Bacteriophage T4 lysis and lysis inhibition: molecular basis of an ancient story

Date

2009-05-15

Authors

Tran, Tram Anh Thi

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Abstract

T4 requires two proteins: holin, T (lesion formation and lysis timing) and endolysin, E (cell wall degradation) to lyse the host at the end of its life cycle. E is a cytoplasmic protein that sequestered away from its substrate, but the inner membrane lesion formed by T allows E to gain access to the cell wall. T4 exhibits lysis inhibition (LIN), a phenomenon in which a second T4 infection occurs ? 3 min after primary infection results a delay in lysis. Mutations that abolish LIN mapped to several genes but only rV encoding the holin, T, and rI, encoding the antiholin, RI, are required for LIN in all hosts which support T4 replication. Antiholin RI inhibits T-mediated lysis by direct interaction with the holin. T has at least one transmembrane domain with its Nterminus (TNTD) in the cytoplasm and C-terminus in the periplasm (TCTD). In contrast, the N-terminus of RI (RINTD) is predicted to function as a cleavable signal sequence allowing the secretion of the RI C-terminal domain (RICTD) into the periplasm. Most of RI mutations which abolish LIN occur in the RICTD, suggesting RI inhibits T-mediated lysis by interacting with T via RICTD. Topological analysis of RI and T showed that fusion of PhoA signal sequence (ssPhoA) to RICTD is necessary and sufficient for LIN and ssPhoA?TCTD interferes with RI-mediated LIN, indicating T and RI interact via periplasmic C-terminal domains. In T4 infection, LIN is observed only when superinfection takes place, indicating either the antiholin or the LIN signal must be unstable. Both RI and RINTD?PhoA are localized to both the inner membrane and the periplasm suggesting that the RINTD is a Signal-Anchor-Release (SAR) domain. Protein stability studies indicated that the SAR domain is the proteolytic determinant of RI, and DegP is the protease that is responsible for RI degradation. To date, how TNTD participates in lysis and LIN is not known. Modifications and deletion of the N-terminus of T change the lysis time, indicating this domain is involved the in timing of lysis. GFP fusion to holin T allowed microscopic visualization of fluorescent patches on the membrane at the time of lysis.

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