A short version of the amyloid-like protein TasA fibrillates and supports biofilm formation in Bacillus cereus

The formation of bacterial biofilms is a doable thanks to the assembly of an extracellular matrix that provides to the entire community with i) an outstanding architectonic structure and ii) protection to the cells from external aggressions. In Bacillus subtilis, a structural element dedicated to the formation of the extracellular matrix is the amyloid-like protein TasA. To form fibers, TasA needs the participation of the protein TapA. Indeed, a tapA mutant resembles phenotypically to a tasA mutant, which is wrinkle-less pellicles or colonies with no distinguishable morphological features. tasA is widely spread within the Bacillus genus, but tapA is absent in the heterogeneous group of Bacillus cereus which includes environmental and pathogenic members; some of them are responsible for important food intoxication outbreaks. Then, we asked whether TasA would still retain functionality in biofilm formation in B. cereus. Comparative genomic analysis showed a region in B. cereus containing two orthologous of tasA, tasA and calY, and the orthologous of sipW, that encodes a signal peptidase. Our mutagenic studies revealed that the entire region was relevant for biofilm formation, and electron microscopy proved the major propensity of TasA than CalY to form fibers in the cell surfaces. These findings also indicated that in B. cereus as opposed to B. subtilis, an accessory TapA protein is not necessary for the fibrillation of TasA. Indeed, the heterologous expression of this region of B. cereus restored the capacity of a B. subtilis tasA operon mutant or a single tasA mutant to form pellicles. These pellicles stained with the amyloid dye Congo Red and the cells were decorated with fibers, both findings suggestive of an amyloid-like nature of the B. cereus TasA. Intriguingly, in a B. subtilis tapA mutant, only the entire region of B. cereus fully rescued pellicle formation, fibrillation or Congo Red staining, to a lesser extent did sipw-tasA, and no restoration was observed with sipW-calY. These observations led us to speculate that TapA might cross seed the fibrillation of TasA or CalY in B. subtilis. In summary, TasA is relevant for biofilm formation in these two bacterial species, which appears to be governed by its polymerizing nature. The fact that we count with two bacterial species containing versions of TasA with subtle differences will be of great value in our studies of the mechanistic of polymerization of these bacterial amyloid-like fibers and their contribution to the assembly of the extracellular matrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Compounds produced by two robust Bacillus amyloliquefaciens biocontrol strains involved in antimicrobial activity and plant-growth promotion

Several members of the Bacillus genus are potential candidates to be used as biological control agents to combat pests or plant diseases. The bacterial attributes associated to Bacillus behaviour are mainly: the production of antimicrobial compounds, the plant-growth promotion capability and the induction of systemic resistance in plant host. In previous works, we have demonstrated this multifaceted biocontrol activity of B. amyloliquefaciens CECT8237 (UMAF6639) and CECT8238 (UMAF6614) strains, which contributes to plant protection against bacterial and fungal pathogens. In order to identify the bacterial features responsible for the outstanding biocontrol activity of these strains, their genomes were sequenced and analysed. Firstly, those features previously described for other Bacillus to be involved in the biocontrol activity were localized: i) Biosynthetic genes of secondary metabolites. Apart from the lipopeptides, formerly detected, we have demonstrated the production of other additional compounds that might participate in the antibiosis activity; ii) Biosynthetic genes of the volatile compounds 2,3-butanediol and acetoin, both involved in the induction of plant defence responses. Secondly, genetic singularities non-conserved within the Bacillus genus, which might contribute to the biocontrol ability of B. amyloliquefaciens CECT8237 and CECT8238 were identified in both genomes. Among them, we highlight two genomic regions hypothetically implicated in the production of non-characterized secondary metabolites. Ongoing studies are focused on elucidating the functionality of these uncharacterized regions, leading to a better understanding of the mechanisms of action involved in the robust biocontrol skills of these strains.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The expression profile of secondary metabolites in biofilms of B. amyloliquefaciens CECT 8237 biocontrol strain

