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Updated: Apr 9, 2026
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Published on: June 11, 2015
Genetic analysis of F1 cluster phages that infect Mycobacterium smegmatis identifies two distinct holin-like proteins
Richard S Pollenz1, Kira Ruiz-Houston2, Wynter Dean1
1Department of Molecular Biosciences, University of South Florida, Tampa, Florida, United States of America.
Abstract:
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.
Insights
Bacteriophage lysis requires two holin-like proteins, LysF1a and LysF1b, for efficient bacterial lysis. LysF1b is essential for lysis, while LysF1a requires LysF1b to function effectively in Mycobacterium smegmatis lysis.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophages utilize lysis systems, including holins and endolysins, to lyse host cells.
- The F1 cluster phages infecting Mycobacterium smegmatis possess conserved lysis cassettes with two holin-like genes (lysF1a, lysF1b) and two endolysin genes.
- Holins are transmembrane proteins that form pores in the bacterial cytoplasmic membrane, crucial for phage-induced lysis.
Purpose of the Study:
- To investigate the roles of LysF1a and LysF1b proteins in the lysis process of Mycobacterium smegmatis by F1 cluster phages.
- To determine the functional relationship between LysF1a and LysF1b during phage-mediated bacterial lysis.
- To characterize the lysis phenotypes associated with deletions of lysF1a and lysF1b genes.
Main Methods:
- Gene deletion analysis of lysF1a and lysF1b in F1 cluster phages infecting Mycobacterium smegmatis mc2 155.
- Phenotypic characterization of phage lysis, including plaque size, lysis timing in liquid culture, and response to energy poisons.
- Isolation and characterization of lysis recovery mutants through genome sequencing.
- Assessment of lysis complementation in M. smegmatis strains lacking lipomannan and lipoarabinomannan.
Main Results:
- Deletion of lysF1b resulted in severe lysis defects, including reduced plaque size and altered lysis timing.
- Phages lacking both lysF1a and lysF1b exhibited similar lysis defects to those lacking only lysF1b.
- Phages with only lysF1b were lysis-competent and sensitive to energy poisons, unlike those with lysF1a deletions.
- Lysis recovery mutants contained point mutations in lysF1a, restoring plaque size but reducing burst size and enabling premature lysis.
- Host cell wall composition (lipomannan/lipoarabinomannan) did not fully rescue the lysis defect of lysF1b deletion phages.
Conclusions:
- Both LysF1a and LysF1b proteins are essential for efficient bacterial lysis mediated by F1 cluster phages.
- LysF1a does not function independently as an antiholin; its lytic activity requires the co-expression and function of LysF1b.
- The distinct membrane topologies and roles of LysF1a and LysF1b highlight a complex, cooperative lysis mechanism in these mycobacteriophages.
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