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Mechanistic and functional studies of Bacillus biofilms assembly on plants, and their impact in sustainable agriculture and food safety

Periodic Reporting for period 3 - BacBio (Mechanistic and functional studies of Bacillus biofilms assembly on plants, and their impact in sustainable agriculture and food safety)

Reporting period: 2018-03-01 to 2019-08-31

Major concerns in agrobiotechnology are: First, the management of plant microbial diseases continuously reduce the yield of the crops. The way these diseases are treated implies the use of pesticides (fungicides, bactericides, nematicides), however the abuse of this approach is threatening the health of the environment. Second, the presence and prevalence of microbes in fruits and vegetables that are causative agents of diseases in humans. This is a major problem that not only have a negative impact in public health but also in contamination of the alimentary industry. All these issues contravene the concept of sustainability, that is leading the politics in agriculture and food industry, and obligate to emphasize the development of novel and successful strategies that reduce environmental damage, public health but maintaining quality and benefit of the produces. Is it posible to contribute to the plant health with the use of beneficial microbes and reducing the use of pesticides, and at the same time avoid the protection of human pathogens? To address this question, we propose to investigate the way these microbes interact with plants and with other microbes that may occupy the same habitat. To do so, in our project we are working with the beneficial bacteria Bacillus subtilis, and with the phylogenetically related species Bacillus cereus, which include pathogenic strains. Both microbes are able to form biofilms, bacterial communities attached to surfaces, that serve to protect bacterial cells from external aggressions, among others functionalities. The formation of biofilms is part of the life cycle of bacteria, and studies with different species have contributed to our understanding of this fascinating bacterial behavior.
In order to propose feasible strategies oriented to promote biofilm formation and therefore a positive effect of beneficial bacteria, and at the same time, banner or impede the establishment of the pathogenic, is important to know the bacterial factors involved in such developmental program and look for specific targets.
Considering the relevance of certain proteins in the assembly of biofilms, and most explicitly, amyloid proteins, in our project we are interested in the following specific aims:
1.- To understand the process of amyloidogenesis, process of fibrillation of amyloids. This aim possess a strong component of basic research, but also an applied branch. To cover this study, we propose to move alternatively from in vitro conditions to plants, using a variety of techniques, including chemistry, biophysics or cell biology.
2.- To know how the plant modulates the formation fo biofilms, with special emphasis in the effect on amyloids, and reciprocally, which is the response of the plant to the establishment of such communities. The question supporting this aim is how beneficial and a human pathogenic bacteria communicates with plants, leading to the establishment or banishment of the bacterial community.
3.- To investigate how the two bacterial species communicate with each other or even other species that may co-exist in the same habitat. We are basically investigating three ecologically relevant interactions: beneficial-beneficial bacteria, beneficial-human pathogenic, and human pathogen-plant pathogen.
All these aims are expected to expand our view of the complexity of the ecological niche represented by the plant phyllosphere, and how the way the different microbes adapt to live and communicate with each other results in different outputs, for the plant and each bacterial species.
In the first period of the project we have dedicated our effort especifically in: i) to really understand the way the amyloids proteins polymerize, ii) the role of biofilms in the interaction of the two bacteria species to plant phyllosphere, and iii) the interaction of beneficial-beneficial bacteria.
i) From the studies on the different amyloids from B. subtilis and B. cereus, we have seen differences on their tendency to fibrillate. In parallel to this estructural and biochemical studies, we are also looking for biomolecules that may target efficiently amyloid proteins, or additionally other components of the extracellular matrix of these two bacterial species. We do count with a battery of molecules that might be active selectively against or contributing to fibrillation of these proteins, as well as biofilm formation of each bacterial species. If that were the case, we would have a combination of molecules that could be use to potentiate the beneficial microbe instead of the pathogenic. In parallel, some the molecules are active in preliminary data against the peptide responsible for Alzheimer, which would help to export our finding to other areas of research as biomedicine.
ii) It is interesting the way the components of the extracellular matrix participate in colonization or persistence of bacterial cells on the phyllosphere compared to the rhizosphere. The results obtained so far, let us to propose a complementary role for the extracellular matrix, and therefore, the need to look for those other bacterial factors that might be contributing to relevant steps in bacterial fitness as adhesion. We also found and can conclude in opposition to a general belief, not always based on experimental data, in vitro biofilm experiments are not conducive to make predictions on bacterial behavior in plant phyllosphere. We consider that a bacteria so adapted to live in a variety of niches can not delegate the success of colonization to one or two bacterial factors, therefore we are facing a more complex bacterial process that needs more intense and specific research. In addition we have found the interaction beneficial bacteria-a fungal plant pathogen-plant to be more complex and fascinating that only production of antimicrobials. Additional factors potentate this direct antagonism, and open the door to explore complementary mode of actions including the immune system of the plants.
iii) The research we have done in this aim are supporting our view of the biofilms, and more specifically the extracellular matrix in the bacterial fitness. The interaction of two bacterial species that beneficially contribute to the plant health, two biocontrol agents, let us to avoid the concept of bacterial inhibition, because the final outcome can be modulated by changing certain environmental factors and micronutrients. In general there exists a respect for the space each bacteria species is occupying, and only under certain conditions, the status quo is broken. Which is the final impact of these microbial interactions on the protection of plants against pathogens is under investigation.
Our project feeds from the principle of merging disciplines, which is foreseen to expand exponentially our knowledge on the questions we are trying to answer. The central aspect of our project is dedicated to really understand how bacteria transit from a free style of life to an organized community, and how relevant is in the interaction with plants. One of the most attractive element involved in the assembly of biofilms are the amyloid proteins, which are widely spread in nature, and playing a variety of functions, therefore, intensive research is dedicated to uncover the way they form fibers and contribution to adaptation, response or interaction with surfaces or other organisms. To really decipher differences on fiber formation, is necessary to combine organic and analytical chemistry with biophysics or biochemistry. We are now positioned to really visualize at high level of resolution the structure of the protein fibers of both bacterial species under study. This is one of the most potent findings because it will decisively contribute to the understanding of the growing family of functional amyloids. We hope that our research will have an impact not only in agro-biotechnology but also in parallel areas of knowledge as biomedicine, given that amyloids are also studied and are involved in a variety of phenomena including human diseases or human-microbe interactions.
We expect to be able to really translate the knowledge accumulated in in vitro biofilm studies to a real scenario as plant surfaces, and thus to contribute decisively to the fundamentals of microbial ecological in plants. Whether or not biofilms are relevant, it is clear that bacteria utilizes a variety of factors to live and communicate with plants, and we expect to increase our knowledge on this aspect. In addition, our research should provide with interesting insights on what is the role of the plant, or how the plant respond to such a external bacterial signals. As humans, the plants posses an immunological system that respond to external aggressions or changes, one of them the bacterial communities that live in association with. The final outcome, disease development, or pathogen establishment, will be the result of a complex network of interactions, and dialogues between all the players: plant pathogens, beneficial, human pathogens. The combination, again of disciplines as microbiology, plant physiology with cutting-edge analytical chemistry techniques and diverse OMICs will decisively contribute to success in these studies and provide interesting conclusion and novel hypothesis. In all these studies we are moving from bacterial population behavior, to really visualize the existence of changes, to further embark in more detailed and specific studies at cellular or individual levels, given that in certain scenarios is not the entire population but some individuals the responsible for the observed phenomena.
In summary our project try to respond basic questions on microbial cell biology and physiology, to really understand in a more holistic perspective how they do coordinate to generate a response in the context of the interaction with plants, a knowledge that would be granted to: 1) the development of more robust and feasible biological control strategies oriented to reduce the use of chemicals either alone or in combination with other management strategies in the context of sustainable practices and 2) contribute to eradicate or impede the establishment of human pathogen communities in ready to eat vegetable or fruits and responsible of human diseases.