Genetic Engineering of Lysogenic-Lytic Switch Genes Improves Burkholderia Phage Killing Efficacy.

Burkholderia pseudomallei, the causative agent of melioidosis, presents significant challenges in both treatment and environmental decontamination. Bacteriophages, or phages, are increasingly being explored as potential diagnostic, therapeutic, and biocontrol agents against this bacterial pathogen. Our recent investigation has shown that most B. pseudomallei genomes contained prophage(s) associated with specific tRNA gene loci, prompting us to explore these detectable prophages as sources of temperate phages for further applications. Transcriptomic profiling of B. pseudomallei Bp82, a model strain that possesses three different prophages, revealed high expression levels of the integrase and certain transcriptional regulatory genes within its prophages during normal exponential growth. Using one of its temperate phages, namely φBP82.2, a P2-like phage, as a model, we investigated the lysogenic-lytic control mechanisms. Mutagenesis of the integrase gene, phiBP82.2_gp51, did not improve killing activity compared to the wildtype phage. In contrast, deletion of phiBP82.2_gp38, a putative transcriptional regulatory gene, and two downstream hypothetical protein genes, phiBP82.2_gp36 and phiBP82.2_gp37, resulted in significant lytic improvement. We conclude that these genes play a crucial role in the lysogenic-lytic switch of φBP82.2, suggesting a new avenue for engineering temperate phages for future applications.