Sociedad Mexicana de Bioquímica, A. C.

Transcription

1 Sociedad Mexicana de Bioquímica, A. C. Courtesy of Jorge A. Girón (University of Florida) Program & s First Meeting of Biochemistry and Molecular Biology of Bacteria San Miguel Regla, Hidalgo March 22 25, 2010

2 Organizing Committee Edmundo Calva Guadalupe Espín Dimitris Georgellis Bertha González José Luis Puente Local Committee Gabriel Betanzos UAEH Technical Edition: and Typesetting: Maria Teresa Castillo Technical Support: Andrea Ortiz Image Cover: Nayeli Quinto Published in México, 2010 Facultad de Medicina, UNAM. Taller de Impresos. Circuito Escolar s/n, Ciudad Universitaria, D. F.

3 Primer Congreso Rama de Bioquímica y Biología Molecular de Bacterias Sociedad Mexicana de Bioquímica, A.C de marzo de 2010 San Miguel Regla, Hidalgo Bienvenida los privilegiados tienen la obligación de regresar algo al país que les ha permitido obtener esa posición. Porque para qué sirve la experiencia, el conocimiento, el talento, si no se usa para hacer de México un lugar más justo? Para qué sirve la educación si no se ayuda a los demás a obtenerla? Para qué sirve ser habitante de un país si no se asume la responsabilidad compartida de asegurar vidas dignas allí? Denisse Dresser, 2009 Los conceptos de Denisse Dresser dan el colofón a nuestras palabras de bienvenida. Porque para cualquier región del mundo, para cualquier país, nuestra ciencia con todas sus abstracciones, pasiones y recompensas no tiene ningún sentido si no incide en una sociedad más educada y más sensible. El hacer buena ciencia es nuestra responsabilidad primaria, pero no es suficiente. También es indispensable crear las oportunidades para otros, desde aquellos que desean seguir una carrera científica hasta los individuos de la sociedad civil, que aprenderán de la ciencia su rigor intelectual y de los científicos su pasión y creatividad. Esto es lo que nos mueve a añadir a nuestras múltiples ocupaciones la organización de un congreso como éste. Hemos querido conjuntar un grupo cohesivo, en donde se discutan con intensa profundidad los problemas científicos que nos apasionan. Es por ello que no hay conferencias simultáneas, ni los trabajos están ordenados por áreas del conocimiento. Creemos que todos debemos aprender de todo. Creemos que la evolución hacia una ciencia propia ya que hemos alcanzado los niveles de una ciencia de calidad internacional- no se dará hasta que entre nosotros encontremos nuestros estilos, nuestra originalidad, como en cualquier otro aspecto de la cultura mexicana que ha alcanzado gran repercusión a nivel mundial. También pensamos que dentro de este espíritu nacionalista debe haber una visión global. Hemos querido que nos acompañen bacteriólogos extranjeros del más alto nivel, que no sólo vengan a impartir una conferencia magistral sino que genuinamente interactúen con todos nosotros, especialmente con los estudiantes y particularmente con aquellos que no han tenido muchas oportunidades de salir al extranjero. Así, hemos insistido en que los resúmenes sean en el idioma inglés y que, de preferencia, también lo sean los carteles y las transparencias de las presentaciones orales. Es de importancia estratégica que nuestro congreso se convierta en uno de los más atractivos para los bacteriólogos de cualquier parte del mundo -pues tenemos todo para lograrlo- en donde los visitantes sepan que no sólo vendrán a compartir su ciencia sino que también se llevarán un aprendizaje valioso. Más aún, un congreso como éste deberá preparar a nuestros principiantes en las lides de lo que implica un congreso en el extranjero. iii

4 De esta manera, nuestra mejor retribución será que el entusiasmo por entusiasmar a los demás, entusiasme a las nuevas generaciones de bacteriólogos mexicanos a fin de que mantengan el espíritu de esta tradición por muchos años venideros; no sólo basándose en lo que se logre en esta ocasión sino aportando de su propia visión y creatividad. Y, finalmente, cerrando con Dresser: La convicción inquebrantable de mejorar a México. De restañar a la República. De volver a México un país de ciudadanos. Un lugar poblado por personas conscientes de sus derechos y dispuestos a contribuir para defenderlos. Dispuestos a llevar a cabo pequeñas acciones que produzcan grandes cambios. Dispuestos a sacrificar su zona de seguridad personal para que otros la compartan. Esto es, los invitamos a seguirnos saliendo de nuestra zona de seguridad, para estar dispuestos a llevar a cabo pequeñas acciones que produzcan grandes cambios. Bienvenidos! Edmundo Calva Guadalupe Espín Dimitris Georgellis Bertha González Pedrajo José Luis Puente iv

5 Funding Institutions v

6 Sponsors vi

7 Table of Contents Schedule... vii Program of Events Monday... 3 Tuesday... 4 Wednesday... 8 Thursday s Plenary Sessions Oral Presentations Poster Sessions Author Index vii

8 March 22-25, San Miguel Regla, Hidalgo MONDAY MARCH. 22 TUESDAY MARCH. 23 WEDNESDAY MARCH 24 THURSDAY MARCH 25 8:00 9:00 BREAKFAST BREAKFAST BREAKFAST REGISTRATION 14:00 TO 18:30 9:00 10:40 10:40 11:00 ORAL SESSION I CHAIR: GABRIEL GUARNEROS ORAL SESSION IV CHAIR: CARLOS CERVANTES ORAL SESSION VII CHAIR: DAVID ROMERO COFFEE BREAK COFFEE BREAK COFFEE BREAK OPENING CEREMONY 18:45 19:00 11:00 12:40 ORAL SESSION II CHAIR: OTTO GEIGER ORAL SESSION V CHAIR: AGUSTINO MARTÍNEZ ORAL SESSION VIII CHAIR: FERNANDO NAVARRO 12:40 13:00 COFFEE BREAK COFFEE BREAK COFFEE BREAK ALEJANDRA BRAVO INSTITUTO DE BIOTECNOLOGÍA, UNAM 13:00 14:00 PASCALE COSSART INSTITUTE PASTEUR ROB EDWARDS SAN DIEGO STATE UNIVERSITY ORAL SESSION IX CHAIR: MIGUEL CASTAÑEDA MECHANISM OF ACTION OF BACILLUS THURINGIENSIS INSECTICIDE CRY PROTEINS OPENING TALK 19:00 20:00 THE BACTERIAL PATHOGEN LISTERIA MONOCYTOGENES: A UNIQUE MODEL IN INFECTION BIOLOGY USING METAGENOMICS TO UNDERSTAND THE MICROBIAL WORLD 14:00 16:00 LUNCH LUNCH LUNCH 16:00 17:00 ORAL SESSION III CHAIR: CARMEN GÓMEZ ORAL SESSION VI CHAIR: YOLANDA LOPEZ VIDAL RETURN 17:00 18:00 RICHARD R. BURGESS UNIVERSITIY OF WISCONSIN TONY ROMEO UNIVERSITY OF FLORIDA DINNER WELCOME COCKTAIL 20:00 22:00 BACTERIAL SIGMA 70: DISCOVERY TO THE PRESENT POST-TRANSCRIPTIONAL REGULATION BY THE CSR SYSTEM COFFEE BREAK COFFEE BREAK 18:00 20:00 POSTER PRESENTATIONS POSTER PRESENTATIONS 20:00 22:00 DINNER AND STRING QUARTET MEXICAN PARTY

9 Program

10 Monday March 22, :00 18:30 Registration 18:45 20:00 Opening Ceremony, and Opening Talk 19:00 20:00 Opening Lecture Alejandra Bravo Mechanism of action of Bacillus thuringiensis insecticide Cry proteins Instituto de Biotecnología UNAM Chair: Bertha González-Pedrajo 20:00 22:00 Dinner and Welcome Cocktail All oral presentations will be held in the Progreso Room The poster presentations will be held in the Conde Room 3

11 Tuesday March 23, 2010 Oral Session I Chair: Gabriel Guarneros Peña 9:00 9:20 Gabriel Guarneros Peña A novel role for the translation termination factors: rescuing of stalled ribosomes at sense codons CINVESTAV IPN 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 César Augusto Aguilar Martínez Preliminary analysis of mutations originated in regulatory genes during an adaptive evolution process for fast growing on glucose in a Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system Instituto de Biotecnología UNAM Susana Brom Klanner Conjugative Transfer in Rhizobium Centro de Ciencias Genómicas UNAM Miguel Ángel Cevallos Gaos In vitro and in vivo analyses of the mechanism of action of an antisense RNA that controls the replication of a repabc plasmid Centro de Ciencias Genómicas UNAM Laura Elena Córdova Dávalos Expression of the protein glycosylation system of Mycobacterium tuberculosis in Streptomyces coelicolor. Instituto de Investigaciones Biomédicas UNAM 10:40 11:00 Break 4

12 Oral Session II Chair: Otto Geiger 11:00 11:20 11:20 11:40 11:40 12:00 12:00 12:20 12:20 12:40 Otto Geiger Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation Centro de Ciencias Genómicas UNAM Ariadnna Cruz Córdova Longus a type IV PILI of enterotoxigenic Escherichia coli is involved in the adherence to intestinal epithelial cells Hospital Infantil de México Federico Gómez Diana Patricia Cruz Pulido Analysis of the function of diguanylate cyclase genes in biofilm formation in Klebsiella pneumoniae Molecular Genetics Group. Corporación CorpoGen, Bogotá Miguel Ángel De la Cruz The two-component system CpxAR negatively regulates SPI-1 and SPI-2 expression in Salmonella enterica serovar Typhimurium Instituto de Biotecnología UNAM Edgardo Escamilla Marván The Fermentative Oxidation of Ethanol in Gluconacetobacter diazotrophicus is a Two Step Pathway Catalysed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADH) Instituto de Fisiología Celular UNAM 12:40 13:00 Break 5

13 Plenary Session I Chair: José Luis Puente 13:00 14:00 Pascale Cossart The bacterial pathogen Listeria monocytogenes: a unique model in infection biology Unité des Interactions Bactéries-Cellules, INSERM. Institut Pasteur 14:00 16:00 Lunch Oral Session III Chair: Carmen Gómez Eichelmann 16:00 16:20 Carmen Gómez Eichelmann Role of DnaK and ClpB in gyrase reactivation during the heatshock response in E. coli K12 Instituto de Investigaciones Biomédicas UNAM 16:20 16:40 16:40 17:00 Ma. del Rocío León Kempis Strain-specific gene expression of DnaK in Gallibacterium anatis Boehringer Ingelheim Vetmedica S. A. de C. V. Ricardo González Chávez The physiological stimulus for the BarA sensor kinase Instituto de Fisiología Celular UNAM 6

14 Plenary Session II Chair: Edmundo Calva 17:00 18:00 Richard Burgess Bacterial sigma 70: discovery to the Present University of Wisconsin 18:00 20:00 Poster Session Odd numbers 20:00 22:00 Dinner 7

15 Wednesday March 24, 2010 Oral Session IV Chair: Carlos Cervantes Vega 9:00 9:20 Carlos Cervantes Vega Functional Analysis of Bacterial Chromate Transporters IIQB - Universidad Michoacana 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 Areli Hernández Valdez Development of an in vivo model for the study of the tryptophan operon expression in Escherichia coli CINVESTAV IPN Unidad Monterrey J. Antonio Ibarra A new member of the HilA-regulon network in Salmonella enterica SEROVAR Typhimurium SL1344 Rocky Mountain Laboratories, NIAID, NIH Juan Ramón Ibarra Rodríguez DNA Integrity Scanning Protein (DisA) plays a role as a DNA damage checkpoint during germination/outgrowth of Bacillus subtilis spores Department of Biology, University of Guanajuato Eva Jacinto Loeza A complex secondary structure (Pseudoknot) in Pth mrna promotes -1 frameshifting CINVESTAV IPN Unidad Zacatenco 10:40 11:00 Break 8

