Abstract
Mycobacteriophage Mcgavigan could be a promising candidate for use as a preventative agent against infections with Mycobacterium avium subsp. paratuberculosis. Bioinformatic analysis of the Mcgavigan genome revealed the presence of an operon containing a "Bxb1-like" repressor. The operon may have been acquired by the phage through horizontal gene transfer with a Bxb1-like mycobacteriophage in its evolutionary past. We sought to investigate the function of the acquired repressor as a potential regulator of lysogeny or as a source of heterotypic superinfection immunity. Recombineering with CRISPR counter-selection was employed to achieve a clean deletion of the Bxb1-like repressor from Mcgavigan's genome. Integrase was also deleted as a means of creating a lytic-only phage for comparison purposes and the elimination of lysogeny with this edit was confirmed. To test phenotypic changes which resulted from these deletions, several parameters such as burst size, latency period, and killing efficiency were measured for each knockout mutant and lysogeny was tested. The integrase deletion mutant had complete lysogeny abolishment and performed similarly to wild-type phage on all measured parameters. The deletion of the Bxb1-like repressor did not affect the lysogenic capability of the phage. Whereas Mcgavigan lysogens are typically immune to superinfection from Terelak, a mycobacteriophage related to Bxb1, lysogens created from Mcgavigan with the Bxb1-like repressor deletion were completely resensitized to heterotypic superinfection by Terelak. This suggested that this repressor was acquired by Mcgavigan through horizontal gene transfer for the purposes of superinfection immunity against cluster A1 mycobacteriophages and was not used for maintenance of lysogeny.