNA virusinfections. PLoS Pathogens.5 (11), e1000658.Domingo, E. (2009). Quasispecies. From molecular Darwinism to viral diseases. Contributions to Science.5(2), 161-168.Ojosnegros, S., Beerenwinkel, N., Antal, T., Nowak, M.A., Escarms, C. and Domingo, E. (2010). Competition-colonization dynamics in an RNA virus.Proc. Natl. Acad. Sci. USA. 107(5), 2108-12.Domingo, E. (2010). The great evolutionary potential of viruses. The 1918 u as a paradigm of disease emergence. In: M.I. Porras and R. Davies,ed. Emerging Infection, Emergent Meanings: The Spanish Inuenza Pandemic of 1918-1919. University of Rochester Press, Rochester, New York.107, 2108-2112.Domingo, E. (2010). Mechanisms of viral emergence. Veterinary Research, 41(6): 38.Martn, V. Abia, D., Domingo, E. and Grande-Prez, A. (2010). An interfering activity of LCMV associated with enhanced mutagenesis. J.Gen. Virol., 91, 990-1003.Ojosnegros, S., Beerenwinkel, N. and Domingo, E. (2010). Competition-colonization dynamics: an ecology approach to quasispecies dynamics andvirulence evolution in RNA viruses. Communicative & Integrative Biology,3(4), 333-6.Domingo, E., Perales, C., Agudo, R., Arias, R., Escarms, C., Ferrer-Orta, C. and Verdaguer, N. (2010). Mutation, quasispecies and lethal mutagenesis.In: E. Ehrenfeld, E. Domingo and R.P. Roos, eds. The Picornaviruses. ASM Press, Washington, D.C., pp.197-211.Manrubia, S.C, Domingo, E. and Lzaro, E. (2010). Pathways to extinction: beyond the error threshold.Phil. Trans. R. Soc. B., 365(1548), 1943-52.

Ferrer-Orta, C., Sierra, M., Agudo, R., de la Higuera, I., Arias, A., Prez-Luque, R., Escarms, C., Domingo, E. and Verdaguer, N. (2010). Structure offoot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin. J. Virol.,84(12), 6188-99.Bordera, A.V., Lorenzo-Redondo, R., Pernas, M., Casado, C., lvaro, T., Domingo, E. and Lpez-Galndez, C. (2010). Initial tness recovery of HIV-1is associated with quasispecies heterogeneity and can occur without modications in the consensus sequence. PLoS One,5(4), e10319.Martn-Acebes, M.A., Herrera, M., Armas-Portela, R., Domingo, E. and Sobrino, F. (2010). Cell density-dependent expression of viral antigens duringpersistence of foot-and-mouth disease virus in cell culture. Virology,403, 47-55.Rodrguez-Calvo, T., Ojosnegros, S, Sanz-Ramos, M., Garca-Arriaza, J., Escarms, C., Domingo, E. and Sevilla, N. (2010). New vaccine designbased on defective genomes that combines features of attenuated and inactivated vaccines. PLoS One,5(4), e10414.Arias, A., Perales, C., Escarms, C. and Domingo, E. (2010). Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus.PLoS One,5(5), e10735.Agudo, R., Ferrer-Orta, C., Arias, A., de la Higuera, I., Perales, C., Prez-Luque, R., Verdaguer, N. and Domingo, E. (2010). A multi-step process ofviral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape. PLoS Pathog. 6(8) pii: e1001072.Perales, C., Lorenzo-Redondo, R., Lpez-Galndez, C., Martnez, M. A., and Domingo, E. 2010. Mutant spectra in virus behavior. FutureVirology5(6), 679-698.

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Patents

N. Sevilla, E. Domingo, C. Escarms, S. Ojosnegros, J. Garca-Arriaza, M. Sanz-Rojo, T.Rodrguez (2009) Vacuna atenuada para la ebre aftosa. PCT1641.49. Entidades titula-res: CSIC (70%), Instituto Nacional de Investigaciones Agrarias (30%).

E. Domingo, R. Agudo, H. Tejero, S.C. Manrubia, C. Perales (2009) Tratamiento antiviral.P200930482. Entidades titulares: CSIC (41%), Instituto Nacional de Tcnica Aeroespa-cial (18%), Universidad Complutense de Madrid (18%), Centro de Investigaciones Bio-

mdicas en Red de Enfermedades Hepticas y Digestivas (CIBERehd) (23%).

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Research Summary

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Publications

Patents

Doctoral Theses

Samuel Ojosnegros Martos(2009) Dinmica evolutiva de virus RNA. Universidad Aut-noma de Madrid. Director: Esteban Domingo Solans.

Rubn Agudo Torres(2009) Caracterizacin de las protenas del virus de la ebre afto-sa implicadas en respuesta a mutagnesis letal por anlogos de nucletido. UniversidadAutnoma de Madrid. Director: Esteban Domingo Solans.

Marta Sanz-Ramos Rojo(2009) Dinmica de cuasiespecies del virus de la ebre aftosain vivo. Universidad Autnoma de Madrid. Director: Esteban Domingo Solans.

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CBMSO2009-2010

B9

Research Summary

Gene expression in Streptomycesand yeasts

Staff

Publications

Patents

Doctoral Theses

Research Summary

Streptomycesand yeasts produce a high number of molecules with biotechnological value.Our objective is to study the expression molecular mechanism of proteins implicated in

some of this biosynthetic process, and to obtain new molecules with possible industrialapplicability. During the last two years, we have been studying the biosynthetic clusterof the nucleoside antibiotic A201A (ata) from Streptomyces capreolus, and some yeastproteins with glycosyltransferase activity able to produce prebiotic oligosaccharides.

The glycosyl residues constitute about 44% of the A201A total mass, which must be relatedwith the biological-pharmacological properties of the antibiotic. We have characterizedsome genes involved in the incorporation and modication of these residues using differentheterologous expression systems. The biosynthesis of novel molecules with antibioticactivity will be attempted based on the low substrate specicity previously described forthe related glycosyltransferases.

We are studying several proteins from the Xanthophyllomyces, Schwanniomyces and

Rhodotorulayeasts genera, which produce different types of prebiotic oligosaccharides.All the analysed proteins are hydrolases that show transferase activity, included withinfamily 32, 31, 1 and 2 of the glycosylhydrolases. To know the structure-function-specicity

relationships of these proteins are one of our objectives. We are carrying out structuralstudies of these proteins (3-D structure of same of them has been resolved), residuesubstitutions, and applying directed molecular evolution techniques in order to increase/modify their transglycosylase activity, and to improve their biotechnological utility.

International patents of most of these proteins have been obtained and a method for itsimmobilization on solid support has been developed. We seek to scale up to industriallevel the enzyme production and the generated products.

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CBMSO2009-2010

B9

Research Summary

Gene expression in Streptomycesand yeasts

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Research Summary

Figure 1.(A) Close up view of theactive site in Schwanniomyces

occidentalis Ffase in a complexwith fructose, 1-kestose(green; left) and the 6-kestose(yellow; right). (B) The fructan1-exohydrolase IIa from Cichoriumintybus (CiFEH) complexed with1-kestose and (C) the invertasefrom Thermotoga maritima

complexed with rafnose. In (A),the active-site of Ffase (blue)is also shaped by the adjacent

subunit (orange).

Figure 2.Oligomerizationpattern of the Ftase

from Schwanniomycesoccidentalis on a this

yeast culture.

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CBMSO2009-2010

B9 Gene expression in Streptomycesand yeasts

Research Summary

Publications

Patents

Doctoral Theses

Staff

Group Leader:Antonio Jimnez / Mara Fernndez Lobato

Graduate Students:Miguel de Abreu FelipeMiguel lvaro BenitoDolores Linde Lpez

Patricia Gutirrez AlonsoBrian Molloy Galiana

Undergraduate Students:David Gonzlez PrezMarta Estvez CanalesHugo Muoz

Technical Assistance:Asuncin Martn Redondo

Visiting Scientists:Vctor Cifuentes (Chile)Marcelo Baeza (Chile)

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Publications

Linde, D., Macias, I., Fernndez-Arrojo, L., Plou, F.J., Jimnez, A. and Fernndez-Lobato, M. (2009)Molecular and biochemical characterization of an fructofuranosidase fromXanthophyllomyces dendrorhous.App Env Microbiol.75(4), 1065-1073.

