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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 ssPhoAΦTCTD 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 RINTDΦPhoA 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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