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
, ϕPYB5, 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 ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.
Genome-wide identification of genetic requirements ofPseudomonas aeruginosaPAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing
