Genetic analysis of F1 cluster phages that infect Mycobacterium smegmatis identifies two distinct holin-like proteins that regulate the host lysis event.

Phages Girr and NormanBulbieJr (NBJ) infect Gram-positive Mycobacterium smegmatis mc2 155. Both phages contain conserved lysis cassettes that harbor two endolysin genes (lysin A and lysin B) and two genes encoding transmembrane domain (TMD) holin-like proteins. The first holin-like protein, termed LysF1a is 88 amino acids, has two TMDs and a predicted N-in-C-in membrane topology. The second, termed LysF1b, has a single N-terminal TMD and a predicted N-out-C-in topology making it distinct from the type III holins or spanins in size and membrane topology. Deletion of lysF1b results in severe lysis defect phenotypes manifest by reduced plaque size and changes to lysis timing in liquid culture. Deletion of both lysF1a and lysF1b genes results in phages that show the same lysis phenotypes as the single lysF1b deletion. Phages with only lysF1b are lysis competent and trigger lysis prematurely when exposed to energy poisons while phages with lysF1a or lysF1a/lysF1b deletions do not trigger prematurely. Deletion of genes upstream of the lysis cassette did not impact lysis phenotypes. Lysis recovery mutants were isolated from phages lacking the lysF1b gene and these mutants generated wild type plaque size but triggered lysis prematurely and showed ~65% reductions in burst size. Genome sequencing identified different point mutations that mapped to TMD1 or the C-terminal region of the lysF1a gene. Infection of an M. smegmatis strain that does not produce lipomannan and lipoarabinomannan by either wild type phages or phages carrying the lysF1b deletion showed modest plaque size increases but did not fully complement the lysis defect of phages lacking the lysF1b gene. Collectively, the findings show that both LysF1a and LysF1b proteins are required for efficient bacterial lysis by these F1 cluster phages. LysF1a does not function as a pure antiholin but requires the expression of the LysF1b protein for efficient lysis functioning.