IV Jornadas Latinoamericanas de Bacteriófagos

Introduction: This study investigates protein-protein interactions during bacteriophage infection, focusing on modern isolate phages ΦXacm4-11, ZC01 and ZC03 and their bacterial hosts Xanthomonas citri and Pseudomonas aeruginosa, respectively. Using the adenylate cyclase (AC)-based bacterial two-hybrid (BACTH) system, we aim to identify bacterial proteins interacting with phage proteins, shedding light on infection steps and resistance mechanisms. The findings will aid in developing antimicrobial and biocontrol strategies. Objectives: Construct BATCH-based genomic libraries (preys) for X. citri strain 306, P. aeruginosa strain PA14, and gene-specific vectors (baits) for ΦXacm4-11, ZC01, and ZC03, and identify protein-protein interactions between ΦXacm4-11/X. citri and ZC01/ZC03/P. aeruginosa. Materials and methods: Bacterial and phage genomic DNA were extracted using commercial kits. The pKT25 and pUT18 family vectors from the BACTH system, stored in E. coli DH5α, were used to construct libraries. Phage genes were amplified by PCR using CloneAmp HiFi PCR Premix (Takara). Vectors were extracted, digested with BamHI, XbaI or SmaI restriction enzymes, and dephosphorylated with FastAP. X. citri genomic DNA was fragmented with Sau3AI, while ΦXacm4-11 genes were digested with BamHI, XbaI or SmaI and purified. T4 DNA Ligase was used for ligation, inserting X. citri DNA into pUT18 family vectors and phage-derived genes into pKT25. Transformation into E. coli DH5α was performed via electroporation. Clone verification was conducted by colony PCR. Results: The genomic DNA of X. citri and ΦXacm4-11 were successfully isolated and purified. Partial digestion with Sau3AI optimized fragmentation, obtaining 250-2500-bp fragments, was determined within 30-45 min. PCR amplification of phage genes successfully generated target fragments for cloning. Colony PCR confirmed the correct insertion of fragments into vectors, ensuring adequate genomic coverage.
Conclusions: The bacterial and phage genomic DNA was successfully cloned into the appropriate vectors, ensuring correct insertion. These results validate the experimental strategy for future research on protein-protein interactions.