Sociedad Mexicana de Bioquímica, A. C.

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1 Sociedad Mexicana de Bioquímica, A. C. Program & Abstracts Second Meeting of Biochemistry and Molecular Biology of Bacteria Huatusco, Veracruz November 7 11, 2011 I

2 Organizing Committee Guadalupe Espín Dimitris Georgellis Agustino Martínez Christian Sohlenkamp Local Committee Omar Elind Arroyo Helguera Rocío Coutiño Ramírez Rebeca García Román Hilda Montero Ladrón de Guevara Cristina Ortíz León Clara Luz Sampieri Ramírez Roberto Zenteno Cuevas Instituto de Salud Pública Universidad Veracruzana Technical Edition: and Typesetting: Maria Teresa Castillo Technical Support: Lucero López, Omar Chávez Image Cover: Nayeli Quinto SODIO Published in México, 2011 II

3 Segundo Congreso Rama de Bioquímica y Biología Molecular de Bacterias Sociedad Mexicana de Bioquímica, A.C de noviembre de Huatusco, Veracruz Bienvenida Bienvenidos al Segundo Congreso de Bioquímica y Biología Molecular de Bacterias!. Pasaron casi 20 meses desde la primera reunión de nuestra rama celebrada en San Miguel Regla, Hidalgo. Un incentivo para dar luz a este congreso, fue el de querer conjuntar un grupo cohesivo en donde se discutieran con intensidad y profundidad los problemas científicos que nos apasionan. Según la percepción de muchos de los asistentes de aquel congreso, la primera reunión fue un gran paso en esta dirección y por ende un gran éxito. Sin embargo, todavía queda un largo camino delante de nosotros como comunidad para acercarnos a las ideas plasmadas hace 20 meses por el Comité organizador del primer congreso. Les invitamos a todos ustedes a que participen con mucho entusiasmo en las diferentes actividades de este congreso para lograr que éste sea una opción preferida para científicos estudiando bacterias. Como bien decía en la bienvenida del congreso pasado: 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 mucho años venideros;. El 11 de febrero del 2011 falleció el Dr. Fernando Bastarrachea Aviles quien fue uno de los pioneros en estudiar la genética molecular bacteriana en México, y cuyo trabajo, rigurosidad cientifica y personalidad influyeron de manera decisiva en el desarrollo de esta diciplina en el Pais. Para conmemorar su papel en la el desarrollo de la microbiología molecular en México, le rendimos homenaje en la sesión IX, en la cual algunos de sus ex alumnos exponen. También en el año 2011 se celebran los 50 años del nacimiento del concepto del operón en la regulación de la expresión génica. No se dedicó una sesión especial a este tema; este concepto se extiende por un gran número de trabajos presentados en este congreso. Bienvenidos! Guadalupe Espín Dimitris Georgellis Agustino Martínez Christian Sohlenkamp III

4 Prólogo de la primera edición 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 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. IV

5 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 V

6 Funding Institutions & Sponsors VI

7 Table of Contents Program of Events Monday... 3 Tuesday... 4 Wednesday... 8 Thursday Abstracts Plenary Sessions Oral Presentations Odd Poster Sessions Even Poster Sessions Author Index VII

8 PROGRAM

9 Monday November 7, :00 18:00 Registration 18:45 19:00 Opening Ceremony, and Opening Talk 19:00 20:00 Opening Lecture Valeria Souza Why so many bacterial species? The roles of food, sex and travel in determining the microbial diversity of our planet Instituto de Ecología UNAM Chair: Guadalupe Espín 20:00 22:00 Dinner and Welcome Cocktail All oral presentations will be held in the Magno Orquídeas Room All poster presentations will be held in the Palapa 3

10 Tuesday November 8, 2011 Oral Session I Chair: José Luis Puente 9:00 9:20 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 César Augusto Aguilar Martínez Characterization of mutations effects in regulatory genes originated through an adaptive evolution process by rapid growth on glucose in an Escherichia coli strain lacking the PTS system Instituto de Biotecnología UNAM Morena Avitia Cao Romero Population genetics of Bacillus sp nov from Cuatro Cienegas Coahuila, Mexico Instituto de Ecología UNAM Víctor Antonio Becerra Rivera The over-expression of cytochrome c 550 from Bacillus subtilis has effects on the activity of respiratory complexes FES Iztacala UNAM Susana Brom Klanner The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain Centro de Ciencias Genómicas UNAM Rosina Cabrera Ruiz Overexpression and Purification of quorum sensing transcriptional regulator NprR of Bacillus thuringiensis Centro de Investigación en Alimentación y Desarrollo 10:40 11:00 Break 4

11 Tuesday November 8, 2011 Oral Session II Chair: Luis Eguiarte Fruns 11:00 11:20 11:20 11:40 11:40 12:00 12:00 12:20 12:20 12:40 Martha Irais Camacho Hernández The global regulators Hfq and CsrA are required for proper uvry translation Instituto de Fisiología Celular UNAM Miguel Ángel Cevallos Gaos The origin of replication of a repabc plasmid Centro de Ciencias Genómicas UNAM Mayra Elizeth Cobaxin Cárdenas Effect Of In Vitro Culture Conditions On The Expression Of Virulence Factors In Vibrio cholerae El Tor Facultad de Medicina - UAEM Susana de la Torre Zavala Gene expression of Pht cluster genes and a putative nonribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola CINVESTAV IPN. Unidad Irapuato Alma Laura Díaz Pérez Structural, phylogenetic and functional analysis of the isocitrate lyase from Pseudomonas aeruginosa Instituto de Investigaciones Químico-Biológicas UMSNH 12:40 13:00 Break 5

12 Tuesday November 8, 2011 Plenary Session I Chair: Dimitris Georgellis 13:00 14:00 Roberto Kolter Disassembly of Bacillus subtilis biofilms Harvard Medical School 14:00 16:00 Lunch Oral Session III Chair: Herminia Loza Tavera 16:00 16:20 Ma. Dolores Rodríguez Torres Metabolic diversity among Bacillus isolates from the sediment of the Churince system in Cuatro Ciénegas, Coahuila CINVESTAV IPN. Unidad Irapuato 16:20 16:40 16:40 17:00 Katy Juárez López Integration Host Factor is a global regulator required for electron transference in Geobacter sulfurreducens Instituto de Biotecnología UNAM Paula Figueroa Arredondo Vibrio cholerae cytolysin VCC probably causes pyroptosis to THP-1 macrophages by activating MAPKs signaling pathway Escuela Nacional de Medicina y Homeopatía IPN 6

13 Tuesday November 8, 2011 Plenary Session II Chair: Agustino Martínez 17:00 18:00 Jeff F. Miller Diversity-Generating Retroelements University of California 18:00 20:00 Poster Session Odd numbers 20:00 22:00 Dinner 7

14 Wednesday November 9, 2011 Oral Session IV Chair: Edmundo Calva Mercado 9:00 9:20 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 Rodolfo García Contreras Quorum Quenching Quandary: Resistance to Antivirulence Compounds Instituto Nacional de Cardiología Ignacio Chávez Jaime García Mena Study of protein-protein interaction in the RNA Degradosome of Escherichia coli upon conditional mrna degradation CINVESTAV IPN Unidad Zacatenco Andrea González González Genetic diversification and genome size diversity of Mexican Escherichia coli Instituto de Ecología UNAM Bertha González Pedrajo Molecular mechanisms participating in the biogenesis of the type III secretion system of enteropathogenic Escherichia coli Instituto de Fisiología Celular UNAM Manuel Alejandro González Vera Transcriptional and posttranscriptional controls involved in the synthesis of flagellin in Rhodobacter sphaeroides Instituto de Investigaciones Biomédicas UNAM 10:40 11:00 Break 8

15 Wednesday November 9, 2011 Oral Session V Chair: Gabriela Olmedo Álvarez 11:00 11:20 11:20 11:40 11:40 12:00 12:00 12:20 12:20 12:40 Isabel M. López Lara FadD is required for utilization of endogenous fatty acids released from membrane lipids Centro de Ciencias Genómicas UNAM Mildred Castellanos Escamilla RpoS regulator of stationary phase, controls transcriptional expressions of phbr gene and phbbac operon witch are responsible for PHB production in Azotobacter vinelandii Instituto de Biotecnología UNAM Rafael Jiménez Mejía Analysis of the environmental conditions involved in regulation of chr genes from Burkholderia xenovorans Instituto de Investigaciones Químico-Biológicas UMSNH Gamaliel López Leal Analysis of the Rhizobium etli stimulons under heat shock and saline stress by RNA-Seq Centro de Ciencias Genómicas UNAM Edgardo Galán Vásquez The regulatory network of Pseudomonas aeruginosa and its comparison with other bacterial networks CINVESTAV IPN. Unidad Irapuato 12:40 13:00 Break 9

16 Wednesday November 9, 2011 Plenary Session III Chair: Guadalupe Espín 13:00 14:00 Stephen Trent Remodeling of the Bacterial Cell Surface: Modification of Lipopolysacchardies University of Texas 13:40 16:00 Lunch Oral Session VI Chair: Mayra de la Torre Martínez 16:00 16:20 Ana Elisa Martínez del Campo Chemotaxis signaling pathway of the flagellar system 2 from Rhodobacter sphaeroides Instituto de Fisiología UNAM 16:20 16:40 16:40 17:00 Eva Martínez Peñafiel Overexpression of 4.3 protein of phage mep021 induces pleiotropic effects like inhibition of cell division, release of basal haemolysin HlyE and cell death CINVESTAV IPN. Unidad Zacatenco Martin Peralta Gil Characterization of multiple arrangements of binding sites of transcription factors in Escherichia coli K-12 and their mechanisms of action in gene regulation Centro de Ciencias Genómicas UNAM 10

17 Wednesday November 9, 2011 Plenary Session IV Chair: Christian Sohlenkamp 17:00 18:00 Peter Greenberg Sociomicrobiology: Quorum sensing in Pseudomonas aeruginosa University of Washington 18:00 20:00 Poster Session Even numbers 20:00 22:00 Dinner 11

18 Thursday November 10, 2011 Oral Session VII Chair: Jesús Campos García 9:00 9:20 9:20 9:40 9:40 10:00 10:00 10:20 10:20 10:40 Daniel Segura González Effect of phag gene expression on polyhydroxyalkanoate structure synthesized by a recombinant strain of Azotobacter vinelandii Instituto de Biotecnología UNAM David Zamorano Sánchez Identification of FikR, the response regulator that completes the fixl-fixk cascade in Rhizobium etli CFN42 Centro de Ciencias Genómicas UNAM Roberto Zenteno Cuevas Characterization of the molecular mechanisms producers of drug resistance in Mycobacterium tuberculosis Instituto de Salúd Pública UV César Quiñones Valles Regulatory circuits controlling asymmetrical cell division in bacteria CINVESTAV IPN. Unidad Irapuato Eria Alaide Rebollar Caudillo Evolutionary history and niche differentiation of Exiguobacterium, a halophilic bacterial genus living at Cuatro Cienegas Basin, Mexico Instituto de Ecología UNAM 10:40 11:00 Break 12

19 Thursday November 10, 2011 Oral Session VIII Chair: Heliodoro Celis Sandoval 11:00 11:20 Gloria Alejandra Sarmina Leonel Characterization of a null mutant in the mazg gene of Rhodobacter sphaeroides Instituto de Fisiología Celular UNAM 11:20 11:40 Omar Alejandro Sepúlveda Robles Genetic diversity of phages of Pseudomonas aeruginosa: isolation, characterization and identification of new species CINVESTAV IPN. Unidad Zacatenco 11:40 12:00 Hortencia Silva Jiménez Construction of a prototype sensor kinase from the hybrid kinase TodS of Pseudomonas putida DOT-T1E Consejo Superior de Investigaciones Científicas 12:00 12:20 Miguel Ángel Vences Guzmán Function of ornithine lipids in Agrobacterium tumefaciens Centro de Ciencias Genómicas UNAM 12:20 12:40 Roche Diagnostics Metagenomics and Microbial Diversity Technical Talk 12:40 13:00 Break 13

20 Thursday November 10, 2011 Plenary Session V Chair: Dimitris Georgellis 13:00 14:00 Susan Gottesman Regulating with small RNAs in E. coli National Cancer Institute 14:00 16:00 Lunch Oral Session IX In Memorian FERNANDO BASTARRACHEA Chair: David Romero 16:00 16:30 16:30 17:00 17:00 17:30 17:30 18:00 Luis Servín González Genetics and genomics coming together to understand the methylspecific restriction system of Streptomyces coelicolor Instituto de Investigaciones Biomédicas UNAM Laura Camarena Motility in Rhodobacter sphaeroides is mediated by a complex rotation system Instituto de Investigaciones Biomédicas UNAM Gloria Soberón Chávez The RhlR regulon in Pseudomonas aeruginosa quorum sensing response. Instituto de Investigaciones Biomédicas UNAM David René Romero Camarena Gene Conversion in Rhizobium etli: role of systems for initiation and migration of the Holliday junction Centro de Ciencias Genómicas UNAM 14

21 ABSTRACTS PLENARY SESSION

22 Opening Lecture. November 7, 2011 Why so many bacterial species? The roles of food, sex and travel in determining the microbial diversity of our planet Valeria Souza and Luis E. Eguiarte Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, CU, AP , Coyoacán The microorganisms that inhabit modern-day microbial mats coexist in space due to the differences in oxygen regime, permitting the co-occurrence of aerobic microbes, different types of photosynthesis and ancient anoxic methanogens, within the same architectural complex. Although microbial mats and microbialites once had broad geographical distribution, they now are only found in extreme environments, where algae cannot prosper. This is the case of the extremely oligotrophic oasis of Cuatro Ciénegas Basin (CCB) in Coahuila Mexico. By describing the microbial diversity in CCB we detected that bacterial clonal lineages were diversifying locally, generating a large total diversity. Moreover, molecular evolution analyses indicate that they are closely related to marine microorganisms that once shared the common ocean of Panthalassa. Further exploration of this link was done through comparative genomics, finding that CCB represents a lost world were ancient lineages persisted despite the fact that the ocean left the region ca. 35 million years ago. In order to explore a possible ecology of the past, we carried out detailed comparative metagenomic analyses of 4 microbialites with different N:P content, observing that each one was unique in its taxonomical composition. However, all of them converged to an amazing array of metabolic pathways that had the potential of performing the majority of the biogeochemical cycles and also of degrading most of the known compounds described in KEGG database. Our inference is that in the Precambriam ocean, microbes survived different environmental catastrophes in part due to their community metabolic cohesion and redundance. Our data and analyses for Cuatro Cienegas and their extrapolation to earlier Earth suggests that it is precisely the low Phosporous (P) content that increase reproductive isolation by limiting all the mechanisms of HGT. Low HGT is followed by local diversification of small population sizes and high metabolic coherence were communities have been competing and coexisting for so long that newcomers are not welcome. On the other hand, in model systems such as E. coli, is precisely the abundance of food that allows ample space for sex and travel, maintaining an enormous cosmopolitan genetic pool that has taken a very long time to diversify and speciate from it sister genes Salmonella. 17

