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

Periodic Reporting for period 4 - BacBio (Mechanistic and functional studies of Bacillus biofilms assembly on plants, and their impact in sustainable agriculture and food safety)

Okres sprawozdawczy: 2019-09-01 do 2021-02-28

Major concern in agrobiotechnology are the management of plant microbial diseases. 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. Besides, 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. 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 that serve to protect bacterial cells from external aggressions, among others functionalities.
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, it is important to know the bacterial factors involved in such developmental program and look for specific targets.
In our project we are interested in the following specific aims:
1.- To understand the process of amyloidogenesis. 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.
3.- To investigate how the two bacterial species communicate with each other or even other species that may co-exist in the same habitat.

1. We have been able to describe at atomic level the main differences of the amyloid protein of the two related Bacilli (B. subtilis and B. cereus), which appears to determine their functionality in the ecology on plants.
2. We have also found a diversification of functions of the ECM in the interaction with the environment. This interaction is chemical (signaling) or structural, and permits the establishment of stable bacterial communities on plants, or a beneficial inter-kingdom communication.
3. We have discovered which appears to being the main function of the B. subtilis amyloid, to provide integrity to the cell membrane when cells enter in stationary phase of growth, besides their role in biofilm assembly. These two functions affect the persistence and antagonistic interaction with fungal pathogens.
5. Different plant surfaces possess distinctive morphological and chemical features that modulate the ecology of Bacilli and the expression of the genes related to biofilm.
6. The ability of B. cereus to induce injury in intestine, is alternatively based on vegetative cells are able to persist on vegetables. This is possible to the maintenance of other ecological skills as biofilm formation.
These findings represent a breakthrough on the real implication of bacterial biofilms in the ecology of bacterial in plants, and how they can be handled to improve the beneficial contribution to the health of the crops. Thus, from an applied perspective, our findings can be easily translated to the improvement in the implementation of microbes and derived molecules in the frame of sustainable programs of production of crop protection. The study on B. cereus has revealed that beyond spores and toxins it is important to pay attention to vegetative cells which can cause a plethora of symptoms in humans, from diarrhea or vomit to death, and therefore make necessary to revise the procedure used for the detection.
These findings has been disseminated in the research community in the form of research manuscripts, or presentation in seminars and conferences. In the other hand, we have contributed also to their dissemination to the general public by participating in different diffusion media. Some examples are:
Participation in the European researcher' night (2018-2019). https://lanochedelosinvestigadores.fundaciondescubre.es/investigador/diego-f-romero-hinojosa/
Media:
Diario sur: https://www.bacbiolab.com/wp-content/uploads/2020/11/cronica-universitaria-27-10-2020-1.pdf
Cordis-Europa: https://cordis.europa.eu/project/id/637971
Programa tesis: http://www.canalsur.es/television/programas/tesis/detalle/62.html?video=1443163&sec=
Our website:https://www.bacbiolab.com
Social media: tweeter: diegoromerohi

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. 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. The combination of organic and analytical chemistry with biophysics or biochemistry has let find differences on fiber formation. We believe that our research has a notable impact in cell biology and structural biology, and agro-biotechnology or biomedicine.
We have been able to validate the knowledge accumulated in in vitro biofilm studies to plants, decisively contributing to the fundamentals of microbial ecological. It is clear that bacteria utilizes a variety of factors to live and communicate with plants, and we have expand our knowledge on this aspect. In addition, our research is also demonstrating how relevant is the plant host not only immunologically, but also metabolically and structurally (topology). 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 have decisively contributed 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.
In summary our project has offer responses to 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.

Melon plant

Electron microscopy of bacterial cells on a trichome

Biofilm of B. cereus stained with Congo red

electron microscopy image of amyloid fibers

Periodic Reporting for period 4 - BacBio (Mechanistic and functional studies of Bacillus biofilms assembly on plants, and their impact in sustainable agriculture and food safety)

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