16 Oral Session V Chair: Agustino Martínez Antonio 11:00 11:20 11:20 11:40 11:40 12:00 12:00 12:20 12:20 12:40 12:40 13:00 Agustino Martinez Antonio Structural and Functional Organization of the Escherichia coli Genome CINVESTAV IPN Unidad Irapuato Paula Figueroa Arredondo Vibrio cholerae cytolysin VCC vacuolating effect is not affected by ATPase inhibitors Escuela Nacional de Medicina Homeopática IPN Varinia López Ramírez Description of bacterial DExD/H box RNA helicases and their Evolutionary history in Firmicutes and γ-proteobacteria CINVESTAV IPN Unidad Irapuato Luary Carolina Martínez Chavarría The SirA/BarA two-component system activates a regulatory cascade that controls expression of the SPI-1 and SPI-2 virulence regulons via HilD Instituto de Biotecnología UNAM Alejandra Prieto Davó Phage Dynamics in Anoxic Oceans: Viral Metagenomics of the Oxygen Minimum Zone off Iquique, Chile San Diego State University Break 9

17 Plenary Session III Chair: Guadalupe Espín 13:00 14:00 Rob Edwards Using metagenomics to understand the microbial world San Diego State University 14:00 16:00 Lunch Oral Session VI Chair: Yolanda López Vidal 16:00 16:20 Yolanda López Vidal Tuberculosis Facultad de Medicina, UNAM 16:20 16:40 16:40 17:00 Liliana Medina Aparicio The Cas-CRISPR immune system is transcriptionally regulated by the global regulatory proteins H-NS, LRP, LeuO and a 5 untranslated regulatory region in Salmonella enterica serovar Typhi Instituto de Biotecnología UNAM Cinthia Núñez López The CbrA/CbrB Two-Component System Negatively Controls Alginate Synthesis in Azotobacter vinelandii Instituto de Biotecnología UNAM 10

18 Plenary Session IV Chair: Dimitris Georgellis 17:00 18:00 Tony Romeo Post-transcriptional regulation by the Csr (Rsm) system University of Florida 18:00 20:00 Poster Session Even numbers 20:00 22:00 Dinner 11

19 Thursday March 25, 2010 Oral Session VII Chair: David Romero Camarena 9:00 9:20 David Romero Camarena Gene conversion in Rhizobium: roads towards equality Centro de Ciencias Genómicas UNAM 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 Jesús Piña Guillén Function and expression of rpos gene in Pseudomonas stutzeri A15 in the presence rice plant CICM BUAP Ruth Reyes Cortés Characterization of a new Escherichia coli surface-factor involved in bacteriophage mep213 infection CINVESTAV IPN Unidad Zacatenco Gabriel Rincón Enríquez Implication of the regulator IscR in bacterial phytopathogenesis CIATEJ Guadalajara Adriana Patricia Rodríguez Hernández Subgingival microbiota associated with generalized chronicperiodontitis and T2DM of Mestizos and Otomies Mexican subjects Facultad de Odontología UNAM 10:40 11:00 Break 12

20 Oral Session VIII Chair: Fernando Navarro García 11:00 11:20 11:20 11:40 11:40 12:00 12:00 12:20 12:20 12:40 12:40 13:00 Fernando Navarro García EspC translocation into epithelial cells by enteropathogenic Escherichia coli requires a concerted participation of type V and III secretion systems and leads to cytoskeleton disruption CINVESTAV IPN Andrea Sabido Ramos Construction and characterization of a system for the expression of chromosomal genes in Escherichia coli Instituto de Biotecnología UNAM Christian Sohlenkamp Modification of membrane lipids in Rhizobium tropici under acid stress Centro de Ciencias Genómicas UNAM Luz Elena Vidales Rodríguez Role of error prevention guanine oxidized system in stationaryphase mutagenesis of Bacillus subtilis Universidad de Guanajuato Magdalena Wiesner Characterization of IncA/C plasmids strongly associated with the Mexican Salmonella Typhimurium ST213 clone Instituto de Biotecnología UNAM Break 13

21 Oral Session IX Chair: Miguel Castañeda 13:00 13:20 Miguel Castañeda Lucío The roles of GacA, RpoS, RsmA, RsmB and RsmC in encystment and in the post-transcriptional regulation of the alginate biosynthetic gene algd in Azotobacter vinelandii IC BUAP 13:20 13:40 13:40 14:00 Yanet Romero Ramírez The Two-component system GacS/GcA, the Sigma Factor, the posttranscriptional Rsm System and the transcriptional regulator specific ArsR Modulate the Encystment response in Azotobacter vinelandii Instituto de Biotecnología UNAM Abraham Medrano López Functional Characterization of Ler, the Positive Regulator of Virulence Genes in Enteropathogenic Escherichia coli (EPEC) Instituto de Biotecnología UNAM 14

