Molecular mechanism of dual-start holins and anti-holins in virus induced host cell lysis

The holin/endolysin concept of phage induced host cell lysis specifies that holins implement cell lysis indirectly by formation of a non-specific lesion in the cytoplasmic membrane through which the endolysin is released to the periplasm at the exact time determined in the developmental program of the phage. Despite the regulatory events governing expression of holin genes at the transcriptional and translational levels, holins appear to be primarily regulated at the level of protein function. Intrinsic timing functions in the lambda S holin have been ascribed to its N- and C-terminus and to its transmembrane domains. The lambda S holin represents the prototype of holins with a dual-start motif. These holin genes code for a lysis-inhibitor (S107) and lysis-effector (S105) differ only by two amino-acid residues at the beginning of the longer product, the two proteins habe opposing function in lysis: S105 serves as the lethal hole-former, whereas S107 acts as an inhibitor of S105. Thus, intrinsic timing functions as well as the dual-start motif. Using genetic, biochemical and recombinant DNA methodologies, we will test whether the operational distinction between S105 and S107 is the difference in the translocation kinetics of their N-termini across the cytoplasmic membrane. In other phage the inhibitor of holin function appears to be encoded by separate genes. We have identified a putative anti-holin from Lactobacillus gasseri phage Phiadh. We will test whether it restricts a possible second function of the Phiadh holin: activation of the membrane associated Phiadh endolysin. In further studies we will attempt to establish an inducible promoter/repressor system from the temperature sensitive Lactobacillus casei phage PhiFSWT1. We will first identify the PhiFSWT1 holin/endolysin genes and then sequence towards the region responsible for the genetic switch. Using promoter test vectors we will try to identify a promoter(s) and a cognitive repressor. If the system behaves as expected, i.e. repression of a reporter function at the permissive temperature and expression at the elecated temperature, the promoter/repressor cassette will be inserted into a vector for Lactobacilli and we will test whether it can be used for controlled expression of the Phiadh or PhiFSWT1 holin/endolysin cassette. If the holin/endolysin cassette can be maintained in such plasmids, further studies will be performed to test the biotechnological applicability of the holin for the release of intracellular products. Furthermore, we will perform functional studies of the Phiadh and PhiFSWT1 lysis genes in the homologous host strains.