23 Plenary Session I. November 8, 2011 Roberto Kolter Harvard Medical School Disassembly of Bacillus subtilis biofilms Most microbes can switch between unicellular and multicellular lifestyles. Depending on environmental conditions, there are times where each of these lifestyles can provide a selective advantage and thus aid in the survival of the species. As a consequence, most microbes have evolved the capacity to respond to environmental cues by switching lifestyles. The most common form of multicellularity among bacteria is the formation of cellular aggregates that are held together by an extracellular matrix. Such aggregates are generally found associated to surfaces and are referred to as biofilms. Depending on the setting where biofilms form, they can have very different consequences for human life. The biofilms that form on the surfaces of the human body are largely mutualistic and most of the time protect the human. However, when normal human defenses are breached, pathogens can colonize surfaces forming biofilms that are the foundation of chronic infections. Because of the importance of biofilms on human health it is therefore important to understand the processes that lead to their disassembly. We are studying these processes in the model bacterium Bacillus subtilis and we have learned much about the molecules involved in biofilm disassembly. What we have learned from these studies can be extended to important pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. We are hopeful that from the basic knowledge we have obtained we will be able to develop strategies to control biofilms that are problematic in clinical settings. 18

24 Plenary Session II. November 8, 2011 Diversity-Generating Retroelements Huatao Guo, Mari Gingery, Diego Arambula, Elizabeth Czornyj and Jeff F. Miller Dept. of Microbiology, Immunology and Molecular Genetics UCLA Host-parasite interactions are often driven by mechanisms that promote genetic variability. In the course of our studies on bacterial pathogenesis, we discovered a group of temperate bacteriophages that generate diversity in a gene that specifies tropism for receptor molecules on host Bordetella species, which cause respiratory infections in humans and other mammals. This microevolutionary adaptation is produced by a diversity-generating retroelement (DGR) that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats, one serving as an donor template (TR) and the other as a recipient of variable sequence information (VR). Recent work has focused on the genetic basis of diversity-generation. The directionality of information transfer is determined by the initiation of mutagenic homing (IMH) sequence present at the 3 end of VR. We have demonstrated that DGR function occurs through a TR-containing RNA intermediate by a unique targetprimed reverse transcription mechanism that precisely regenerates target sequences. This non-proliferative, copy and replace mechanism enables repeated rounds of protein diversification and optimization of ligand-receptor interactions. The potential utility of DGRs is illustrated by the identification of over 150 related elements in bacterial, phage, and plasmid genomes. DGRs are present in human pathogens (Treponema, Legionella spp.), human commensals (Bacteroides, Bifidobacterium spp.), green sulfur bacteria (Chlorobium, Prosthecochloris spp.), cyanobacteria (Trichodesmium, Nostoc spp.), magnetotactic bacteria (Magnetospirillum spp.), and many other diverse species. DGRs comprise a new family of retroelements with the potential to confer powerful selective advantages to their host genomes. 19

25 Plenary Session III. November 9, 2011 Remodeling of the Bacterial Cell Surface: Modification of Lipopolysacchardies M. Stephen Trent The University of Texas at Austin Bacteria synthesize remarkable surface structures that are often required for growth and infection of the human host. In response to extracellular stimuli, gram-negative organisms frequently remodel lipopolysaccharide (LPS) structures to promote their survival. Often these modifications occur on lipid A, a saccharolipid that anchors LPS in the outer membrane. Notably, lipid A is the endotoxic portion of LPS that is recognized by the TLR4-MD2 receptor of the mammalian innate immune system. Enzymatic remodeling of the lipid A provides resistance to cationic antimicrobial peptides that bind to the anionic surface of bacteria and promotes immune evasion by modulating TLR4-MD2 recognition. The level of diversity seen in LPS modification systems is quite extraordinary and our laboratory has focused on the characterization of modification machinery from a number of organisms including Escherichia coli, Campylobacter jejuni, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, and others. More recently, we have discovered that LPS modification machinery can serve in the assembly of additional bacterial structures such as flagella and peptidoglycan. Understanding how bacteria remodel their cell surface is of fundamental importance and may yield new therapeutic strategies for intervention in bacterial infections. 20

26 Plenary Session IV. November 9, 2011 E. Peter Greenberg University of Washington, Seattle USA Sociomicrobiology: Quorum sensing in Pseudomonas aeruginosa For most of the last century biologists held the belief that except for rare exceptions bacteria were not social organisms. This belief has given way to a new understanding that bacteria are like other creatures. As a result of selection, social activities have evolved in many bacterial species. This has led to the development of an emerging discipline that now counts hundreds of scientists as practitioners; sociomicrobiology. The rationales for studying the social behavior of bacteria are as follows: Studies of bacterial sociality can significantly increase our understanding of sociobiology in general. If one hopes to understand bacterial activities in any comprehensive way it is critical to include social activities in the equation. Finally, we have observed that bacterial social activities are important for successful infections by a variety of pathogenic bacteria. Therefore, it may be possible to develop new medicines as a result of sociomicrobiology investigations. Our studies of bacterial social activity began about thirty years ago with the beginnings of an understanding that bacterial cells produced pheromones that enabled cell-to-cell communication. This form of communication allows individuals of a given species to monitor the abundance of that species, and at sufficiently high population densities the community responds by activating specific genes that result in production of public goods (eg extracellular enzymes, antibiotics, light production in special cases). We thus termed this activity quorum sensing and response. This presentation will cover recent investigations of quorum sensing signal receptors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. There will be a particular on new information regarding the key receptor LasR 21