22 v Courtesy of Guadalupe Espin (IBT-UNAM) s Plenary Session

23 OPENING LECTURE, MONDAY MARCH 22, 2010 Mechanism of action of Bacillus thuringiensis insecticide Cry proteins Mario Soberón and Alejandra Bravo Instituto de Biotecnología, Universidad Nacional Autónoma de México Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces crystalline inclusions during the sporulation phase that are formed by Cry protoxins. These toxins have high insecticide activity and are very specific, innocuous to humans and biodegradable, and have been used as bio-insecticides in agriculture during the past 50 years. Our laboratory has been devoted to the understanding of their functions at a molecular level. When susceptible insects ingest these crystals they are solubilized in the intestine s alkaline medium. Soluble protoxins are activated through proteolytic digestion after which sequential binding to different receptors located in the intestinal epithelium cell microvilli takes place. These receptors are a cadherin-like protein (Bt-R1) and an aminopeptidase (APN). This interaction provokes a toxin conformational change that facilitates further proteolytic cleavage of the domain I amino-terminal helix α-1 resulting in toxin oligomerization. This produces a 200-fold increase in affinity of the toxin for the second receptor, APN, which is located at membrane microdomains, where the lipid composition favors insertion of the prepore and thus pore formation. The pore produced by the toxin is poorly selective, thus enabling the passage of several ions inside the cell and causing depolarization of the plasma membrane and passage of water until the cell lysis leading to larvae s death. The study of resistant insects by several groups has shown that the most common resistance generating mechanism is that insects lose the first receptor, cadherin. Our experiments have led us to determine that it is possible to skip the interaction with the first receptor by eliminating the same region of the amino-terminal end that is removed upon contact with the first receptor, thus allowing the toxin to continue its mechanism of action. Modified Cry proteins are toxic both for normal and for resistant insects. These data represent a feasible future strategy to control resistance in the field. I will present experimental evidence indicating that upon toxin oligomerization conformational changes occur at the exposed regions of domains II and III, thus allowing for exchange between one receptor and the other. In addition, I will discuss our recent observations regarding the intracellular responses triggered by these toxins in larvae intestinal cells. We have observed that MAPK p38 participates in the defense response and that when the expression of this protein is silenced via an interfering RNA, Cry-toxin-hypersensitive larvae are produced. 17

24 PLENARY SESSION I, TUESDAY MARCH 23, 2010 The bacterial pathogen Listeria monocytogenes: a unique model in infection biology P. Cossart Unité des Interactions Bactéries-Cellules, INSERM U604, INRA USC2020, Institut Pasteur, 28 rue du Dr Roux Paris, France The food borne pathogen L. monocytogenes discovered by Murray in 1926 is responsible for a severe infection with various clinical features (gastroenteritis, meningitis, meningoencephalitis and materno foetal infections) and a high mortality rate (30%). The disease is due to the ability of Listeria to cross three host barriers during infection: the intestinal barrier, the placental barrier and the blood brain barrier. It is also due to Listeria capacity to survive in macrophages and to enter into non phagocytic cells, such epithelial cells. Recovery from infection and protection against reinfection are due to a T-cell response, explaining why Listeria has since many years has become a model in immunology. Nearly three decades of molecular biology and cell biology approaches coupled to genetic and post-genomic studies have promoted Listeria among the best models in infection biology. In depth studies of the mechanism of entry into cells has help unraveling how Listeria crosses the intestinal and placental barrier. Unsuspected concepts in cell biology were discovered. Post-genomic studies have recently allowed to unveil the Listeria transcriptional landscape during switch from saprophytism to virulence. The talk will give an overview highlighting recent results in the framework of wellestablished data 18

25 PLENARY SESSION II, TUESDAY MARCH 23, 2010 Bacterial sigma70: Discovery to the Present Richard R Burgess Department of Oncology, University of Wisconsin-Madison, Madison, WI USA It has been 41 years since the discovery of the first transcription factor, the sigma70 subunit of E. coli RNA polymerase. The earlier part of this talk will recount the scientific and technical context and events surrounding this discovery from the personal point of view of the discoverer. This will be followed by a summary of the some of the key steps in the growth of our knowledge of sigma factors. There are seven different sigma factors in E. coli, each with ability to direct the RNA polymerase to a different class of promoter by way of specific interaction with the promoter DNA sequences. These sigma factors bind to the core RNA polymerase made up of subunits beta, beta prime, alpha, and omega to form the RNA polymerase holoenzyme. Each different holoenzyme interacts specifically with its cognate promoters, and the amount and activity of the various sigmas provides an important means of global regulation of bacterial transcription. In particular, two recent studies will be presented briefly: the first on biochemical measurements of the binding of various sigmas to core and their implication for mechanisms by which the various sigmas compete for binding to core; the second on determination of the regulons of sigma32, sigmaf and sigma54 19

26 PLENARY SESSION III, WEDNESDAY MARCH 23, 2010 Using metagenomics to understand the microbial world Rob Edwards Departments of Computer Science and Biology, GMCS411 San Diego State University Campanile Dr, San Diego Advances in sequencing technology have made it quick and cheap to sequence samples directly from the environment. We are starting to learn how microbial systems respond to changes, and the complex interplay of viruses, bacteria, archaea, and eukaryotes in these systems. Microbial sequences from around the globe, and around our own bodies are now available, and these sequences tell us how microbes are impacting our every day life. I will show how phages, viruses that infect bacteria, are affecting the bacteria, and how they affect us. 20

27 PLENARY SESSION IV, WEDNESDAY MARCH 23, 2010 Post-transcriptional regulation by the Csr (Rsm) system Tony Romeo Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA Bacteria respond rapidly to changing environmental and physiological conditions to survive stresses, maintain homeostasis and growth, and compete with other species. Coordination of gene expression and metabolism during such times involves pathways and cascades of factors, as well as signaling networks that span multiple regulatory circuits and determine global phenotypic properties (lifestyles). The Csr (Rsm) system in Escherichia coli is a regulatory system comprised of four components: a dimeric RNA binding protein, CsrA, which regulates mrna translation and stability, two noncoding regulatory RNAs, CsrB and CsrC, that bind multiple copies of CsrA and thereby antagonize its activity, and CsrD, a GGDEF- EAL domain protein that governs the turnover of CsrB/C by RNase E. These factors function within a complex feedback repression loop in which CsrA indirectly activates a two-component signal transduction system, BarA-UvrY, which activates transcription of CsrB/C regulatory RNAs. Similarly, CsrA represses csrd expression, which leads to stabilization of CsrB/C RNAs. This circuitry fine tunes CsrA activity in the cell, and regulates expression of numerous pathways and processes through effects on structural and regulatory genes. The workings of this system and its effects on biofilm development, motility, carbon flux and other systems will be discussed. 21

