Genetic insights into novel lysis suppression by phage CSP1 in Escherichia coli

Lysis inhibition (LIN) in bacteriophage is a strategy to maximize progeny production. A clear plaque-forming mutant, CSP1C, was isolated from the turbid plaque-forming CSP1 phage. CSP1C exhibited an adsorption rate and replication dynamics similar to CSP1. Approximately 90% of the phages were adsorbed to the host cell within 12 min, and both phages had a latent period of 25 min. Burst sizes were 171.42 ± 31.75 plaque-forming units (PFU) per infected cell for CSP1 and 168.94 ± 51.67 PFU per infected cell for CSP1C. Both phages caused comparable reductions in viable E. coli cell counts at a low multiplicity of infection (MOI). However, CSP1 infection did not reduce turbidity, suggesting a form of LIN distinct from the well-characterized LIN of T4 phage. Genomic analysis revealed that a 4,672-base pairs (bp) DNA region, encompassing part of the tail fiber gene, CSP1_020, along with three hypothetical genes, CSP1_021, CSP1_022, and part of CSP1_023, was deleted from CSP1 to make CSP1C. Complementation analysis in CSP1C identified CSP1_020, CSP1_021, and CSP1_022 as a minimal gene set required for the lysis suppression in CSP1. Co-expression of these genes in E. coli with holin (CSP1_092) and endolysin (CSP1_091) resulted in lysis suppression. Lysis suppression was abolished by disrupting the proton motive force (PMF), supporting their potential role as antiholin. Additionally, CSP1_021 directly interacts with holin, suggesting that it may function as an antiholin. These findings identify new genetic factors involved in lysis suppression in CSP1, providing broader insights into phage strategies for modulating host cell lysis.