27 Plenary Session V. November 10, 2011 Regulating with small RNAs in E. coli Susan Gottesman Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD E. coli grows in a variety of environments, and thus must adjust its metabolism and regulation as it moves from environment to environment. Transcriptional regulation is at the core of these changing responses, but it is becoming increasingly clear that other layers of regulation exist. I will discuss the small non-coding RNAs (srnas) that regulate mrna stability and translation. The best-studied family of srnas binds the RNA chaperone Hfq and pairs with target mrnas. Expression of the srnas are highly regulated, and in many cases the srnas act as bridges between different regulatory cascades. For instance, the Arc two-component system negatively regulates ArcZ, which in turn positively regulates the RpoS sigma factor and negatively regulates the regulator of motility, flhdc. We use a combination of microarrays, bioinformatics, and screens of srna libraries to identify which srnas regulate which mrnas, with the aim of understanding how the srnas modulate regulatory networks. I will describe our recent findings in dissecting these networks. 22

28 Plenary Sessions NOTES 23

29 Plenary Sessions NOTES 24

30 ABSTRACTS ORAL PRESENTATIONS

31 Oral Session I. Tuesday Characterization of mutations effects in regulatory genes originated through an adaptive evolution process by rapid growth on glucose in an Escherichia coli strain lacking the PTS system César Aguilar*, Adelfo Escalante, Noemí Flores, Ramón de Anda, Guillermo Gosset, Francisco Bolívar Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología (UNAM). Apdo. Postal Cuernavaca/Morelos 62250, México. *Corresponding author: Tel , Fax: 52(777) , address: The intracellular availability of phosphoenolpyruvate (PEP) is very controlled; now a days engineering of metabolic pathways has succeeded in increasing this availability by eliminating in a strain of Escherichia coli (JM101) the ptshlcrr operon, which codes to the phosphotransferase system (PTS), which consumes more than 50% of PEP originated from glycolysis (Flores, N. et. al 2005). From the strain PTS -, and by a process of selective pressure for rapid growth in glucose, we generated the PB12 strain, which partially recovered the ability to assimilate glucose (PTS - Glc + ) and the ability to grow efficiently using glucose as the only carbon source. Through metabolic engineering, we were able to redirect part of PEP not used by PTS to the synthesis of aromatic compounds with high yield in PB12 strain (Escalante, et al. 2010; Cortés-Tolalpa et al. 2011). It has been shown that strain PB12 overexpressed most of the genes in the glycolytic pathway, as well as genes involved in the metabolism of ppgpp (Flores, N. et. al. 2005, 2007, 2008). In order to know accurately the changes arising during the evolution process that gave rise to PB12 strain, it was sequenced by two methods: using a comparative sequencing performed by Roche-Niblegen and by a second method performed with the Genome Analyzer system GAIIx company (IBT, UNAM). Several point mutations were found including mutations in genes involved in regulation (arcb, bara, rna, rpod, rssa, yjju and ypda genes). Also the absence of a chromosomal fragment comprising 10.3 Kb's long where 12 genes are located (including relevant ones such as the rpph, muth and galr genes) was detected. This work try to explain the possible consequences of each of the mutations in the regulatory genes originated in the strain PB12 during the evolution process thorough a performed set of experiments, including complementation, inactivation and RT-qPCR experiments. Results show the relevance of certain mutations on growth recovery in strain PB12, and explain some of the metabolic characteristics of this evolved strain and furthermore the importance of eliminating chromosomal region as important part of the adaptation process, which is speculated to be among the first mutational events occurring during the evolution process of this strain. 27

32 Oral Session I. Tuesday Population genetics of Bacillus sp nov from Cuatro Cienegas Coahuila, Mexico Avitia Cao Romero Morena, Souza Saldívar Valeria, Eguiarte Fruns Luis Instituto de Ecología, Universidad Nacional Autónoma de México Correo electrónico: Dirección postal: Apartado Postal Ciudad Universitaria, UNAM México, D.F. Circuito exterior s/n anexo al Jardín Botánico Exterior Population genetics studies of bacterial species have focused mainly on pathogenic and symbiotic organisms. However there are only a few works exploring the population structure of free-living bacteria. Cuatro Cienegas Basin (CCB) is located in Coahuila, Mexico, and it is a place with great diversity of aquatic environments. These environments are characterized by the presence of complex bacterial communities called microbial mats, as well as diverse planktonic communities with high beta diversity. Within that ample microbial diversity, few genera are abundant and diverse in many sites. One of these groups with broad distribution is Bacillus. Previous studies of Bacillus from Cuatro Cienegas have allowed us to understand part of the evolutionary history of this group and some aspects of its adaptation to the particular environmental conditions of this basin. The aim of the present work was to explore the population structure of a new particular lineage of free-living bacteria from the Bacillus genus, which is widely distributed throughout the CCB. We described the evolutionary forces that have been shaping such populations in contrast with other lineages with restricted distribution. The multilocus analysis of this group of microorganisms showed that this population has a clonal structure and its distribution does not seem to correlate with environmental factors. 28