The contribution of Bacillus amyloliquefaciens CECT 8237 (UMAF6639) strain to the plant protection against bacterial and fungal pathogens is mainly based on: i) the production of antimicrobial compounds, ii) the plant-growth promotion capability and iii) the induction of systemic resistance in plant host. In previous works, we demonstrated the relevant implication of the three families of lipopeptides in the biocontrol activity and biofilm formation on melon leaves. The analysis of the genome sequence revealed features previously identified in other Bacillus strains, such as genes related to biofilm formation, phytostimulation and induction of systemic resistance in the host plant, and novel genomic regions non-conserved within the Bacillus genus, and therefore with potential genes implicated in the biocontrol activity. Considering the relevance of biofilm formation and production of secondary metabolites in biocontrol, we analyzed the expression profile of several secondary metabolites produced by CECT 8237 in biofilm inducing conditions. To do so, we optimized an in situ detection method based on MALDI-TOF analysis of secondary metabolites within the bacterial colony and in supernatants and pellicles of B. amyloliquefaciens biofilms. We found a major accumulation of these secondary metabolites in the core and middle area of the colony and in the spent medium compared to pellicle. Further studies will help elucidating the real implication of these molecules in the bacterial ecology or in its mechanisms of defence, against competitors, and/or offense, against pathogens and its possible relation with the niche they occupy. This work was supported by grants from the Plan Nacional I+D+I (AGL-2012-31968) and Incentivos a Proyectos de Excelencia de la Junta de Andalucía (P10-AGR-57-97), both cofinanced by FEDER funds (EU). This work was also supported by grant from Koppert B.V. (8.06/60.4086) and European Research Council-Starting Grant BacBio ERC637971.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Standardization of a new photodiagnosis method based on LEDs for patients with solar urticaria sensitive to visible light

Standard methods for photodiagnosis of solar urticaria are based in exposure of patient skin to different polychromatic UV and visible sources where minimal urticarial doses for different spectral bands (UVB and UVA) are established. Classical photodiagnosis devices are based in solar simulation and use of UVB and UVA enhanced fluorescent lamps. In case of visible US photodiagnosis, US patient skin is exposed for 15 min to a slight projector, provided with halogen lamp, at a distance of 15 cms and presence of erythema and/or wheals is determined as positive reaction. Slights projector is from several years almost out of market due to use of new projection digital technologies and new visible light emerging technologies are good candidates for their substitution as photodiagnosis tool. The objective of the present work is to analyze photodiagnosis of visible light solar urticaria with using a LED device in comparison to normal slight projector exposure protocol. A total of twenty patients, from 7 different photodiagnosis units have participated in the study. Patients, with SU positive to visible light (with or without to UV radiation) following the standard photodiagnosis protocols were included in the study. Slight projector used in all photodiagnosis units were of similar characteristics and irradiance at 15 cm distance, as well as total dose of visible light after 15 min were calculated for each halogen lamp device. LED exposure was performed in parallel in a closed zone of the back of the patients. For LED photodiagnosis a prototype from University of Málaga (Spain) has been developed consisting in a black box provided with 4 holes of 12 mm diameter in which each hole white warm of a LED of 1 W is emitted. Thus, each LEDs dose is controlled independently and the device allows establishing, as well as for UVB and UVA normal protocols a MUD also under visible light. In that case, maximal visible light dose is reached in less than 5 min compared to 15 min under exposure to slight projector. All patients were positive to LED warm visible light with presence of erythema and / or wheals in parallel to the exposure to the slight projector. A MUD to visible light has been established with significant variations between patients which reveals different grade to visible light sensibilization. In conclusion, a new technology of illumination based in LEDs can be used in photodiagnosis of SU.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Endurance of quantum coherence due to particle indistinguishability in noisy quantum networks

Quantum coherence, the physical property underlying fundamental phenomena such as multi-particle interference and entanglement, has emerged as a valuable resource upon which modern technologies are founded. In general, the most prominent adversary of quantum coherence is noise arising from the interaction of the associated dynamical system with its environment. Under certain conditions, however, the existence of noise may drive quantum and classical systems to endure intriguing nontrivial effects. In this vein, here we demonstrate, both theoretically and experimentally, that when two indistinguishable non-interacting particles co-propagate through quantum networks affected by non-dissipative noise, the system always evolves into a steady state in which coherences accounting for particle indistinguishabilty perpetually prevail. Furthermore, we show that the same steady state with surviving quantum coherences is reached even when the initial state exhibits classical correlations.Comment: arXiv admin note: substantial text overlap with arXiv:1709.0433