28 Courtesy of Guadalupe Espin (IBT-UNAM) s Oral Presentations

29 ORAL SESSION I. TUESDAY A novel role for the translation termination factors: rescuing of stalled ribosomes at sense codons 1 Vivanco-Domínguez S. 1 Bueno-Martínez J. 2 León-Avila G. 3 Nobuhiro I. 1 Magos- Castro M.A. 3 Kaji H. 4 Kaji A. and 1 Guarneros G. 1 Dept. of Genetics and Mol. Biol., CINVESTAV, Av. IPN 2508, Col. San Pedro Zacatenco, C.P , Mexico D.F. 2 Dept. of Zoology, ENCB-IPN., Mexico. 3 Dept. of Biochemistry and Mol. Biol., Thomas Jefferson University. 4 Dept. of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia. svivanco@yahoo.com.mx During the translational reading of mrna, ribosomes may stall due to starvation for one or more components essential for translation. Accumulation of the stalled ribosomal complexes, that contain peptidyl-trna and mrna, affects the efficiency of protein synthesis and the cell viability. In this work we asked whether protein factors, known to help ribosome release at the translation termination stage, contribute to release ribosomes stalled at sense codons as a mechanism to safeguard genetic expression. To analyze this question, an enriched preparation of stalled ribosomes was generated by translation of an abundant mrnas carrying tandems of AGA codons read by a trna which seems to turn over slowly in the ribosome. Under these conditions, the levels of specific peptidyl-trna accumulated were estimated in cell mutants for the investigated factor genes. Our results strongly indicate that proteins RF3 and RRF, which are usually active after translation termination, help to release stalled ribosomes at sense codons. Furthermore, proteins RF1 and RF2, definitely acting at translation termination, also help to release stalled ribosomes. We propose that the proteins of the canonical translation termination system also participate in the release of ribosomes stalled at nontermination codons. 25

30 ORAL SESSION I. TUESDAY Preliminary analysis of mutations originated in regulatory genes during an adaptive evolution process for fast growing on glucose in a Escherichia coli strain lacking the phosphoenolpyruvate : carbohydrate phosphotransferase system Aguilar Martínez CA, Gosset Lagarda G, Bolívar Zapata F. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM. Apdo. Postal Cuernavaca / Morelos (México). galnex@ibt.unam.mx The PEP is a precursor of several molecules important to society, however, the intracellular availability of this precursor has always been controlled and the engineering of metabolic pathways has succeeded in increasing it availability by eliminating in a strain of E. coli the ptshlcrr operon, which codes for the PTS system and consumes over 50% of PEP originating from glycolysis. From this strain PTS - called PB11, and by a process of selective pressure for rapid growth on glucose, strain PB12 was generated. In order to know in detail, the changes during the process of evolution, the genome of strain PB12 was sequenced. Because of this study was found several mutations that arose in this process; among them, three mutations originated in genes involved in regulation (bara, yjju and rssa). To identify possible effects of these three mutations in strain PB12 as well as the relevance of them, complementation experiments were performed. The presence of wild genes caused decreases in specific growth rate ( ) of strain by supplementing with bara and yjju genes. Furthermore, inactivation of genes yjju and rssa in PB11 strain resulted in increases in greater than 20%. Also analyzed the possible effect of the mutation in the gene bara, and its implications on Csr system. The results indicate lower concentrations of CsrB in PB12 strain, which could explain why in this strain glycolysis is favored. On the other hand, PB11 apparently present CsrB concentrations significantly greater compared with the strain PB12, a situation that could explain in turn, the physiological status of the strain PB11, since in this strain, favors gluconeogenesis. 26

31 ORAL SESSION I. TUESDAY Conjugative Transfer in Rhizobium Brom Susana, Cervantes Laura, Ponce Yaxal, Bustos Patricia, Santamaría Rosa Isela, Dávila Guillermo, and Romero David Centro de Ciencias Genómicas. UNAM. Av. Universidad 1001, Cuernavaca, Mor., México Rhizobium etli strain CFN42 is able to develop a symbiotic relationship with bean. Genes involved in establishment of the symbiosis are located on the symbiotic plasmid (psym). Conjugative transfer of the psym of CFN42 is achieved through cointegration with an endogenous self-transmissible plasmid (p42a). Cointegration may be mediated by IntA catalyzed site-specific recombination, or by homologous recombination involving RecA. We have assembled a collection of bean-nodulating Rhizobium strains from diverse geographical origins, as well as some strains isolated from other legumes. We have used this collection to analyze the transmissibility of their psyms, and the dependence of the transfer of these psyms on endogenous or exogenous selftransmissible plasmids (Stps). We found different scenarios: 1) strains similar to CFN42, with psym transfer depending upon the presence of endogenous Stps. Furthermore, p42a from CFN42 and the Stps from these strains were able to substitute each other. 2) Strains where the psym was not transmissible, but its transfer could be induced by p42a. 3) Strains with non-transmissible psyms, which remained unable to be mobilized, even in the presence of p42a. Furthermore, the transfer frequency of p42a decreased significantly when introduced into these strains, suggesting the presence of a strong inhibitor of conjugative transfer. 4) The Stp from one strain (see abstract by Laura Cervantes et al.) showed incompatibility with the R. etli psym, hybridization experiments indicated that this Stp contained a large segment of the R. etli psym, including the origin of replication, but not the region related to symbiosis, suggesting that this plasmid may have been generated through recombination of a psym and a Stp plasmid. The mechanism by which this Stp promotes psym transfer may be different from that described previously. Acknowledgments: To José Luis Fernández for excellent technical help. To DGAPA for PAPIIT grant IN