33 Oral Session I. Tuesday The over-expression of cytochrome c 550 from Bacillus subtilis has effects on the activity of respiratory complexes Becerra Rivera Victor Antonio, Cabellos Avelar Tecilli y Gutiérrez-Cirlos Madrid Emma Berta Unidad de Biomedicina, FES Iztacala UNAM, Av. De los Barrios #1. Los Reyes Iztacala. Tlalnepantla, Edo. de México. c.p , Tel: , ext Fax: Cytochrome c-type is a small mobile protein that transports electrons from complex III to the terminal oxidase in the intermembrane space. It may be present in two forms: as soluble periplasmic proteins or attached to the membrane with a hydrophobic N-terminal extension. The mechanism of action of the later is still unknown. The information available from these proteins is limited in Gram-positive bacteria, in contrast to the amount of information available in Gram-negative bacteria and eukaryotes, therefore studies of this cytochrome in Gram-positive bacteria as Bacillus subtilis are of extreme importance. The c-type cytochrome in B. subtilis is encoded by the gene ccca and is called cytochrome c 550. This protein of 13 kda is composed of a α helix as a transmembrane binding domain and a heme domain. It is located on the outer surface of the plasma membrane, as all bacterial c-type cytochromes. In this work, we cloned and over expressed the membrane-bound cytochrome c 550 to evaluate its effect on the respiratory complexes in the bacterium Bacillus subtilis grown in LB medium and MSR (3% succinate) and harvested at three different times (5, 9 and 23 hr of growth). We found that cells grown in MSR medium give a higher yield as well as over-expression of cytochrome c 550 demonstrated by spectrophotometric analysis of the isolated membranes. The maximum concentration obtained for cytochrome c 550 was similar (36.7 and 40.4μM) for LB and MSR respectively. It is important to note that it is required of smaller volume of cells grown in LB than MSR to obtain this concentracion. Polyacrylamide gels using lithium dodecyl sulfate showed the kinetics of expression of cytochrome c 550, which increased according to time. We observed that in LB medium, expression of cytochrome c 550 increases from 5 to 9 hr and then decreases at 23 hr. Also the enzymatic activity of the four respiratory complexes was measured spectrophotometrically. We found that the activity of complex I, increases significantly after 9 hr of growth in the case of LB medium but in MSR it increases at 23 hr. Complex II shows a significant increase in both media at the three different times of harvesting. Complexes III and IV also show a significant increase in the two media but only at 9 hr. Finally, complex IV activity increased significantly only between 5 and 9 hr of growth in both media. Thus, this paper introduces the expression of proteins of the respiratory chain of B. subtilis, which varies depending on the carbon source where it grows. 29

34 Oral Session I. Tuesday The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain Susana Brom, Laura Cervantes, Patricia Bustos, Lourdes Girard, Rosa Isela Santamaría, Guillermo Dávila, Pablo Vinuesa, and David Romero Centro de Ciencias Genómicas. UNAM. Av. Universidad 1001, Cuernavaca, Mor., México. Tel. (777) Beans and their symbiont Rhizobium etli originated in Mesoamerica, while soybean and its symbiont Sinorhizobium fredii evolved in East Asia. S. fredii strains (e.g. GR64) have been isolated from bean nodules in Europe, suggesting the occurrence of conjugative transfer events among introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pret42d) requires cointegration with the endogenous self-transmissible plasmid pret42a. With the purpose of further understanding the generation of diversity among bean nodulating strains, we studied the plasmids of S. fredii GR64 (psfr64a and psfr64b). Plasmid psfr64b corresponds to the symbiotic plasmid, although it allows beannodulation, it differs from typical R. etli symbiotic plasmids, concerning various features, such as the replication functions, and reiteration of nifh genes. Regarding conjugative transfer, plasmid psfr64a was found to be self-transmissible, and required for transfer of the symbiotic plasmid (psfrgr64b). We obtained the complete sequence of psfr64a, finding 166 ORFs. The plasmid showed three large segments of different evolutionary origins; one of them presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; another one harbored 51 ORFs with highest similarity to genes from pret42d, including the replication, but not the symbiosis genes; as a result, psfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, although it did not contribute to symbiosis. The last sector contained 36 ORFs with highest similarity to genes localized on pret42a, 20 of them involved in conjugative transfer. Nevertheless, pret42a was unable to substitute psfr64a for induction of psym transfer, furthermore pret42a transfer frequency was significantly diminished in GR64 background. Our results indicate that S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We suggest that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour, exposing their adjustment to diverse host environments. Acknowledgments: To DGAPA for PAPIIT grant IN

35 Oral Session I. Tuesday Overexpression and Purification of quorum sensing transcriptional regulator NprR of Bacillus thuringiensis a Rosina Cabrera-Ruiz, b Adriana Guzmán-Soto, a Refugio Robles-Burgueño, a Jorge Rocha-Estrada, c Gabriel Guarneros y a Mayra de la Torre a Centro de Investigación en Alimentación y Desarrollo, A. C., Departamento de Ciencia de los Alimentos, Hermosillo Sonora 83304, buniversidad de Sonora, Departamento de Medicina, Hermosillo Sonora 83000, ccinvestav, Departamento de Genética y Biología Molecular, México DF Bacterial cells use quorum sensing mechanism (QS) to interact with each other and coordinate gene expression in response to cell density. RNPP is a family of QS intracellular-receptors that bind directly to its peptide effector. One of the proteins that integrate this family is the Neutral Protease Regulator (NprR). In Bacillus subtilis and Bacillus stearothermophilus, NprR was described as activator of a neutral protease and it has been recently related to sporulation and expression of cry genes in the entomopathogenic bacterium Bacillus thuringiensis (Bt). However, NprR has not been characterized, this work represent the first step to know the molecular mechanism and function that this protein performs as a QS transcriptional regulator. We overproduced the protein NprR using a heterologous expression system (Escherichia coli BL21), at the same time, to avoid the rapid degradation or aggregation of the recombinant protein we co-expressed two teams of chaperones (DnaK-DnaJ-GrpE y GroES-GroEL) to assist protein folding and this leads to increased production of active proteins. The protein was purified by affinity chromatography using Ni-NTA and its purity was analyzed by SDS-PAGE at 10%. This expression system allowed the overexpression of NprR as a soluble protein. 31

36 Oral Session I. Tuesday NOTES 32

37 Oral Session II. Tuesday The global regulators Hfq and CsrA are required for proper uvry translation Martha I. Camacho, Adrián F. Álvarez y Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiología Celular. Universidad Nacional Autónoma de In prokaryotic cells, the sensing and processing of environmental signals depends mainly on two-component signal transduction system (TCS). These systems consist of a membrane-bound histidine kinase (HK) and a cytosolic response regulator (RR). The BarA/UvrY TCS of Escherichia coli consists of BarA as the HK, and UvrY as its cognate RR. BarA has been shown to autophosphorylate in response to extracellular acetate or other aliphatic carboxylic acids, and transphosphorylate UvrY. UvrY-P, in turn, acts as a transcriptional regulator that directly activates the expression of the noncoding RNAs (srnas) CsrB and CsrC. These srnas act as antagonists of CsrA, an RNA binding protein with profound effects on many physiological processes, such as central carbon metabolism, virulence, motility and biofilm formation. Curiously, csrb transcription, which directly depends upon UvrY, is not activated in a csra mutant strain, suggesting that CsrA may affect directly or indirectly UvrY expression. Here, we present the results of experiments aiming at elucidating the regulatory interactions of BarA/UvrY and CsrA/CsrB. A simple model for this regulatory circuitry is presented and discussed. 33

38 Oral Session II. Tuesday The origin of replication of a repabc plasmid Miguel A. Cevallos, Ramón Cervantes Rivera, Francisco Pedraza López, Gabriela Pérez Segura Programa de Genómica Evolutiva; Centro de Ciencias Genómicas UNAM. Avenida Universidad s/n, col. Chamilpa, CP: Cuernavaca, Morelos, (777) repabc operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 -proteobacterial genera and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repa, repb, and repc. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repb-repc genes modulates repc expression. To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repabc operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repc open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repc revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not enter an R. etli strain containing p42d, but similar constructs that carried repc from Sinorhizobium meliloti psyma or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC SymA was able to replicate in the presence of p42d. Conclusions: RepC is the only element encoded in the repabc operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriv of this plasmid resides within the repc gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in this phenotype. 34

39 Oral Session II. Tuesday Effect Of In Vitro Culture Conditions On The Expression Of Virulence Factors In Vibrio cholerae El Tor Cobaxin Cárdenas Mayra Elizeth 1, 2 and Sánchez Castillo Joaquín 2 Facultad de Ciencias 1 and Facultad de Medicina 2, UAEM. Cuernavaca Morelos, Mexico. Phone: (777) Ext Cholera, which is characterized by voluminous watery diarrhea, is produced when the gram-negative curved bacillus Vibrio cholerae colonizes the upper small intestine of its human host. Two major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP), play major roles in the pathogenesis of this infection. The two major biotypes of V. cholerae, classical and El Tor each require ToxR and ToxT for activation of virulence factors, and potential regulatory sequences are largely conserved between the two biotypes. Nevertheless, the mechanism controlling expression of toxt appears to be biotype specific, because classical strains express CT and TCP under a wide range of experimental conditions, while El Tor strains require specific growth conditions, termed AKI, for detectable expression of the ToxR regulon. Such conditions comprise a biphasic culture where vibrios are first statistically grown for a 4-h period and then shifted to shaking. However, previous work indicated that CT synthesis is induced in single phase still cultures if the surface area of the exposed culture is increased and shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype by enhancing ToxT activity. On the other hand, proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression and at the molecular level CT expression is controlled by AphB, which induces expression of TcpP, TcpP activates synthesis of ToxT and ToxT directly enhances transcription of cistrons encoding CT. It has recently been shown that the activity of AphB is increased under low oxygen concentrations and the activity of ToxT is induced by exogenous bicarbonate. Both of these responses are compatible with stimulation of CT synthesis by AKI conditions. In an attempt to more precisely define how growth conditions may function to induce expression of CT, in this work we followed the kinetics of consumption of dissolved oxygen during bacterial growth; results suggest a working model that may account for induction of CT expression under both AKI and non-aki conditions. 35

40 Oral Session II. Tuesday Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacAin Pseudomonas syringaepv. phaseolicola. De la Torre-Zavala, Susana 1, Hernández-Flores, José Luis 1, Aguilera Selene 1, Hernández-Morales, Alejandro, Alvarez-Morales, Ariel 1 1 Departamento de Ingeniería Genética de Plantas. CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, carretera Irapuato-León. Irapuato, Gto. México. C. P Tel.: +52 (462) ext. 439, Pseudomonas syringaepv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway produced at low temperatures (18-20 C). Phaseolotoxin [N -(N -sulphodiaminophosphinyl)- ornithyl-alanyl-homoarginine] biosynthesis and regulation have not been determined, although it has been demonstrated that genes withing the Pht cluster are required for phaseolotoxin production. To achieve full pathogenicity and virulence, the bacterium expresses numerous pathogenicity and virulence factors that lead to a successful invasion and infection. Signal transduction pathways coupling the sensing of an environmental signal to modulation of the expression of the relevant target genes frequently involve the use of two-component systems, consisting of sensor histidine kinases coupled to their respective response regulators to modulate de expression of transcriptional factors that activate functionally-related genes. Among these, the GacS/GacA two-component system, which is widespread in Pseudomonas spp. and other Gram-negative bacteria, plays a major role in the regulation of important pathogenicity and virulence factors. However, it was previously reported that GacS did not exert control over phaseolotoxin production. In the present study we hybridized a genomic microarray of P. syringaepv. phaseolicola NPS3121 to compare transcriptional profiles from wild type strain and a gaca - null mutant with a Tox - phenotype. Our results show that GacA controls expression of genes within the Pht cluster as well as another group of clustered genes located in a different region in the bacterial chromosome and it contains at least one gene that we unambiguously showed to be directly involved in phaseolotoxin biosynthesis. Our results suggest that this cluster is a new pathogenicity island containing genes whose regulation is also under the GacA regulatory cascade. 36

41 Oral Session II. Tuesday Structural, phylogenetic and functional analysis of the isocitrate lyase from Pseudomonas aeruginosa Díaz-Pérez 1, A. L., C. Díaz-Pérez 1, C. R. Sosa-Aguirre 1, and J. Campos-García 1 * 1 Instituto de Investigaciones Químico-Biológicas, UMSNH. Edificio B-3, Ciudad Universitaria, CP 58030, Morelia, Mich., México. Isocitrate lyase (ICL) is an enzyme that plays an essential role in Pseudomonas aeruginosa when grows on fatty acids, acetate, acyclic terpenes and amino acids, such as leucine, channelizing their final metabolites to the glyoxylate pathway [1]. In P. aeruginosa, ICL is encoded by the acea gene (ORF 2634) [1]. In the present work we carried out the molecular modeling, phylogeny and functional analysis by site directed mutagenesis of P. aeruginosa ICL. The phylogenetic analysis suggests that ICL superfamily is divided in two families, the AceA (constituted for 5 subfamilies) and the PrpB ICLs. The ICL from P. aeruginosa belongs to subfamily 3, showing the characteristic Q IE N Q V X DE K Q CGH QD GK fingerprint. Molecular modeling of ICL shows two particular domains, named D3 and D4, which are missing in the subfamily 1, subfamily 2 and PrpB s. Alignment of fingerprint and active site showed that Q211, N214 and Q221 were unique for subfamily 3. ICL from P. aeruginosa was expressed as His-tag recombinant protein showing a molecular mass of 68 kda. The recombinant ICL at 30 o C and ph 7.5 as optimal conditions shows a Michaelis-Menten behavior with kinetic constants of V max of 29 mmolmin -1 mg -1 and K m of 0.82 mm for isocitrate. Site directed mutagenesis for residues Q211H, N214D, E219A, and Q221K, conserved in the ICL subfamily fingerprint, indicated that N214 is essential for enzymatic activity at least in P. aeruginosa. E219A not shows effect, while the change Q221K caused an increment in ICL substrate affinity. Mutations in Q211H and Q221K caused shift in optimal temperatures toward the thermofilic region, which was implicated with a reduced thermostability of ICL at 45 o C, suggesting that these residues are implicated in the thermodynamic properties of ICL. 37

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