Regulation und Mechanismus der vom Phagen T4 induzierten Lyse von Wirtszellen

authored by

Jan Michel Frederik Schwarzkopf

supervised by

Thomas Brüser

Abstract

After Infection with virulent phages, the bacterial host cells are lysed to release new phage particles. In case of phages with double-stranded DNA genomes, this is achieved by the mura-lytic activity of endolysins. Their access to the cell wall is mediated by holins, which form lesions in the cytoplasmic membrane. The protein T of Escherichia coli phage T4 is one of the canonical holins that enable passage of cytoplasmic endolysins through the membrane. Because canonical holins form aggregates and membrane lesions with a diameter up to 1 μm in the ab-sence of endolysins, it is believed that endolysins are released non-specifically through large membrane holes. After adsorptions of secondary phages on the host cell, lysis is delayed by phage T4. The interaction of T with antiholin RI is essential for this lysis inhibition (LIN). A crystal structure of an in vitro formed T2-RI2 tetramer only comprised soluble regions of the proteins and provided no explanation how the complex is anchored in the membrane. In this study, LIN was reconstituted in E. coli by a vector-based system that was used to analyze RI variants in vivo. Although RI has a signal peptide and is released into the periplasm, LIN was also mediated by a membrane-anchored variant. Furthermore, it was shown that only one interface of the crystal structure is relevant in vivo. Thus, T is inhibited by RI in a heterodimer. By examining the abundance of RI variants after the inhibition of translation, it was found that the signal peptide cleavage site of RI functions to regulate the concentration of this antiholin. Fluorescence-labeled holin T was analyzed by microscopy over time and it was shown that the release of endolysins precedes the formation of large aggregates. By functional analyses of T variants, which were generated by missense mutations and truncations, it was found that the cytoplasmic helix of the holin is essential for the formation of membrane lesions. In agreement with this, structural models of T complexes by AlphaFold 3 predicted a transmembrane orien-tation of this helix and thereby the hydrophilic inside of a pore. In addition, the AlphaFold 3 models were supported by site-specific in vivo cross-links. The analyses of T indicate that the holin mediates lysis by the formation of pores, which emerge from small assemblies.

Organisation(s)

Institute of Microbiology

Type

Doctoral thesis

No. of pages

172

Publication date

24.06.2025

Publication status

Published

Electronic version(s)

https://doi.org/10.15488/19180 (Access: Open)