32 ORAL SESSION I. TUESDAY In vitro and in vivo analyses of the mechanism of action of an antisense RNA that controls the replication of a repabc plasmid Miguel Ángel Cevallos Gaos, M. A., Cervantes Rivera, R. Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, México mac@ccg.unam.mx repabc plasmids, commonly found in alpha-proteobacteria, are characterized by the presence of a repabc operon which carries all the elements required for plasmid replication and segregation. In general, repabc operons consist of three protein-encoding genes, an antisense RNA (ctrna) gene, and a centromere-like region (pars). The products of the two first genes, repa and repb, in conjunction with the pars region compose the segregation system, and repc, the last gene of the operon, encodes the initiator protein. The ctrna gene is located in the large repb-repc intergenic region, and encoded on the strand complementary to that encoding the operon mrna. The symbiotic plasmid, formally pcfn42d (p42d), of Rhizobium etli CFN42 carries a repabc operon whose expression depends on a single promoter. The transcription of all repabc operons is negatively autoregulated by RepA and RepB, and in all of them the ctrna (55-59 nt) is a negative posttranscriptional regulator of repc, the gene encoding the initiator protein. These molecules also act as strong incompatibility factors as it was shown that genes encoding ctrnas introduced in trans displace their cognate plasmids. In this work, we determined the in vitro secondary structures of: 1) the ctrna encoded in the repb-repc intergenic region of the R. etli CFN42 symbiotic plasmid p42d; 2) the repabc mrna (the target) in the repb-repc intergenic region (minc ) of the same plasmid; 3) the complex formed between the ctrna and minc, using a combination of RNA probing assays and a in silico RNA secondary structure prediction algorithm (Mfold). Also, we calculated: the kinetic parameters of hybrid formation between minc, the ctrna and various ctrna mutants; and the half-life of the ctrna. Further, we conducted an in vivo incompatibility analysis of mutant ctrnas. Our findings indicate that the interaction between the ctrna and repabc mrna is very fast, and initiates at the non-paired 5 end of the ctrna and a loop in the target mrna. This system operates by a novel mechanism, contrasting with the loop-loop kissing reported in other replication plasmid systems 28

33 ORAL SESSION I. TUESDAY Expression of the protein glycosylation system of Mycobacterium tuberculosis in Streptomyces coelicolor Córdova Dávalos, LE., Espitia Pinzón, C., Servín González, L. Departamento de Biología Molecular y Biotecnología. Instituto de Investigaciones Biomédicas. UNAM. Ciudad Universitaria, D.F., México Until recently, reports of glycoproteins in bacteria were scarce; however, their existence is now fully accepted and the description of bacterial glycoproteins has increased in recent years. In Mycobacterium tuberculosis some glycoproteins are important for pathogenesis, and they are also used for the diagnosis of tuberculosis. Since it is technically difficult to work with M. tuberculosis, we have chosen Streptomyces, a related actinomycete, as a host for expression of mycobacterial glycoproteins. Members of this genus are also able to express, secrete and glycosylate proteins. The aim of this work was to demonstrate that the products of the S. coelicolor genes SCO3154 (protein mannosyltransferase or Pmt) and SCO1423 (polyprenyl-monophosphate-mannose synthase or Ppm) and their M. tuberculosis homologues (the products of genes Rv1002c and Rv2051c, respectively) are responsible for glycosylation of the APA protein and the C31 phage receptor. S. coelicolor null mutants of SCO3154 and SCO1423 were obtained by PCR-targeting; no glycosylation activity was observed in these mutants. These were complemented with their homologues from M. tuberculosis. Ppm of M. tuberculosis was able to complement the S. coelicolor null mutant lacking Ppm ( SCO1423), restoring glycosylation of APA and the C31 phage receptor. On the other hand Pmt of M. tuberculosis could not restore protein glycosylation to the S. coelicolor mutant lacking Pmt ( SCO3154), even though it was expressed and correctly localized in the cytoplasmic membrane; this lack of complementation is most likely due to the inability of M. tuberculosis Pmt to interact with the Sec protein secretion system of S. coelicolor. 29

34 ORAL SESSION II. TUESDAY Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation Geiger O, Zavaleta-Pastor M., Sohlenkamp C., Medeot D., López-Lara I. M. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos, CP 62251, México. otto@ccg.unam.mx Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE), rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids by lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids PE and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol (DAG), demonstrating that SMc00171 functions as a phospholipase C. DAG in turn is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate (Pi) can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphatelimiting conditions, membrane phospholipids provide a pool for metabolizable Pi, which in turn can be used for the synthesis of other essential phosphorus-containing biomolecules. To our knowledge, this is the first example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous pre-existing membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria. 30

35 ORAL SESSION II. TUESDAY Longus a type IV PILI of enterotoxigenic Escherichia coli is involved in the adherence to intestinal epithelial cells Ariadnna Cruz 1*, Sara A. Ochoa 1, Karina Mazariego-Espinosa 1, Juan Xicohtencatl- Cortes 1 1 Laboratorio de Bacteriología Intestinal, Hospital Infantil de México Federico Gómez, Dr. Márquez 162, Col. Doctores, Delegación Cuauhtémoc, México D.F., México. ariadnnacruz@yahoo.com.mx Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler s diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus and ECP, are two pili produced by ETEC. These bacteria grown in Pleuro-pneumonialike organism (PPLO) at 37 C and 5% CO 2 produced longus and ECP, showing that the assembly and expression of both pili depends on the composition of the media and of strict regulatory and environmental conditions. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analysis were done, observing similar levels of adherence with values of x 10 4 in HT-29, x 10 4 in CaCo-2, and x 10 4 in T84 cells. Meanwhile, the E9034A lnga strain showed a reduction of 40% in HT-29, 42% in Caco2, and 50% in T84 cells compared to the wild-type strain. In experiments performed with non-intestinal cells (HeLa and HEp-2 cells), differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A, E9034 ecpa, E9034A lnga(plnga) strains were 30-35% more adherent in intestinal cells than in non-intestinal cells. Twitching motility experiments were performed, showing that ETEC E9034A, E9034A ecpa and E9034A lnga(plnga) strains had the capacity to form spreading zones while ETEC E9034A lnga and E9034A lnga/ ecpa do not. In addition, our data suggest that longus from ETEC participle in the colonization of human colonic cells. 31

36 ORAL SESSION II. TUESDAY Analysis of the function of diguanylate cyclase genes in biofilm formation in Klebsiella pneumoniae Cruz Pulido DP., Lozano Riveros M., Huertas Valero MG and Zambrano Eder M.M. Molecular Genetics Group. Corporación CorpoGen, Bogotá, Colombia Corresponding author: María Mercedes Zambrano, Corporación Corpogen, Carrera 5 No. 66A-34, Bogotá. Colombia. Phone: (57) Fax (57) Postal Code: mzambrano@corpogen.org Klebsiella pneumoniae is an opportunistic pathogen commonly associated with nosocomial infections, many of which are related to the use of indwelling medical devices that can harbor bacteria growing as biofilms on their surfaces. To learn about the mechanisms involved in biofilm formation in K. pneumoniae, we analyzed mutants and did comparative genomics of diguanylate cyclases (DGCs) genes implicated in the synthesis of the second messenger c-di-gmp, which regulates important bacterial functions during biofilm formation, such as the synthesis of adhesins and exopolysaccharides. Two previously identified K. pneumoniae insertional mutants defective in biofilm formation (M39 and M14) were found to be affected in the DGC genes yfin and ycdt, respectively. However, M39 forms a very robust biofilm while M14 shows a very low level of biofilm formation when compared to the parental control strain. We analyzed expression of yfin and of a putative cellulose synthase gene (cel1) using qrt-pcr and found that cel1 was over-expressed two-fold whereas the DGC gene yfin was over-expressed eight-fold in M39 compared to M14 and the control strain. Bioinformatic analysis of cel1 showed that it has a PilZ domain, which is known to bind c-di-gmp. Thus the phenotype in M39 could be explained by overexpression of yfin, which increases the intracellular levels of c-di-gmp that in turn can activate the cellulose synthase by binding its PilZ domain. This result also indicates that the yfin gene product might be important at a transcriptional level in cellulose production in K. pneumoniae. We also analyzed three different genomes of K. pneumoniae (two clinical and one environmental isolate) to determine the number, distribution and organization of DGC genes. We found that the environmental strain has the largest amount of DGC gene copies (23), and that although most of the genes are highly conserved, some of these copies are found only in some strains and might be relevant for phenotypic differences. 32

37 ORAL SESSION II. TUESDAY The two-component system CpxAR negatively regulates SPI-1 and SPI-2 expression in Salmonella enterica serovar Typhimurium De la Cruz MA, Bustamante VH, Martínez-Chavarría, LC., Calva E., and Puente JL. Instituto de Biotecnología UNAM Cuernavaca, Morelos, México Salmonella enterica evolved to be a facultative intracellular pathogen mainly by incorporating, at different evolutionary times, pathogenicity islands SPI-1 and SPI-2, both encoding a type three secretion system (T3SS). The acquisition of these islands through horizontal gene transfer events, imposed the need of adapting preexisting and novel regulatory mechanisms to appropriately control the spatiotemporal expression of genes within the islands response to specific signals found at different niches within the host. In this respect, the two-component system CpxAR allows Salmonella enterica to perceive alterations in the external environment or damage to cellular components, in order to set the appropriate physiological changes to prolong survival. The CpxAR system consists of a histidine kinase, CpxA, and a response regulator, CpxR. The Cpx pathway is activated by alterations in cell envelope composition, which could be induced by overexpressing the NlpE lipoprotein or other misfolded envelope proteins, as well as by ph changes. In this work, we found that expression of SPI-1 genes hild and hila and SPI-2 genes ssrab and ssag genes are significantly reduced in a cpxa mutant growing in LB medium (LB), whereas the cpxr and cpxra mutants had a phenotype similar to that seen in the wild-type. These data indicated that in the absence of CpxA, the main phosphatase of CpxR-P, high levels of CpxR-P repressed SPI-1 and SPI-2 in LB, where both regulons are controlled by HilD. This observation was corroborated by overexpressing CpxR, which abolished the expression of SPI-1 and SPI-2 genes in LB. Consistently, activation of the Cpx pathway by overexpressing NlpE repressed the expression of the SPI-1 and SPI-2 regulons in a CpxR-dependent manner. Interestingly, SPI-2 repression by CpxR was also seen in N-minimal medium, indicating that CpxR also repressed SPI-2 in a HilD-independent manner. In summary, our observations suggest that in order to down regulate T3SS expression to prevent cell damage, assembly of T3SS apparatuses causes cell envelope stress, and thus activation of the CpxAR system, once enough needle-complexes have been assembled. 33

38 ORAL SESSION II. TUESDAY The fermentative oxidation of ethanol in gluconacetobacter diazotrophicus is a two step pathway catalysed by a single enzyme: Alcohol-Aldehyde Dehydrogenase (ADH) E. Escamilla 1, S. Gómez 1, M. Contreras 1, R. Arreguín 2, and G. Mendoza 3. 1 Instituto de Fisiología Celular, 2 Instituto de Química and 3 Facultad de Medicina. Universidad Nacional Autónoma de México. Ciudad Universitaria, CP-04510, AP-70242, México D.F. eescami@ifc.unam.mx Gluconacetobacter diazotrophicus is a N 2 -fixing endophyte originally isolated from sugar cane. The oxidation of ethanol to acetic acid is located in the periplasmic space and according with the dogma, catalyzed by two membrane bound enzymes complexes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). These enzymes are usually recognized as quino-haemproteins that contain PQQ and cytochromes c as prosthetic groups. Here we present evidence showing that the well known membrane bound Alcohol Dehydrogenase (ADH) of Ga. diazotrophicus is indeed a double function enzyme, able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilise a reaction mechanism where the substrate ethanol is subjected to a two step oxidation to produce acetic acid without releasing the acetaldehyde intermediary to the media. Under physiological conditions, the mechanism proposed would permit the massive conversion of ethanol to acetic acid, usually seen in acetic acid bacteria, without the inconvenient transient accumulation of the highly toxic acetaldehyde. Thus, the role of ALDH remains as an open question. Grants: CONACyT and PAPIIT-UNAM IN

