Person: Dorado Morales, Pedro
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Dorado Morales
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Pedro
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ProducciĆ³n Agraria
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0000-0001-5760-1999
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811141
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Publication Open Access Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis(Society for Applied Microbiology, 2020) Dorado Morales, Pedro; MartĆnez, Igor; Rivero Buceta, Virginia; DĆaz, Eduardo; BƤhre, Heike; Lasa Uzcudun, ĆƱigo; Solano GoƱi, Cristina; Ciencias de la Salud; Osasun ZientziakBacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.Publication Open Access The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers(Nature Research, 2020) Taglialegna, Agustina; Matilla Cuenca, Leticia; Dorado Morales, Pedro; Navarro, Susanna; Ventura, Salvador; Garnett, James A.; Lasa Uzcudun, ĆƱigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun ZientziakFunctional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.Publication Open Access Systematic reconstruction of the complete two-component sensorial network in staphylococcus aureus(American Society for Microbiology, 2020) RapĆŗn Araiz, Beatriz; Haag, Andreas F.; Gil Puig, Carmen; Dorado Morales, Pedro; Lasa Uzcudun, ĆƱigo; Ciencias de la Salud; Osasun ZientziakIn bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen. IMPORTANCE Bacteria are able to sense environmental conditions and respond accordingly. Their sensorial system relies on pairs of sensory and regulatory proteins, known as two-component systems (TCSs). The majority of bacteria contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Traditionally, the function of each TCS has been determined by analyzing the changes in gene expression caused by the absence of individual TCSs. Here, we used a bacterial strain deprived of the complete TC sensorial system to introduce, one by one, the active form of every TCS. This gain-of-function strategy allowed us to identify the changes in gene expression conferred by each TCS without interference of other members of the family.Publication Open Access Genetic tools derived from Staphylococcus aureus for biotechnological applications in Gram-positive bacteria(2021) Dorado Morales, Pedro; Lasa Uzcudun, ĆƱigo; Solano GoƱi, Cristina; Ciencias de la Salud; Osasun ZientziakStaphylococcus aureus is a versatile human pathogen that has emerged as one of the most successful infectious agents of recent times, able to cause a range of diseases including skin and soft tissue infections, endocarditis, sepsis, pneumonia, osteomyelitis, bacteremia, and abscesses in organ tissues. Besides its clinical relevance, S. aureus has served as a model to study fundamental cellular processes, such as biofilm formation, the regulatory functions of small RNAs or growth and division of spherical cocci. Based on the accumulated knowledge of S. aureus biology, the availability of database resources and the advances in high-throughput genome sequencing, in this work we have aimed at developing new genetic tools derived from S. aureus for biotechnological applications in Gram-positive bacteria.Publication Open Access Fitness cost evolution of natural plasmids of staphylococcus aureus(American Society for Microbiology, 2021) Dorado Morales, Pedro; GarcillĆ”n-Barcia, MarĆa Pilar; Lasa Uzcudun, ĆƱigo; Solano GoƱi, Cristina; Ciencias de la Salud; Osasun ZientziakPlasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids.