Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into λΔSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Ho1118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to λ S. Therefore, different N-terminally modified Hol118 variants were tested. However, in contrast to λ S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing. In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support λ R-mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the λ S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by λ.