Transformación de Podosphaera xanthii mediante Agrobacterium tumefaciens y su aplicación para el estudio de efectores

Los oídios son patógenos biotrofos obligados que requieren células vivas para completar su ciclo de vida. Esto se debe a que estos hongos patógenos desarrollan una estructura especializada en el interior de las células de la planta, denominada haustorio, estableciendo una íntima relación con su hospedador. El haustorio es el responsable de la toma de nutrientes y el intercambio de factores con la planta. Estos factores son los denominados efectores, pequeñas proteínas secretadas por el patógeno responsables de la inhibición o modificación de la defensa innata de la planta. El análisis de estos efectores ha focalizado un gran interés en los últimos años, haciéndose necesaria la puesta a punto de técnicas para la manipulación genética de estos patógenos. Hasta la fecha, se han desarrollado varios métodos para transformar hongos filamentosos, sin embargo, debido al estilo de vida de estos patógenos, estas estrategias de transformación o no son adecuadas o no han dado resultados satisfactorios. Recientemente, se han desarrollado metodologías para el estudio y análisis de efectores usando la planta como vector para la expresión de construcciones para el silenciamiento o la sobreexpresión de estas proteínas, sin embargo, la localización y el mecanismo de secreción de los efectores sigue siendo desconocido. En este trabajo hemos desarrollado un método para la transformación de Podosphaera xanthii, el principal agente causal del oídio de las cucurbitáceas, usando Agrobacterium tumefaciens como vector de transformación. Mediante este método, hemos obtenido transformantes estables para una variedad de construcciones: i) plásmidos expresando el gen egfp bajo el control de promotores constitutivos o inducibles de Aspergilus nidulans, ii) plásmidos expresando el casete de resistencia a higromicina B o un alelo de β-tubulina de P. xanthii que confiere resistencia a fungicidas MBC. Tras la transformación, las colonias de los transformantes fueron analizas mediante microscopía confocal, resultando ser estables tras sucesivas generaciones cuando se aplicaba el marcador de resistencia y presentando el mismo aspecto que las colonias no transformadas del parental. Además, los análisis moleculares mostraron la presencia del T-DNA en las colonias transformadas de P. xanthii. El objetivo de este trabajo es poner a punto un método para el estudio de los posibles efectores de P. xanthii. Por ello, se llevaron a cabo estudios de microscopía confocal con fusiones traduccionales de efectores candidatos de P. xanthii fusionados a GFP. Entre los efectores ensayados, destacó la presencia de una amplia familia de efectores localizados en la papila y en pequeñas vesículas del haustorio, así como un efector de localización en la membrana del hongo. Estos resultados demuestran la fiabilidad de este método para la transformación de P. xanthii y abre una nueva vía para el estudio de efectores candidatos directamente en el patógeno sin mediar la planta.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech. Plan Nacional de I+D+i del Ministerio de Economía y Competitividad (AGL2013-41939-R), cofinanciado con fondos FEDER (UE)

Adhesin proteins in biofilms of the food-borne pathogen Bacillus cereus

One of the greatest challenges of the food industry is to reduce food poisoning caused by food-borne pathogenic bacteria. Bacillus cereus is one the recurrent bacterial pathogens responsible of many outbreaks. Biofilm formation and sporulation are thought as the most important reservoirs of B. cereus. Biofilms are bacterial communities difficult to eradicate from biotic and abiotic surfaces. They are usually composed of exopolysaccharides and proteins. In our study, we are interested in the characterization of the molecular bases leading to biofilm formation in B. cereus during the interaction with plants. To start this analysis we have focused on the structural components of the extracellular matrix of these biofilms. Using as a model the well-studied B. subtilis, we show in this work the presence in B. cereus of an orthologous of tasA. TasA forms amyloid-like fibers in the extracellular matrix of B. subtilis. We demonstrate that the heterologous expression of this allele in a B. subtilis strain lacking the entire tasA operon and unable to form biofilm, restores the assembly of biofilm formation. Biochemical and morphological studies of this protein let us think on a possible amyloid-like nature of this protein, which could be involved in cell-cell interaction and biofilm formation as it does in B. subtilisUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Una región genómica implicada en la formación de fibras tipo amiloide en biofilms de Bacillus cereus

