Localization and Regulation of the T1 Unimolecular Spanin.

Spanins are bacteriophage lysis proteins responsible for disruption of the outer membrane, the final step of Gram-negative host lysis. The absence of spanins results in a terminal phenotype of fragile spherical cells. The phage T1 employs a unimolecular spanin gp that has an N-terminal lipoylation signal and a C-terminal transmembrane domain. Upon maturation and localization, gp ends up as an outer membrane lipoprotein with a C-terminal transmembrane domain embedded in the inner membrane, thus connecting both membranes as a covalent polypeptide chain. Unlike the two-component spanins encoded by most of the other phages, including lambda, the unimolecular spanins have not been studied extensively. In this work, we show that the gp mutants lacking either membrane localization signal were nonfunctional and conferred a partially dominant phenotype. Translation from internal start sites within the gp coding sequence generated a shorter product which exhibited a negative regulatory effect on gp function. Fluorescence spectroscopy time-lapse videos of gp-GFP expression showed gp accumulated in distinct punctate foci, suggesting localized clusters assembled within the peptidoglycan meshwork. In addition, gp was shown to mediate lysis in the absence of holin and endolysin function when peptidoglycan density was depleted by starvation for murein precursors. This result indicates that the peptidoglycan is a negative regulator of gp function. This supports a model in which gp acts by fusing the inner and outer membranes, a mode of action analogous to but mechanistically distinct from that proposed for the two-component spanin systems. Spanins have been proposed to fuse the cytoplasmic and outer membranes during phage lysis. Recent work with the lambda spanins Rz-Rz1, which are similar to class I viral fusion proteins, has shed light on the functional domains and requirements for two-component spanin function. Here we report, for the first time, a genetic and biochemical approach to characterize unimolecular spanins, which are structurally and mechanistically different from two-component spanins. Considering similar predicted secondary structures within the ectodomains, unimolecular spanins can be regarded as a prokaryotic version of type II viral membrane fusion proteins. This study not only adds to our understanding of regulation of phage lysis at various levels but also provides a prokaryotic genetically tractable platform for interrogating class II-like membrane fusion proteins.