A cytoplasmic antiholin is embedded in frame with the holin in a Lactobacillus fermentum bacteriophage

Abstract

In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum, ψPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5, hyb5157-465 and hyb5209-328, were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli. Surprisingly, host cell lysis was delayed by Hyb5157-465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5157-465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5157-465, implying that the interaction between Hyb5157-465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro, Hyb5157-465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5157-465 and Hyb5. All the results suggest that Hyb5157-465 is an antiholin of Hyb5 that is involved in lysis timing.