BACTERIAL PHYSIOLOGY

Author: MALLORQUÍ FERNÁNDEZ NOEMÍ.
Year: 2003.
University: GIRONA [www.udg.es].
Place of defense: CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS DE LA UDG.

Summary: This paper focuses on the study paartir different levels of carotenoids species of brown Greens Sulfur Bacteria (GSB, the English Green Sulfur Bacteria). The overall objective has been to find out what the function of these pigments enestos microorganisms as well as broaden the knowledge of its structure and entrcciones withthe otres pigments of the photosynthetic apparatus. It first designed a method cromotografía liquid high rasolución (HPLC) for anilizar to more accurately and quickly carotenides of different strains of GSB (Chapter 3). This method is characterized by a purificaciín precvia of extracts pigment through columns of alumina to remove cateroclorofilas (BLLS). This permitó analyzed with a high resolution and etan only 45 minutes. Of the various chromatographic career carotenoids and their precursors, as well as configuaraciones trans and cis their isómeros.El second genetic methods used consisted of a modification of the method of Borrego and Garcia-Gil (1994) and allow the separation requires all kinds of pigments, both from culrivos pure as sample complexity. A concrete example was the ancient sediments in the area of Lake Banyoles. In these sediments (0,7-1,5 million years antigà ¼ age) detectron, among other pigment, carotenoids specific species of brown GSB which confirmed the presence deestas bacteria in the lake area in Banyoles ya des Pleistocene lower. This first chapter also analyzed the carotenoid of Cholrobium (Chl.) phaeobacteroides CL 1401 through liquid chromatography coupled to spectrometry masaa (LC-MS/MS), in order to confirm its identity and its molecular weight. We also analyzed the effect of temperature, light and various oxidizing and reducing agents on the xomposición quantitative and calitativa of carotnoides and BCLS this especie.Estos experiments permitieroon confirm the photosensitive delas BCLS and trans-isomers / cis of various carotenoids are not artifacts produced during handling of the samples but are constituents of the photosynthetic apparatus of these microorganisms. Chapter 4 includes the physiology experiments performed with some species of GSB, focused on elucidating the mechanisms in dynamic synthesis of the different pigments (BCL antenna, BCL ai carotenoids) during the growth of these species. This research also helped monitor variations in the number of CR during the adaptation process luminance. From the cuantificaciín of BCL663, the acceptor first in the electron transport chain in GSB, we calculated the number of RC. The estimate of the relationship RC / clorosoma held, the first from data estequiométricos and biometric present at the bibligrafía and then from the experimental data of this work. The vuen adjustment between different estimates gave strength to estequiométrico calculated value, which averaged about 70 CR by clorosoma. In this chapter of physiology also studied variriones in relations trans / cis among major carotenoids species of brown GSB.Las relations calculated for culrivos not resentaron differences highlighted in terms of lighting conditions of these. In the two conditions studied, high and low light intensity, the value of the relationship trans / cis was aproxomadamente, 2. In clorosomas isolated from different cepar brown GSB also the relationship was approximately 2, both for the isorenierateno (Isr) and the B-isore 8 niearate 5cc not (B-Isr). In natural populations of Chl. Pheobacteroides this relationship was also 2 trans-isomers cis isomer for each, keeping both in depth and constant over time. Finally, in Chapter 5 presents a molecular marker that allowed the identification of specific species of brown GSB. Despite his initial design was based on a gene involved in the synthesis of carotenoids (crtY, which encodes a pair licopeno cyclase) the final sequence from which we have obtained the encebadores selective is ralcionada with the family of protínas of the Policétido-ceto-sintasas (PKT). Nevertheless, the new tool can be very useful for discriminating species brown GSB respect to the green in mixed populations like those found in natural environments and opens the door to future microbial ecology experiments using techniques such as real-time PCR, which would allow selective monitoring of polblaciones species brown GSB in ecositemas natural.

Author: AZUA PEREZ IÑIGO.
Year: 2004.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: The marine aggregates, ubiquitous and of different origin, are enriched in organic matter and heavily colonized by microorganisms. They removed the means by physical disintegration, differential sedimentation and biological decomposition, and participating in the aftermarket, and decomposition of organic matter sedimentation in the sea. The decomposition procariótica includes breathing, biomass production and / or solubilisation aggregate. Both habitats, and added liquid phase, presented two communities procarióticas, living free and pasted with different morphological, physiological and taxonomic. The objective of this paper is to analyze the organic decomposition procariótica of marine aggregates. Consideration is being pinned in prokaryotes and living free primary hydrolytic attack, the incorporation of organic matter released and biomass production. It discusses three types of aggregates: aggregates oligotrophic marine waters and two types of aggregates formed in the laboratory, aggregates and natural fitoplanctónicos. The aggregate fitoplanctónicos, with abundant compounds readily usable, to simulate formed during the massive growth fitoplanctónico. The simulated natural to gregados impoverished and more refractory formed by aggregating different marine particles. Both prokaryotic differ both in the hydrolysis in the making of organic compounds. The free-living expresses enzymes hidrolíticos and transportation systems with greater affinity due to the scarcity of nutrients. These differences explain the kinetics multiphasic observed in marine systems. In aggregate fitoplanctónicos the procariota get advantage of its superior adhesion through hydrolysis, while natural aggregates, poorest, there are no differences between prokaryotes. In the procariota acceded oligotrophic waters presents top takes only little particles settled and presumably newly formed. In aggregate fitoplanctónicos and natural, there is an adaptive strategy, decline in the uptake and increase in the affinity, as it increases the degree of colonization and impoverish quantitatively and qualitatively aggregates. Aggregates are centers of high activity and strong growth prokaryotic only recently formed. As we are colonized and degraded chemical changes occur and cease to be a favorable habitat. In aggregate fitoplanctónicos newly formed by hydrolysis and making processes are coupled. However, during its decomposition is decoupled by decreasing making while remaining high hydrolysis. Due to desacomplamiento is solubilizan organic compounds in the liquid phase fostering the growth of community life free. Thus, marine aggregates act as enzyme reactors that release organic matter during his deposition, which would help explain the growth rates reaching the prokaryotic life free.

Author: JIMÉNEZ BELENGUER ANA ISABEL.
Year: 2004.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Dep. Biotecnologia.
Place of preparation: Universidad Politécnica de Valencia.

Summary: Gender Helicobacter includes 23 species of bacteria microaerófilas. The most studied is Helicobacter pylori, which is responsible for 90% of all peptic ulcers and closely linked with the development of gastric cancer. The mechanisms of transmission of this organism are not entirely certain. Their presence has been detected in the faeces, drinking water, milk and vegetables, but in very few cases has been isolated. It has recently been suggested, moreover, that Helicobacter pylori may be resistant to chlorine and ozone concentrations used for potabilizar water distribution. Furthermore, experimental studies have shown that in terms of environmental stress these microorganisms can enter a phase feasible but not arable (VNC), which, despite not being able to be recovered, preserved various metabolic activities and express virulence factors . The induction of this state VNC, which is accompanied by morphological changes (move to form cocoide) has been proposed as a possible mechanism for survival in the environment, which would encourage its transmission. This paper has studied the survival of a strain of H. Pylori belonging to our collection, in different models at different temperatures aquatic microcosm. It has been found that the cells of H. Pylori kept in phosphate buffer saline PBS to 11Â ° C, are cultivated for 12 days, while in non-chlorinated water at the same temperature, arable remain until 5 days. Also, at lower temperatures the cultivabilidad in PBS persists longer, and cultivable cells are detected until the 22 days. We also noted the existence of helical viable non-arable both surface water and in PBS. In evaluating the survival of the strain HBTC6 H. Pylori in the presence of chlorine has been that while his cultivabilidad is extremely short, just 5 minutes, persist helical forms and cocoides to 24h. Exposure. Throughout the study, all microcosm remained constant various parameters, such as rRNA and DNA content, as well as the production of urease and catalase, enzymes associated with virulence. These results demonstrate the continued presence of viable cells non-arable (VNC) of H. pylori in aquatic systems over long periods. One of the biggest concerns is whether these forms are potentially infective. This involves determining the ability of these cells to be cultivated again. The pursuit of growth media that can retrieve this microorrganismo your state VNC is also crucial to their isolation from environmental samples. In this work we have tested 15 growth media, in order to determine whether H. pylori cells in a state VNC recover their ability to replication. Although it could not be recovered by culture plate, the results show the persistence of viable forms in all of them, even 14 days after its incubation, which indca maintaining a specific metabolic potential.

Author: Hernández Romero Diana.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología. Universidad de Murcia.
Place of preparation: Facultad de Biología.

Summary: The polyphenol oxidasas (PPO) are enzymes that oxidized phenols using oxygen. They are divided into lacasas and tirosinasas and enzymes are of great interest both biotechnology (for use in the degradation products recalcitrant, in the paper industry, the dyes and pigments, etc.) for their involvement in the biosynthesis melaninas that they are generally black or brown pigments involved in many roles, most of them for protection. Have been found in the genome of Ralstonia solanacearum up to four potential genes that may encode enzymes with PPO activity. Among the objectives of the Doctoral Thesis highlights the detection and optimization of these activities PPO in R. Solanacearum, mutagenesis of potential genes responsible for the activities and the purification and characterization of enzymes with PPO activity. Another goal will be based on an evaluation of the effects of the addition of phenols in the physiology of R. Solanacearum as well as the potential impact on the infectivity of the mutants compared with the wild strain. Through the construction of null mutants through homologous recombination we have the mutant gene involved in the various activities that could encode PPO. Once obtained cellular extracts of these mutants look at the loss of a PPO specific activity associated with each of the mutated genes, which however did not agree with the prior endorsement of these gene products so we are proposing a change in the nomenclature of themselves, so that the product of the gene RSc0337 is a tirosinasa, gene RSc1501 is a catechol and oxidase gene RSp1530 encodes a laccase. It dismissed the study of gene RSp0656 by encode a protein of resistance to copper CopA and therefore not encode a PPO. It got purification products protéicos of these genes, as well as a biochemical and kinetic characterization of the same observed in the case of tirosinasa greater affinity for tyrosine that dopa, which led to the development of a patent by our group on the process of obtaining L-dopa using tirosinasa R. Solanacearum. Following the work of the characterization of these enzymes, we can establish a set of criteria that allow structural differentiate the tirosinasas of catechol oxidasas depending on the characteristics of resíduos aminoacídicos close to the centers of copper. Another important result is observed when adicionamos phenolic compounds in crops R. Solanacearum, where we see a greater sensitivity to them by mutants affected in the PPO, which suggests an involvement of these processes in detoxification of these compounds, processes that could have a physiological explanation in defending the bacterium front the attack on the plant that produces these compounds as a response to different stress, such as infection by a pathogen. Additionally, we have also been able to observe the synthesis of melaninas and other pigments associated with the activities PPO in R. Solanacearum. Finally we were unable to demonstrate a loss of infectivity associated with the lack of any concrete PPO, which in principle could be explained by the fact that the infectious process is a multifactorial process where the affectation of a PPO could not ocasonar effect as phenotypically unclear as to be visible on a qualitative test for pathogenicity.

Author: URETA BARRIOS ANA CLAUDIA.
Year: 2005.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRONOMOS.
Place of preparation: ESCUELA TECNICA SUPERIOR DE INGENIEROS AGONOMOS.

Summary: The symbiosis Rhizobium-leguminosas there are two key enzymes involved in the metabolism of molecular hydrogen (H2): nitrogesanas and hydrogenase. The nitrogenasa catalyzes the reduction of atmospheric nitrogen to ammonia and this process involves the production of hydrogen. The hydrogen gas is lost as it is a waste of energy in the process of nitrogen fixation. The hidrogenasas catalyzes the oxidation of hydrogen molecular protenes. Electrons from the oxidation of hydrogen entering the respiratory chain until final acceptor is oxygen, and this process can be coupled to the production of energy (Ruíz-Argà ¼ So et al, 2000). The recycling of hydrogen has been associated with an increase in plant productivity due to increased energy efficiency in the symbiosis Rhizobium-leguminosas. The hydrogenase is an enzyme heterodimérica that this coupled with the membrane and contains an active bimetálico [NiFe] in the largest subunit. The incorporation of nickel in the heart of the hydrogenase active is an essential step in the synthesis of the enzyme. It has been established that the availability of nickel limits the expression of the hydrogenase-level processing subunit of the enzyme. It has been established that the availability of nickel limits the expression of the hydrogenase-level processing subunit of the enzyme in plants grown in vermiculite (Brito et al, 1994), but it is unknown whether this limitation exists under natural conditions of cultivation. The objectives of this thesis are: 1-To analyze the level of nickel soil as a possible limiting factor in the expression dela hydrogenase activity in agricultural soils for this study we explored the effect of the addition of nickel in the soil. We selected 6 agricultural soils representative of the Community of Madrid which identified their physicochemical characteristics. These soils were used as a substrate for the growth of pea (Pisum sativum) inoculated with a strain hydrogenase positive R.leguminosarum. The analysis of hydrogen metabolism in bacteroides of these plants showed that neither present in these soils is not sufficient to recycle 100% of the hydrogen produced in the process of nitrogen fixation. As a strategy for overcoming the limitation nickel was used by a strain more efficient recycling of hydrogen. These results were correlated with tests inmunodetección where we explored the effect of nickel in the prosecution of the largest subunit of the hydrogenase and saw that the processing enzyme depends on the levels of nickel in the soil. The results indicate that low levels of nickel from the soil are a limiting factor for the hydrogenase activity in the symbiosis R.leguminosarum-guisantes. 2-To assess the benefits associated with the presence of hydrogenase in inoculators rizobaianos. To assess the benefits associated with the presence of hydrogenase in inoculators rizobianos, an integrated system hup of Rhizobium leguminosarum bv viciae strains hydrogenase negative noduladoras of time (Vicia sativa) using a minitransposón (TnHB100, Báscones et al, 2000 ). This system has made it possible to integrate into a stable grouping hup into the genome of the bacterium receiving. We selected two strains with inserts located in sites irrelevant genome and analyzed the hydrogenase activity and behavior symbiotic. One of the strains showed significant increases in accumulation of nitrogen associated presence of the recycling capacity of hydrogen. 3-To conduct a comparative analysis of genomes of strains of Rhizobium leguminosarum capable of expressing the system hydrogenase. The comparison was conducted genotypic strains R.leguminosarum UPM791, holder of the system hup and 3841, whose genome has been sequenced, using 8 microarr 426 ays DNA generated for the strain 3841. Prior to the comparative study verified that the system hup is expressed correctly in the strain 3841. The comparison showed that 78% of genes were conserved in both strains, indicating that these microaarays are useful for the analysis of the factors involved in controlling the expression of the hydrogenase of UPM791.