A cytoplasmic antiholin is embedded in-frame with the holin in a bacteriophage.

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, phi PYB5, 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, hyb5(157-465) and hyb5(209-328), 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 Hyb5(157-465) but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5(157-465) 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 Hyb5(157-465), implying that the interaction between Hyb5(157-465) and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro, Hyb5(157-465) and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5(157-465) and Hyb5. All the results suggest that Hyb5(157-465) is an antiholin of Hyb5 that is involved in lysis timing. IMPORTANCE Phage-encoded holins are considered to be the "molecular clock" of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of Lactobacillus fermentum phage phi PYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.