Holins are small hydrophobic proteins causing non-specific membrane lesions at the end of bacteriophage multiplication, to promote access of the murein hydrolase to their substrate. We have established a lambda-delta-Sthf genetic system, which enables functional expression of holins from various phages in an isogenic phage lambda background, and allows qualitative evaluation of their ability to support lysis of Escherichia coli cells. Synthesis of holins is under control of native lambda transcription and translation initiation signals, and the temperature-sensitive CIts857 repressor. A number of different holins were tested in this study. The opposing action of phage lambda S105 and S107 holin variants in lysis timing could be confirmed, whereas we found evidence for a functionally non-homologous dual translational start motif in the Listeria phage Hol500 holin. The largest holin known, HolTW from a Staphylococcus aureus phage, revealed an early lysis phenotype in the lDSthf background, which conferred a plaque forming defect due to premature lysis. Mutant analysis revealed that an altered C-terminus and/or a V52L substitution were sufficient to delay lysis and enable plaque formation. These results suggest that the extensively charged HolTW C-teminus may be important in regulation of lysis timing. Gene 17.5 product of E. coli phage T7, and Gp T from T4 was found to support sudden, saltatory cell lysis in the lDSthf background, which clearly confirms their holin character. In conclusion, lDSthf offers a useful genetic tool for studying the structure-function relationship of the extremely heterogeneous group of holin protein orthologs. MscL, the mechanosensitive channel forming protein, which shares main structural features with holins of was unable to complement the lysis defect of lDSthf, confirming specitivity of holin function. The functional properties of Hol118 holin from Listeria monocytogenes bacteriophage A118 were analysed in detail. The gene was cloned into lDSthf, whose CIts857 repressor allows precise estimation of the cell lysis event mediated by a cloned holin, through the possibility to heat-induce the lytic cycle in lysogenized E. coli. Native hol118 caused relatively late cell lysis, beginning at 90 min after induction. Lysis could not be prematurely triggered with energy poisons, indicating that the energized membrane does not inhibit permeabilization by this holin. Immunological analyses demonstrated that Hol118 appears in the inner membrane fraction of infected cells 20 min after phage multiplication starts in induced E. coli. Hol118 could also be detected in A118-infected Listeria monocytogenes cells. Since hol118 features a dual start, different N-terminally modified Hol118 variants were tested for differences in lytic properties. Changing the ATGs encoding M1 or M4 into CTG had no significant influence on lysis timing, indicating that these alleles do not assume the effector/inhibitor roles described for S. Toeprinting assays of hol118 mRNA revealed use of an additional ATG start codon at position 40, encoding M14. Using in vitro approaches, we were able to demonstrate that a Hol118(83) variant is actually translated from the hol118 transcripts. This N-terminally truncated holin lacks the first predicted transmembrane domain. Although it appears in the cytoplasma membrane, it is functionally deficient and unable to complement R in lDSthf. Changing the M14-encoding ATG into codons not used as translational starts (M14I, M14L) resulted in an accelerated, premature lysis phenomenon, pointing to an inhibitor function of Hol118(83). This hypothesis was further supported by the observation that hol118(83) expressed in trans also inhibited holin function. This suggests that the first transmembrane domain is indispensable for the permeablization process leading to pore formation. Based on our findings, we propose a new model of holin functional regulation, where the intragenic Hol118(83) acts as an functional inhibitor, and therefore constitutes a key part of the lysis clock of A118. The strict regulation and inhibition of poreforming aids to explain the long latend period of Listeria phage A118, where the onset of lysis under optimal conditions takes approximately 70 min, more than twice the time needed by phage l. The functional properties of Hol500 holin from Listeria monocytogenes bacteriophage A500 was also analysed, and compared to Hol118 and to Hol2438 from Listeria innocua. Native hol500 caused cell lysis, beginning at 60 min after induction of lDSthf::hol500. Here, lysis could be prematurely triggered with energy poisons, indicating that the energized membrane inhibits permeabilization by this holin. N-terminally modified Hol500 variants were tested for differences in lytic properties. Changing M14-encoding ATG into ATT resulted in accelerated cell lysis. Toeprinting assays on hol500 mRNA revealed use of M14 as a translational start pointing to the synthesis of a truncated protein from this position. We have shown that Hol118(83), the intragenic inhibitor of Hol118, can also inhibit Hol500 lysis, which further supports our model for regulation of lysis timing in these very similar Listeria holins. Hol2438 differs from Hol500 in the reduced net charge of the C-terminal domain, due to the lack of one lysine residue at the C-terminal end. This difference had a significant influence on lysis timing, confirming the crucial role for the distal part of the C-terminus of Listeria holins tested in this work.
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Holins are small hydrophobic proteins causing non-specific membrane lesions at the end of bacteriophage multiplication, to promote access of the murein hydrolase to their substrate. We have established a lambda-delta-Sthf genetic system, which enables functional expression of holins from various phages in an isogenic phage lambda background, and allows qualitative evaluation of their ability to support lysis of Escherichia coli cells. Synthesis of holins is under control of native lambda transcr...
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