39 ORAL SESSION III. TUESDAY Role of DnaK and ClpB in gyrase reactivation during the heatshock response in E. coli K12 Gómez Eichelmann M.C., Lara Ortíz T., Castro Dorantes J, y Ramírez Santos J. Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM, México. cargom@servidor.unam.mx DNA molecules are under helical tension or DNA supercoiling (SC). In E. coli, the level of SC is regulated mainly by DNA topoisomerases I and II (gyrase). Top I relaxes DNA, while gyrase uses ATP to increase SC. The SC level is modified by stress conditions. For example, a heat shock induces an initial SC decrease, followed by a recovery which depends on proteins synthesized during the first 5 minutes of the stress. Studies on the role of chaperones in this recovery, suggest that DnaK is important to gyrase activity. At high temperatures, proteins unfold and a fraction of them forms aggregates in which GyrA is present. DnaK and ClpB function synergistically to remove and reactivate proteins from the aggregates. In rpoh cells exposed to heat, overproduction of DnaK at non physiological levels, or co-overproduction of DnaK and ClpB at lower levels, prevents protein aggregation. In this work, the role of DnaK and ClpB in gyrase reactivation during heat stress was evaluated. Strain BB7224 rpoh lacking RpoH ( 32 ) and plasmids carring IPTG-inducible dnak and clpb genes were used. To evaluate the SC level, plasmid topoisomers were separated by electrophoresis through 1% agarose gels (10 g/ml chloroquine). Results obtained showed a DNA relaxation after exposure of rpoh cells to 47 0 C, but not the SC recovery observed in wild-type cells. Overproduction of ClpB induced by a high IPTG concentration (200 M) did not allow the SC recovery; however, this recovery was observed when DnaK was over-produced at the same IPTG concentration. The co-overproduction of the DnaK-ClpB bichaperone system in cell conditions in which the overproduction of DnaK or ClpB did not reverse DNA relaxation (100 M IPTG), induced the recovery of the SC. These results show that the DnaK-ClpB system participates in the response to heat, ensuring the reactivation of gyrase during this response. 35

40 ORAL SESION III. TUESDAY Strain-specific gene expression of DnaK in Gallibacterium anatis Schumacher, T. & León-Kempis, M. R. Boehringer Ingelheim Vetmedica S. A. de C. V. Calle 30 no. 2614, Zona Industrial. Guadalajara, Jalisco. C.P G. anatis (Ga) is a Gram negative bacterium that belongs to the Pasteurellaceae family. It is a pathogen of many avian species and the reproductive tract is the main target in infections by Ga, hence having an impact on the egg production in layer hens. No data on proteomics of this microorganism has been generated and just a few members of the Pasteurellaceae family have been looked at. The goal of this study was to compare by means of SD gels followed by Mass Spectrometry (MS) the expression patterns of several Ga isolates from different geographical origins. Strains were grown in tryptose broth and subcellular fractions were obtained: periplasm, inner or outer membrane proteins, cytoplasm and secreted proteins. Such fractions were analysed by 2D gels/ms and the identity of the spots was determined by Peptide Mass Fingerprinting or by N-terminal sequencing. A number of different elongation factors were found in many of the fractions analysed as well as other house-keeping enzymes. Interestingly, DnaK, a member of the heat shock proteins (hsp) was found to be expressed in the periplasm of two out of the six analysed strains. Amongst many of the reported functions of hsp, it is known they work under normal conditions as well as under stress (temperature, ph, po 2, etc). As perspective, it will be interesting to evaluate what the role of DnaK is in Ga and to determine why it is only present in some isolates and see if that confers an advantage in virulence. 36

41 ORAL SESSION III. TUESDAY The physiological stimulus for the BarA sensor kinase Gonzalez Chavez, R., 1 Alvarez, A.F., 1 Romeo, T., 2 and Georgellis, D. 1 Departamento de Genética Molecular, Instituto de Fisiología Celular. Universidad Nacional Autónoma de México México, D. F., México, 1 and Department of Microbiology and Cell Science, P.O. Box , University of Florida, Gainesville, Florida rgonzalez@ifc.unam.mx The BarA/UvrY two-component signal transduction system (TCS) of Escherichia coli consists of the histidine sensor kinase BarA and its cognate response regulator UvrY. BarA (bacterial adaptive response) is a member of the subclass of tripartite sensor kinases, whereas, UvrY is a typical response regulator of the FixJ family. A key role of this TCS and its homologues in other Gram-negative bacteria, such as BarA/SirA of Salmonella, ExpS/ExpA of Erwinia, VarS/VarA of Vibrio and GacS/GacA of Pseudomonas species is to activate expression of noncoding RNAs, e.g. CsrB and CsrC in E. coli. These small regulatory RNAs possess repeated sequence elements that allow them to interact with multiple copies of the RNA binding protein CsrA and thus prevent its interaction with mrna targets. Because CsrA somehow activates csrb and csrc expression via the BarA-UvrY TCS, and thereby feedback inhibits its own activity in the cell, this regulatory circuitry appears to serve as a homeostatic device for fine tuning CsrA activity. Deletion of the bara or uvry gene leads to altered central and secondary carbon metabolism, biofilm development, motility, peptide uptake, hfq expression, and virulence gene expression in E. coli and other species. Despite the important roles played by the BarA/UvrY system, the physiological signals that regulate the activity of the BarA sensor kinase and thereby control the activity of the UvrY response regulator have not been identified for any of the orthologous TCSs. Here, we show that formate and acetate, but also other short chain fatty acids, provide a physiological stimulus for the BarA sensor kinase. 37