Bacillus cereus es una bacteria patógena responsable de numerosos brotes de intoxicación alimentaria. Las esporas y el biofilm son considerados los reservorios más importantes de B. cereus que se pueden encontrar en frutas y verduras frescas contaminadas. Los biofilms son comunidades bacterianas difíciles de erradicar de superficies bióticas y abióticas debido a una matriz extracelular estable que recubre y protege a las células bacterianas. Estas matrices extracelulares suelen estar formadas de exopolisacáridos, proteínas, ADN extracelular, y otros componentes minoritarios. Estudios de biofilms en Bacillus subtilis, especie filogenéticamente relacionada con B. cereus, han demostrado la existencia de fibras tipo amiloide en su matriz extracelular, y su relevancia en el desarrollo de biofilms. El objetivo de este trabajo ha sido investigar la existencia de este tipo de proteínas en B. cereus y su implicación en la formación de biofilms. El análisis comparativo de los genomas de B. subtilis y B. cereus nos permitió identificar una región genómica que contiene dos loci que codifican ortólogos de la proteína tipo amiloide TasA y un ortólogo de la correspondiente peptidasa señal SipW, responsable del correcto procesamiento de TasA previo a su secreción. La mutación de esta región genómica condujo a la incapacidad de B. cereus de formar biofilms, un fenotipo compartido por mutantes en el gen sipW. Los mutantes en tasA o calY no perdieron completamente la capacidad de formación de biofilms, aunque desarrollaron fenotipos diferenciados entre sí. El estudio con microscopía electrónica reveló que TasA, y en menor medida CalY, polimerizan en forma de fibras largas y abundantes en la superficie de las células. La expresión heteróloga de estos loci en una cepa de B. subtilis que carece de los factores necesarios para el ensamblaje de fibras de tipo amiloide de TasA reveló: (i) la implicación de esta región genómica B. cereus en la formación de películas en la interfase aire-líquido y (ii) la capacidad intrínseca de TasA para formar fibras similares a las fibras formadas por su ortólogo en B. subtilis. Los resultados obtenidos en este estudio indican: (i) La implicación de la región genómica que contiene los genes sipW, tasA y calY en la formación de biofilms de B. cereus en superficies abióticas, (ii) las propiedades adhesivas de TasA y CalY, y (iii) la potencial naturaleza amiloide de la proteína TasA de B. cereus como su ortólogo en B. subtilis. Este trabajo ha sido financiado con una ayuda del Plan Nacional de I+D+i del Ministerio de Economía y Competitividad (AGL2012-31968), cofinanciado con fondos FEDER (UE)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The pivotal functionality of the amyloid protein TasA in bacillus physiology and fitness

Biofilms are complex bacterial communities formed on any virtual surface and composed of cells embedded in an extracellular matrix. Studies on Bacillus subtilis have demonstrated this tissue-like structure comprised of diverse exopolymeric substances (eps) including exopolysaccharides, the protein BslA, and TasA and TapA the two main components of the amyloid fibers that confer robustness to the architecture of the biofilm. It has been demonstrated that the genetic pathways involved in formation of biofilms are active in the interaction of B. subtilis with plant surfaces. Indeed, we previously showed that surfactin acts as a self-trigger of biofilm in the plant phylloplane, which connected with the suppressive activity of B. subtilis against phytopathogenic fungi. These findings led us to hypothesize a major contribution of the extracellular matrix in the ecology of B. subtilis in the poorly explore plant phylloplane. In this work, we show that the amyloid protein TasA has a meaningful role in adhesion and biofilm formation over the plant phylloplane, however, despite the inability of the tasA mutant to form a biofilm, it still retained a similar antagonistic activity compared to the wild type strain. An in-depth transcriptomic analysis of the mutant led us to find unexpected variations in the expression levels of over 300 genes, including the overexpression of: i) production of acetoin ii) secondary metabolites and non-ribosomal peptides iii) eps and other biofilm-related components and iv) general stress, among others. These findings suggested that besides the structural role, TasA might have a regulatory function on the physiological stage of the cells. Indeed, an allele of TasA unable to restore biofilm formation allowed us to separate both functions, supporting the importance of this functional amyloid in regulating bacterial physiology and fitness.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech