Production and purification of xanthomonas citri minicells for use in cryoelectron tomography (cryo-ET)

• Grant number: 24/16240-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Microbiology
• Principal Investigator: Germán Gustavo Sgro
• Grantee: Matheus de Lima Ortega
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 22/03018-7 - Structural, molecular and functional characterization of bacteriophages associated with bacteria of medical and agricultural interest, AP.PNGP.PI

Abstract

The creation of minicells for visualizing cellular structures using electron microscopy methodologies (mainly cryo-electron tomography, cryo-ET) is already a well-known and effective approach, allowing greater visualization of details in the tiny pseudo-cells. In X. citri, a bacterium relevant for its impact on citrus agriculture and as an increasingly established model for the study of cellular structures, there are already some strains with this capability. One of the most promising is the ¿minC variant, a deletion mutant of the minC gene involved in cell division. However, due to the modifications in the cell division process of this strain, several difficulties arise in its development: its growth is considerably slower than that of wild-type bacteria, its survival is lower and shorter, and there is not enough knowledge in the literature on how to overcome these obstacles. Thus, there is also no readily available protocol to optimize the obtaining and collection of these minicells produced by X. citri, necessitating the creation of documentation and standardization of the same for future use.Furthermore, with the aim of expanding and favoring the development of methodologies that enhance the use of minicells for structural studies by electron microscopy, we also intend to use molecular biology techniques to develop a series of strains that combine various mutations responsible for minicell creation - these being in genes such as minCDE, mreB, ftsQAZ, plsB, and plsC - so that each one enhances the action of the others for the best possible result at the end of the growth and separation protocol.Thus, at the end of the project, we hope to have greatly facilitated the obtaining of minicells from the modified strains and their respective protocols, allowing future research in the laboratory on a wide variety of topics, from studies of membrane molecular complexes to phage infection in minicells, to develop with greater success.