Gutirrez-Alonso, P., Fernndez-Arrojo, L., Plou, F.J. and Fernndez-Lobato, M. (2009) Biochemicalcharacterization of a b-fructofuranosidase from Rhodotorula graciliswith transfructosylating activity. FEMSYeast Research.9, 768-773.

Polo, A., lvaro-Benito, M., Fernndez-Lobato, M. and Sanz-Aparicio, J. (2009) Crystallization andpreliminary X-ray diffraction analysis of thefructofuranosidase from Schwanniomyces occidentalis. ActaCryst. 65, 1162-1165.

Baeza, M., Retamales, P., Sepulveda D., Lobato P., Jimenez A. and Cifuentes V. (2009) Isolation,characterization and long term preservation of mutant strains ofXanthophyllomyces dendrorhous.J. BasicMicrobiol. 49, 135-141.

Alvaro-Benito, M., Polo, A., Gonzlez, B., Fernndez-Lobato, M. and Sanz-Aparicio, J. (2010) Structuraland kinetic analysis of Schwanniomyces occidentalisinvertase reveals a new oligomerization pattern andthe role of its supplementary domain in substrate binding. J. Biol. Chem.285(18), 13930-14941.

Polo, A., Linde, D., Estvez, M., Fernndez-Lobato, M. Julia Sanz-Aparicio, J.. (2010). Crystallization andpreliminary X-ray diffraction analysis of the fructofuranosidase fromXanthophyllomyces dendrorhous.ActaCryst.66, 1441-1444.

lvaro-Benito, M., de Abreu, M., Portillo, F., Snz-Aparicio, J. and Fernndez-Lobato, M. (2010). Newinsights into the fructosyltransferase activity of Schwanniomyces occidentalis -fructofuranosidase emergesfrom a non-conventional codon usage and directed mutation.App. Env. Microbiol.76(22), 7491-7499.

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Patents

L. Fernndez Arrojo, F. Plou, A. Ballesteros, M. Alcalde, P. Gutierrez Alonso, M. Fernndez-Lobato (2009). Biocatalizador inmovilizado basado en alginato para la biotransformacin decarbohidratos. N P200930001. PCT-ES2010/070104 (24 -2- 2010). Titular: CSIC-UAM.

M. Fernndez-Lobato, M. Alvaro Benito (2009). Fructofuranosidasa mejorada genticamentepara la obtencin del prebitico 6-kestosa. N P0200930929 (29-12-2009). Titular: UAM.

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Doctoral Theses

Brian Molloy Galiana(2010). Expresin heterloga del cluster biosinttico del antibiticoA201A y estudio de las posibles glicosiltransferasas implicadas en su biosntesis.Universidad Autnoma de Madrid. Director: Mara Fernndez-Lobato.

Mara Dolores Linde Lpez(2010). Caracterizacin bioqumica, molecular y estructuralde una b-fructofuranosidasa con capacidad trasferasa de la levadura Phafa rhodozyma

aplicable a la produccin de oligosacridos prebiticos. Universidad Autnoma de Madrid.Director: Mara Fernndez-Lobato.

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CBMSO2009-2010

B10 Virus Engineering

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Research Summary

We use protein engineering techniques for the study of structure-function relationshipsin viruses and their capsids, and for the design of modied viral particles fornanobiotechnological applications (Mateu (2011). Prot.Eng.Des.Sel.24, 53-63).

Scientic relevance and technological implications: A better knowledge of poorlyknown stages of the viral cycle, including virus assemby, conformational dynamics anddisassembly; design of new vaccines, antivirals and nanoparticles for targeted drugdelivery.

Some recent scientic results: i) Using protein engineering, the thermal stability of foot-and-mouth disease virus (FMDV) against dissociation into subunits has been increased.We are currently investigating the molecular basis of such thermostabilization, and thepossibility of using the modied virions as improved vaccines. ii) We have carried out anextensive mutational analysis of compensatory mutations in the FMDV capsid, and arecurrenly investigating the molecular bases of these effects. iii) In collaboration with P.J. dePablo y J. Gmez (Dept. Physics of Condensed Matter, UAM) we have used the minutevirus of mice (MVM) to investigate the role of capsid pores and cavities in the mechanicalproperties of the viral particle, using atomic force microscopy (a technique we are currentlyusing in our lab). We have obtained evidence that the remarkable mechanical properties

found may have been evolutionarily selected to optimize the resistance of the virus againstphysical agents, while at the same time allowing the conformational changes needed forinfectivity. iv) In collaboration with J.L.Neira (CBMC) y M.A.Martnez (IRSI-Caixa), we areinvestigating the capacity of different synthetic peptides and other molecules to inhibit

human immunodeciency virus assembly and infectivity. These studies may be importantfor the design of new anti-HIV agents. v) In collaboration with G.Rivas (CIB), we haveused two model viral systems to show that, in physiological macromolecular crowdingconditions, the inhibitory activity of small-size antiviral compounds may be reduced.

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B10

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Virus Engineering

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Research Summary

Figure 1. Topographic image of an individual MVM

particle in liquid, obtained in our laboratory by atomic forcemicroscopy. Immediately after the image is obtained, themicroscope is used to apply a force on the particle; thisallows the determination of its mechanical elasticity. Figure 2. Location on the FMDV capsid

structure (A) of a collecion of lethalmutations at the interfaces betweenpentameric subunits (B, colored white), andof the different compensatory mutationsthat restored infectivity (C, colored greenor orange).

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Group Leader:Mauricio Garca Mateu

Postdoctoral:Rebeca Prez Fernndez

Graduate Students:Rebeca Bocanegra RojoMilagros Castellanos Molina

Inmaculada Lpez PrezPablo Jos Prez CarrilloVernica Rincn Forero

Technical Assistance:Miguel ngel Fuertes VilladangosAlicia Rodriguez Huete

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Publications

Carrasco, C., Douas, M., Miranda, R., Castellanos, M., Carrascosa, J.L., Mateu, M.G., Marqus, M. andde Pablo, P.J. (2009). Action of capillary forces of water conned at the nanoscale during dessication ofviruses. Proc. Natl. Acad. Sci.USA106, 5475-5480. (A)

Serena, P.A., Douas, M., Marqus, M.I., Carrasco, C., de Pablo, P.J., Miranda, R., Carrascosa, J.L.,Castellanos, M. and Mateu, M.G. (2009). MC simulations of water meniscus in nanocontainers: explainingthe collapse of viral particles due to capillary forces. Phys. Status Solidi C 6, 2128-2132.

Mateu, M.G. (2009). Capsid protein interactions in human immunodeciency virus type 1 assembly. FEBSJ.276, 6098-6109.

Luna, E., Rodriguez-Huete, A., Rincn, V., Mateo, R. and Mateu, M.G. (2009). A systematic study of thegenetic response of a variable virus to the introduction of deleterious mutations in a functional capsid

region. J.Virol.83, 10140-10151.

Martn-Acebes, M., Rincn, V, Mateu, M.G. and Sobrino, F. (2010). A single amino acid substitution in thestructural protein VP3 of foot-and-mouth disease virus can increase acid-lability and confer resistance to

acid-dependent uncoating inhibition. J.Virol.84, 2902-2912.

Domenech, R., Abin, O., Bocanegra, R., Correa, J., Sousa-Herves, A., Riguera, R., Mateu, M.G.,Fernndez-Mega, E., Velzquez-Campoy, A. and Neira, J.L. (2010) Dendrimers bind the homodimerizationinterface of the capsid protein of HIV-1. Biomacromolecules11, 2069-2078.

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Mauricio G. Mateu, member of the Editorial Board of Virus Research

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OEPM Patent n 2 323 929 (Foot-and-mouth disease vaccine). Granted June 2010.

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Eva Luna Garca(2010). Aproximaciones a la obtencin de cpsidas ms estables delvirus de la ebre aftosa y estudio de la generacin de mutaciones compensatorias.Universidad Autnoma de Madrid. Director: Mauricio G. Mateu.

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CBMSO2009-2010

B11 Effects of extrachromosomal elements on behaviour of its host Bacillus

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Research Summary

So-called mobile genetic elements (MGE), e.g. phages, plasmids, transposons and ICEs,can be transferred horizontally between cells and affect the genetic make-up and hencethe behaviour of bacteria. Accordingly, horizontal gene transfer (HGT) has a crucial role inmicrobial evolution and has important implications in a myriad of environmental and public

health problems. For instance, HGT is mainly responsible for the emergence and rapiddispersion of antibiotic resistance.

Little is known, especially in Gram positive bacteria, about the mechanisms by which

MGE exert their behavioural effects on their host or on regulation of their mobility. A betterunderstanding of these issues is warranted to face important threats of for instance antibioticresistance. In our laboratory we study these issues using as host Bacillus subtilisand welimit the MGE to plasmids and phages.

We use B. subtilisbecause (i) it is probably the best studied Gram-positive bacterium; (ii)it is non-pathogenic; (iii) it is amenable to genetic manipulation due to its ability to developnatural competence; and (iv) B. subtilis is related to pathogenic/fastidious bacteria likeBacillus anthracis,B. cereusand, although more distantly, to Listeria monocytogenes.

For some phages it has been described that they alter the behaviour of B. subtilisuponinfection. However, neither sequence nor mechanistic information of how these phages

affect behaviour of their infected host is available. We are studying these focussing on twophages. About 20% of the natural isolates of B. subtiliscontain an endogenous plasmid.Most large plasmids (>10 kb) can be transferred to other cells via the process of conjugation.We are the rst to have sequenced two large Bacillus plasmids and have identied genesinvolved in conjugation as well as others that probably affect behavioural processes of thehost. We are now analyzing these to unravel regulation of the conjugation process and togain insight in the way plasmid-located genes alter the life style of the host.

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Figure 1.Phase contrastmicroscopy image of

sporulatingB. subtiliscells

harbouring conjugative

plasmid pLS20.

Figure 2. Geneticmap of B. pumilus

sporulation inhibiting

plasmid p576.

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Group Leader:Wilfried J.J. Meijer

Graduate Students:Praveen K. SinghGayetri Ramachandran

Undergraduate Students:Esther Serrano

Sandra Ballestero Beltrn

Technical Assistance:Adriana Marulanda Aguirre

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Singh, P. K., Ballestero-Beltrn, S., Ramachandran, G. and Meijer, W.J.J. (2010) Complete nucleotidesequence and determination of the replication region of the sporulation inhibiting plasmid p576 of BacilluspumilusNRS576. Res. Microbiol. 161, 772-782.

Muoz-Espn, D., Daniel, R., Kawai, Y., Carballido-Lpez, R., Castilla-Llorente, V., Errington, J.*, Meijer,W.J.J.*, and Salas, M.* *: contributed equally. (2009) The actin-like MreB cytoskeleton organizes viral DNAreplication in bacteria. Proc. Natl. Acad. Sci. USA.July 27 Epub ahead of print.

Castilla-Llorente, V., Meijer, W.J.J., and Salas, M. Differential Spo0A-mediated effects on transcription andreplication of the related Bacillus subtilis phages Nf and f29 explain their different behaviours in vivo.(2009) Nucleic Acids Res.37: 4955-4964.

Castilla-Llorente, V., Salas, M., and Meijer, W.J.J. (2009) Different responses to Spo0A-mediated suppressionof the related Bacillus subtilisphages Nf and f29. Environ. Microbiol.11: 1137-49.

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CBMSO2009-2010

B12

Research Summary

Human immunodefciency virus reverse transc

03b virology and microbiology

Documents