Bacterial Characterization of Sourdough from a Local Factory

Motivation: Sourdough is the fermentation product of flour and water dough by yeasts and lactic acid bacteria (LAB). Depending on the origin of the microorganisms it can be classified in Type I (spontaneous and with backslopping), Type II (uses a starter culture without backslopping) and Type III (uses a starter culture and backslopping)1. Sourdough-based products have improved properties, which have been attributed to the LAB and their metabolism 2. This study is focused on the characterization of LAB in an industrial sourdough at two different moments. Methods: To isolate LAB from sourdough, independent samples were taken from a homogenized portion. Appropriate dilutions were plated on mMRS agar and grown on aerobiosis at 30ºC. Cell concentration was calculated as CFU/g of sourdough.  Morphologies of Catalase negative isolates were determined by optic microscopy. For molecular identification, DNA was extracted by a method described by Cold Spring Harbour Laboratory3. Fragments from rRNA 16S gene were amplified by PCR and sent for sequencing. Sequences were compared with databases using the BLASTn tool4. Non-pathogen S. aureus and L. innocua were used as indicator strains to detect antimicrobial capacity on BHI medium. Clear zones around colonies were rated as positive results. Results: LAB were present in the two sourdough samples at 2.75*10^7 CFU/g and 4.3*10^7 CFU/g. The dominant morphologies were long, medium and short bacilli. They presented percentages of 41.5%, 18.9% and 39.6% in the first sample and 13.5%, 30.8% and 55.8% in the second, respectively. The dominant species were Lactobacillus crustorum (corresponding to long bacilli), Lb. rossiae (short bacilli) and Lb. plantarum (medium bacilli). Antagonistic activity was detected only against S. aureus just in 5% of candidates from the second sample. Conclusions: LABs present in two sourdough sample´s from the same factory have been characterized. Cell concentrations were similar to that described in the bibliography. Dominant species identified were Lb. crustorum, Lb. rossiae and Lb. plantarum. However, their proportion was different in the two sourdough samples. Antagonistic activity against S. aureus was detected in the second sampl

A new element involved in the regulation of tetralin degradation genes in Sphingopyxis granuli strain TFA

Motivation: Sphingopyxis granuli strain TFA is a gram-negative bacterium able to grow on the organic solvent tetralin as the sole carbon and energy source. Tetralin is a bicyclic molecule, composed of an aromatic and an alicyclic moiety, which is toxic to bacterial cells as it makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes (Sikkema et al., 1992). In our lab, the metabolic pathway and the specific regulation of genes involved on tetralin degradation (thn genes) has been deeply characterized (López-Sánchez et al., 2010 and references therein, Rivas-Marín et al., 2016 and references therein). Regarding the regulation, it is known that structural thn genes are induced in the presence of tetralin by ThnR, a LysR-like transcriptional regulator, and ThnY, a ThnR co-activator. Besides, the expression of thn genes is under carbon catabolite repression (CCR) by preferential carbon sources, such as β-hydroxybutyrate (β-HB) or sebacic acid. However, not very much is known about the regulatory elements involved in this repression. Synthesis of the carbon storage granule PHB is indirectly involved in CCR on thn genes.Methods: Comparison of the global gene expression in tetralin- and β-HB-grown cells revealed the presence of a small non-coding RNA (sRNA), annotated by Infernal Software 1.1, preferentially expressed in β-HB. Northern Blot analysis and β-galactosidase assays of a chromosomally integrated suhB::lacZ transcriptional fusion confirmed the differential expression of this sRNA. Expression of thn genes under CCR conditions in a mutant lacking the sRNA was evaluated using a chromosomally integrated thnC::lacZ translational fusion. Furthermore, putative targets of the sRNA were detected in vitro by IntaRNA software and the predicted interaction was experimentally validated by RNA-RNA EMSA.Results: A differentially expressed sRNA has been identified in TFA as belonging to the Rfam family RF00519 (SuhB) (García-Romero et al., 2016). It is a highly conserved sRNA in α-proteobacteria. Under CCR conditions, thn genes are partially de-repressed in a mutant lacking SuhB. Furthermore, the 5' UTR of thnR mRNA has been identified in silico as a target of SuhB. Direct interaction of SuhB at the thnR ribosomal binding site has been demonstrated. The high level of ThnR in the SuhB mutant indicates a negative role of SuhB on ThnR translation.Conclusions: The available data so far indicate that SuhB is one of the elements involved in CCR of thn genes in Sphingopyxis granuli strain TFA, by blocking the translation of the regulator ThnR.    

The role of the transcriptional regulator, Tex, under stress conditions in Lactococcus lactis

Motivation:Tex, originally described in Bordetella pertussis, is a regulator involved in a variety of transcriptional processes and conserved in a wide range of bacteria. However, very little is known about its function in Lactococcus lactis. Preliminary proteomic data have shown that Tex is more abundant in a L. lactis ΔftsH mutant (ftsH knock-out) under cell wall stress. FtsH is a membrane protease involved in regulation assuring cellular protein stability under stress conditions. Moreover, based on previous knowledge, L. lactis ΔftsH presents some distinct phenotypes such as lysozyme resistance and NaCl sensitivity. A link is proposed between those phenotypes and the high level of Tex, which might be due to a higher half-life of this protein, as a consequence of the lack of FtsH protease. In order to get insights on the role of Tex in L. lactis, this work tries to answer two main questions: (i) Is the abundance of Tex regulated by FtsH? and (ii) are the higher levels of Tex causing the resistance and sensitivityMethods:The gene coding for Tex was amplified by the polymerase chain reaction (PCR), with specific primers designed to clone it under both, a constitutive promoter P32 in pMG36e and the nisin inducible promotor Pnis in pNZ8020. Growth of cells with the empty plasmid (pNZ8020) or overexpressing Tex (plasmid pBL95) in GM17Cm medium was measured by OD at 600 nm in a microplate reader. The L. lactis tex knockout is currently being constructed, by cloning a truncated version of tex in pGhost9, a thermo-sensitive plasmid. Experiments to estimate Tex half-life will be carried out using antigen-antibody tagging in the presence or absence of FtsH.Results: It has not been possible to clone tex gene under the constitutive P32 promoter. L. lactis cells carrying pBL95 (tex gene under Pnis) growing in GM17Cm medium show a delayed growth related to the nisin concentration. The higher the concentration used, the slower the growth rate indicating that Tex overexpression might have a deleterious effect. Regarding to the lysozyme resistance and NaCl sensitivity, Tex abundance in ΔftsH might be the cause of those characteristics and experiments are in progress to probe it.Conclusions:Tex functions remain unknown in Lactococcus lactis but constitutive expression and/or too high levels of Tex seem to be deleterious. Further studies are necessary to analyse its role in the generation of new phenotypes and its regulation by FtsH for a better understanding

Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB

This is an open access article under the terms of the Creative Commons Attribution License.Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes.Work in the authors laboratory was supported by the Spanish Ministry of Economy and Competitivity, grants BIO2011-24003 and CSD2007-00005, and by the Andalusian Government, grants P05-CVI-131 and P07-CVI-2518.Peer Reviewe

Ensamblaje del genoma de Sphingopyxis macrogoltabida estirpe TFA y análisis de la anotación funcional

Motivación: El conocimiento del genoma de Sphingopyxis macrogoltabida estirpe TFA (bacteria de interés en el campo de la biodegradación) es el primer paso para el conocimiento de su mapa metabólico, lo que permitirá establecer las rutas importantes para la biodegradación de contaminantes. Debido a que el resultado de la secuenciación masiva es un genoma fragmentado, es necesario su ensamblaje y anotación como paso previo a la predicción del mapa metabólico. Métodos: La secuenciación del genoma, el ensamblaje y la anotación inicial se han llevado a cabo por la empresa "LifeSequencing". Una anotación adicional que identifica secuencias truncadas se realizó por la empresa "Era7". Para ordenar los contigs (fragmentos), se han utilizado hipótesis basadas en el análisis de los extremos mediante BlastX y/o BlastN y la comparación con bacterias filogenéticamente cercanas, como Sphingopyxis alaskensis. La confirmación del orden y el cierre de gaps (huecos) se ha realizado mediante PCR y secuenciación. Una vez agotadas las hipótesis de unión, se han probado PCRs para todas las combinaciones de unión posibles y buscado los extremos contiguos en cósmidos de una genoteca de TFA, mediante Southern Blot. La anotación funcional ha sido organizada a través del software Pathway-Tools y completada con nuevos genes encontrados en las regiones intergénicas gracias a la herramienta GeneMark. Resultados: El número de contigs se ha reducido de 42 a 5 y los gaps entre ellos han sido secuenciados, obteniendo un genoma parcialmente ensamblado. Se han anotado un total de 115 nuevos genes en las regiones intergénicas y el mapa metabólico en Pathway-Tools se ha completado con la ruta de degradación de tetralina, la cual no fue predicha inicialmente. Como era de esperar, se han detectado pocas reacciones de transporte, lo cual es característico de un organismo oligotrofo como TFA, y un solo operón ribosómico, propio de bacterias de crecimiento lento. Se han identificado genes de plásmidos dispersos en el genoma pero ninguna región que indique la presencia de  un plásmido integrado. Además, a pesar de encontrar genes de fagos en la anotación, no se han detectado profagos en el genoma. Conclusiones: La secuenciación del genoma y su anotación han permitido obtener una visión global de la distribución génica y el mapa metabólico de TFA, determinando sus características más relevantes. Se está trabajando en la identificación de genes codificantes de pequeños RNA para completar la anotación

Caracterización de la proteína Hfq en la bacteria TFA

Las bacterias detectan diferentes señales procedentes del entorno y responden a ellas específicamente variando sus perfiles de expresión génica con el objetivo de adaptarse mejor a los cambios ambientales. Dichos mecanismos de regulación de la expresión génica pueden operar tanto a nivel transcripcional como a nivel post-transcripcional y responden a señales específicas y/o globales para asegurar una correcta expresión génica. Esta regulación post-transcripcional de la expresión génica puede estar mediada por sRNAs (smallRNAs en inglés). Estos sRNAs tienen un papel crucial en el control de las funciones celulares tales como, por ejemplo, el metabolismo central o la virulencia en algunas bacterias patógenas (1).Se ha descrito que la proteína Hfq juega un papel esencial en la actividad de algunos de estos sRNAs con los que interacciona en numerosas bacterias (1, 2). Hfq fue descrita por primera vez en la bacteria E. coli como un factor esencial para la replicación del fago Qβ, un virus de ARN (3). Hfq se engloba dentro de la familia de proteínas Sm-Sm_like que se caracteriza por tener una estructura cuaternaria en forma de anillo hexamérico que le permite la interacción con otras macromoléculas, como serían los sRNAs y los ARN mensajeros diana de los anteriores (1, 2). La bacteria Sphingopyxis macrogolitabida estirpe TFA es una alfa-proteobacteria perteneciente a la familia Sphingomonadaceae capaz de degradar el contaminante ambiental tetralina y utilizarlo como fuente de carbono y energía (4). En dicha bacteria, objeto de estudio en este trabajo, se ha detectado un gen que codifica para una proteína reconocible como hfq pero con una estructura primaria diferente a las descritas hasta el momento en otras bacterias. estas diferencias encontradas a nivel estructural en la proteína junto al hecho de que no haya estudios previos sobre esta proteína en esta familia de bacterias hacen muy interesante su caracterización con objeto de determinar si su mecanismo de actuación en la célula es diferente o no al descrito previamente en otras bacterias

Tuning and validation of an analytical method for the determination of gluten in food samples: The immunochromatographic strips

Motivation: Gluten is a food allergen present in many cereals it is composed for complex mixture of proteins, mainly prolamines and glutenines. (Biesiekierski, J.R, 2017) Prolamins contain immunogenic peptides that are resistant to gastrointestinal digestion, such as peptide 33-mer, and trigger adverse reactions in people with hypersensitivity, causing from the mild allergic reactions to the well-known, celiac disease. Currently, gluten hypersensitivity is one of the most spread digestive disorders in the world. (Ozuna, C., et al, 2016) That's why there is a need to detected foods that contain gluten to provide this information to consumers to their own safety.There are many different techniques to detection gluten very effectively like Enzyme-Linked ImmunoSorbent Assay (ELISA). However, the present work aims to carry out the validation and tuning of a new method faster and simpler: immunochromatographic strips. This is ultimately intended to accredit the method so that the analytical laboratory can offer its customers reliable and reproducible results. Accreditation is "the internationally established tool to build confidence in the proper execution of a certain type of activity." The National Accreditation Entity is responsible for accrediting an analytical method under Regulation (EC) Nº765/2008.Methods: These strips are based on the detection of inmmunogenic peptides. On the support of the strip is a nitrocellulose membrane that containing prefixed antigydine antibodies, which will be colored if the sample analyzed has gliadin. This technique is usually a qualitative metod, unlike the previous ones that is quantitative, however, it's intended to include a strip reader to obtain quantitative values.In this study, different test has had to be carried out to analyse a number of parameters that need to be studied in validation. The parameters studied have been: detection limit, quantification limit, accuracy, precision and robustness.Results:It can be said that this method is able to unequivocally detect the presence of gluten in the sample, as well as provide values of it, however this quantification is not as accurate as other current detection techniques would be.Conslusions:It’s a reliable method that could be competent in the market for a given sector in which it's only interested to know the presence or not of gluten, however there is still a lack of development in terms of the accuracy of the quantification

