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

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

Harnessing the power of microbial metabolism

Microorganisms are rich repositories of genetic material encoding many activities of potential interest. Recent advances make identifying and exploiting the metabolic treasures of uncultured microbes an easier proposition. Improved expression vectors and metagenomic screening techniques make it easier to identify activities of interest. Synthetic biology and efficient genome editing techniques allow microbial genomes to be modified almost without restriction. Computational approaches based on organism-wide analysis of transcription, protein synthesis and metabolic fluxes make it possible to accurately predict the outcome of the metabolic processes and modifications required for optimization. Together these advances represent a major breakthrough in microbial biotechnology that is expected to yield new generations of tailor-made biocatalysts suitable for multiple biotechnological applications.This work was supported by grants BIO2013-42073-P and BIO2014-57545-R from the Spanish Ministerio de Ciencia e Innovación.Peer Reviewe

Involvement of the small RNA suhb on carbon catbolite repression of tetralin degradation genes in Sphingopyxis granuli strain TFA

Resumen del póster presentado al 7th Congress of European Microbiologists, celebrado en Valencia (España) del 9 al 13 de julio de 2017.[Backgrounds]: Sphingopyxis granuli strain TFA is able to use the organic solvent tetralin as the sole carbon and energy source. 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 a preferential carbon source, such as β-hydroxybutyrate (β-HB). [Objectives]: The main objective is to stablish the role of a small RNA (sRNA) in thn genes expression control under CCR conditions. [Methods]: Comparison of the global gene expression in tetralin- and β-HB-grown cells revealed the presence of a sRNA expressed preferentially in β-HB, which was annotated by Infernal software 1.1. Northern Blot and β-galactosidase assays confirmed the differential expression of this sRNA. The effect on CCR over thn genes of mutant lacking the sRNA was evaluated. Furthermore, its putative targets were detected by IntaRNA software and the interaction validated by RNA-RNA EMSA. [Conclusions]: The sRNA is predicted as belonging to the family RF00519 (SuhB), which is highly conserved in α-proteobacteria. The thn genes are partially de-repressed in a mutant lacking SuhB under CCR conditions. Furthermore, the 5' UTR of thnR mRNA is identified as a target of SuhB in which would block the ribosomal binding site. Accordingly, ThnR levels are higher in the mutant strain. The available data so far indicate that SuhB is involved in CCR of thn genes.Peer Reviewe

Involvement of a putative cyclic AMP receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB

Glucose catabolite repression of tetralin catabolic genes in Rhodococcus sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the thnA1 and thnS genes.Work in our laboratory was supported by the Spanish Ministry of Science and Innovation, grants BIO2008-01805 and CSD2007-00005, and by the Andalusian government, grants P05-CVI-131 and P07-CVI-2518.Peer reviewe

Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters

Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.This work was supported by the Spanish Ministry of Economy and Competitivity, Grants BIO2011-24003 and BIO2014-57545-R.Peer Reviewe

Implicación del pequeño ARN SuhB en la represión catabólica de los genes de degradación de tetralina en Sphingopyxis granuli estirpe TFA

Póster presentado a la XI Reunión del Grupo de Microbiología Molecular, celebrada en Sevilla del 6 al 8 de septiembre de 2016.Sphingopyxis granuli estirpe TFA es una bacteria Gram-negativa de la familia de las α- proteobacterias capaz de utilizar el solvente orgánico tetralina como única fuente de carbono y energía. Los genes estructurales (genes thn) se organizan en dos operones divergentes (B y C) que se inducen en presencia de tetralina. Formando parte del operón C se encuentran los genes reguladores thnR, que codifica un regulador tipo LysR, y thnY, que codifica un co-activador de ThnR. En ausencia de tetralina, thnRY tienen una expresión basal desde un promotor interno constitutivo. Además de la inducción en presencia de tetralina, la expresión de los genes thn está sujeta a represión catabólica por fuente de carbono preferencial (RCC). El mecanismo de activación específica se ha estudiado en profundidad, si bien aún no se conocen los elementos implicados en la represión de los genes thn cuando en el medio, además de tetralina, está presente otra fuente de carbono preferencial, como el β-hidroxibutirato (βHB). Los únicos mutantes obtenidos hasta el momento muestran una desrepresión parcial y están afectados en la síntesis del gránulo de reserva de polihidroxibutirato. La comparación de la expresión génica global en células crecidas en tetralina o en β-HB puso de manifiesto la presencia de un pequeño ARN (sRNA) con expresión preferencial en células crecidas en βHB, predicho en el genoma de TFA por el software Infernal 1.1 y perteneciente a la familia RF00519 (SuhB) sólo descrita en α-proteobacterias. En un mutante carente de SuhB los genes thn se encuentran parcialmente desreprimidos en condiciones de represión catabólica. La búsqueda de posibles dianas de SuhB utilizando el software IntaRNA identificó, entre otros, al extremo 5’ del mRNA de thnR, localizándose la interacción de SuhB en la región de unión al ribosoma. El efecto de esta posible interacción sobre los niveles de ThnR se ha analizado mediante Western Blot en condiciones de represión catabólica, detectándose más cantidad de ThnR en la estirpe mutante que en la silvestre. Los datos disponibles hasta el momento indican que SuhB es uno de los elementos que interviene en la represión catabólica de los genes thn en Sphingopyxis granuli estirpe TFA.Peer Reviewe

Genome-scale metabolic reconstruction and analysis of Sphingopyxis granuli strain TFA

Resumen del póster presentado al 7th Congress of European Microbiologists, celebrado en Valencia (España) del 9 al 13 de julio de 2017.[Backgrounds]: Sphingopyxis granuli strain TFA is a Gram-negative bacterium belonging to Sphingomonadaceae family whose most studied metabolic feature is its ability to use the aromatic organic solvent tetralin as the sole carbon and energy source. Regarding other metabolism aspects, this bacterium is able to accumulate poly-hydroxybutyrate (PHB) as carbon reservoir and, recently, it has been described as the first Sphyngopyxis representative able of growing anaerobically using nitrate as final electron acceptor. As several oligotrophic bacteria have been assigned to this genus, it is interesting to study other features of its metabolism that remain poorly or not characterized at all. [Objectives]: The main objective of this work is to know better and deeply the TFA metabolic capabilities through the construction of a metabolic model at a genome scale. [Methods]: Initially, two models of TFA were constructed based on Escherichia coli and Pseudomonas putida models, using the MrBac server. They were merged to obtain the first draft in which each reaction was manually reviewed. In order to complete the model, missing reactions were incorporated to fill the gaps in metabolic pathways thanks to the TFA genome annotation and metabolic/biochemical databases such as KEGG, BRENDA, MetaCyc and BiGG or by similarity search in Uniprot/SwissProt and the Pseudomonas Database. [Conclusions]: The constructed model of TFA metabolism, iIG738, consist of 738 genes, 1392 reactions and 1107 metabolites classified in 95 metabolic subsystems. The reconstruction has highlighted diverse new carbon sources that TFA can use, which have been validated in vivo. Overall, iIG738 constitutes a powefull computational tool to evaluate, at system level, the metabolic traits of the oligotrophic bacterium TFA under a large array of environmental conditions.Peer Reviewe