Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 5 uppsatser.

The N-terminal hydrophobic domain of the phage P1 endolysin Lyz was found to facilitate the export of Lyz in a sec-dependent fashion, explaining the ability of Lyz to cause lysis of E.coli in the absence of the P1 holin. The N-terminal domain of Lyz is demonstrated to be both necessary and sufficient not only for export to the membrane but also for release into the periplasm of this endolysin. We propose that this unusual N-terminal domain functions as a "signal arrest- release" (SAR) sequence, which first directs the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. To understand why release from the membrane is required for the physiological expression of the lytic activity of Lyz, we examined the role of its seven cysteine residues in the biogenesis of the active endolysin. The inactive, membrane-tethered and the active, soluble forms of Lyz differ in their pattern of intramolecular disulfide bonding. We conclude that the release of Lyz from the membrane leads to an intramolecular thiol-disulfide bond isomerization causing a dramatic conformational change in the Lyz protein. As a result, an active site cleft that is missing in nascent Lyz is generated in the mature form of the endolysin. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an SAR sequence is not unique to Lyz. Studies on holin and antiholin indicated that P1 encodes two holins, LydA and LydC. The antiholin LydB inhibits LydA by binding to it directly on the membrane. All above results demonstrate a new paradigm for control of phage lysis, which is, upon depolarization of the membrane by holin function at a programmed time, endolysin is released from the bilayer leading to the immediate lysis of the host.