Use of two-hybrid assay in bacteria to determine protein-protein interactions in bacteriophage infections

• Grant number: 21/15055-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2022
• End date: August 31, 2023
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Germán Gustavo Sgro
• Grantee: Verena Ariak Vieira Seabra
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Type IV pilus (T4P) is a thin, flexible, and resistant filament located on the surface of bacterial cells. This appendix is involved in different bacterial behaviors, including twitching motility, adhesion to surfaces, natural transformation, biofilm formation, protein secretion, and chemotaxis. It has been previously demonstrated that infection of the phytopathogen Xanthomonas citri (Xac) by the bacteriophage ¦Xacm4-11 (Podoviridae family) is T4P-dependent (Dunger et al., 2014). More recently, viral particles have been purified and the structures of both the capsid and the viral tail resolved at resolutions of 3.2 Å and 3.6 Å, respectively, by cryogenic electron microscopy (unpublished data). At the same time, the genomic sequence of this phage was also determined in collaboration with groups from the Institute of Chemistry at USP, allowing us to also have the sequences of the ORFs (Open-Reading Frames) encoded by it (unpublished data). Despite all this, it is still not known which proteins of ¦Xacm4-11 are the ones that interact with the T4P of Xac, and what is the mechanism of recognition and injection of the genome during the infection. Through this project, we intend to identify some of these protein-protein interactions in order to improve our understanding of the phage-mediated infective process. For this, we will use a bacterial two-hybrid tool (BACTH), placing structural proteins of ¦Xacm4-11 on one side, and on the other side, proteins coding for the T4P of Xac. The results obtained will help to better understand the mechanisms of recognition and interaction of bacteria and phage, allowing the exploration of new alternatives for the control of pathogenic bacterial species, such as Xanthomonas citri.Dunger et al., 2014: https://doi.org/10.1094/MPMI-06-14-0184-R.(AU)