The HIRES-SOM Project: Soil organic matter and microbial communities in volcanic materials from La Palma Island assessed by high-resolution techniques: implications for pedogenesis and sustainability

Comunicación oral presentada en el 1st European Meeting on Geomicrobiology of volcanic caves. días 2-3 de marzo de 2023 celebrado en la Casa de la Ciencia-CSIC de SevillaThe HIRES-SOM project is a multidisciplinary project funded by the Ministry of Science and Innovation of Spain, aimed at generating knowledge on soil organic matter (SOM) and microbiota to guide novel strategies for the sustainable management of volcanic soils. The investigation will be conducted within the framework of the recent Tajogaite eruption in La Palma Island, which constitutes an optimal benchmark both for scientific research on freshly erupted materials and for the management of affected soils of high added value. Insights into the generation and resilience of SOM and of the associated microbiota will be explored with advanced biogeochemical, microbiological, mineralogy and modelling techniques [1,2]. The main goals of the project are twofold. On the one hand, it intends to assess the state and evolution of the SOM and microbiota in soils calcined by the lava flows and covered by the pyroclastic ashes. On the other hand, fundamental understanding will be sought about the initial stages of pedogenesis and consolidation of organic matter on the eruptive materials. The project intends to identify primary microbial communities and molecular precursors of organic matter in volcanic ash, lava flows and eruption-affected soils. Moreover, accelerated microbial colonization of volcanic substrates under laboratory conditions will allow monitoring photoautotrophic microorganisms that colonize natural volcanic materials, with the ultimate innovative goal of devising methods for the rapid fertilization of porous pyroclastic ashes by indigenous microorganisms for sustainable soil regeneration. References: [1] Martinez-Haya et al, Energy Fuels 35 (2021) 8699; Talanta 185 (2018) 299; Phys. Chem. Chem. Phys. 22 (2020) 19725. [2] Miller A., et al, Sci Total Environ 426 (2012) 1; Geomicrobiol. J. 31 (2014) 236; Sedimentology 65 (2018) 1482; Sci Total Environ 698 (2020) 134321; Coatings 10 (2020) 1134.The HIRES-SOM project is funded by the Ecological and Digital Transition Programme of the Ministry of Science and Innovation of Spain (grants TED2021-130683B-C21/C22).N

Microbiología Aplicada

Guía docente de la asignatura Microbiología Aplicada.Peer reviewe

El discurso antiterrorista oficial de China como herramienta de legitimación

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Filosofía y Letras, Departamento de Linguística, Lenguas Modernas, Lógica y Fª de la Ciencia y Tª de la Literatura y Literatura Comparada. Fecha de Lectura: 20-12-2022Given the transnational nature of the fourth wave of terrorism and its relationship with religious extremism, the United Nations has proposed since 2006 to strengthen efforts and international consensus to prevent violent extremism and international terrorism. The expansion of China's influence, as well as its increasingly evident implication in non-traditional global threats, raised questions regarding the possible impact of its measures against international terrorism. Therefore, In order to optimize the possibilities and efficiency of international cooperation in this matter, it is crucial to gain a better understanding of China's interpretation and reaction to the threat within its local context, and in accordance with its national priorities. The representation of terrorism in China is framed within a three-dimensional threat called the "Three Evil Forces" (composed of terrorism, separatism and extremism). In accordance with China's conceptualization and reaction to the threat, Critical Terrorism Studies is applied as a theoretical framework to explore its official discourse on terrorism as a legitimizing tool for the Communist Party of China (CPC), both at the national and international levels. In this regard, the conceptual framework responds to a study of the evolution of the main sources of Party legitimacy since the leadership of Mao Zedong. Starting from the theoretical knowledge gathered through the previous literature review, the Critical Terrorism Studies paradigm, and studies on legitimacy in China, the research proceeds with a holistic analysis of the official discourse. Through a synergistic quantitative (Corpus Linguistics) and qualitative (Critical Discourse Analysis) methodology, this thesis presents the main results of a detailed examination of two main sources: the official reports of the National Congresses of the Communist Party of China between 1992 and 2017; and the Xinjiang White Papers between 2002 and 2019. Overall, this research ultimately aims to provide a comprehensive analysis of China's official discourse on terrorism and its direct relationship to maintaining and promoting Party legitimacy as a top priorit