BackgroundSpanins are phage lysis proteins required to disrupt the outer membrane. Phages employ either two-component spanins or unimolecular spanins in this final step of Gram-negative host lysis. Two-component spanins like Rz-Rz1 from phage lambda consist of an integral inner membrane protein: i-spanin, and an outer membrane lipoprotein: o-spanin, that form a complex spanning the periplasm. Two-component spanins exist in three different genetic architectures; embedded, overlapped and separated. In contrast, the unimolecular spanins, like gp11 from phage T1, have an N-terminal lipoylation signal sequence and a C-terminal transmembrane domain to account for the topology requirements. Our proposed model for spanin function, for both spanin types, follows a common theme of the outer membrane getting fused with the inner membrane, effecting the release of progeny virions.ResultsHere we present a SpaninDataBase which consists of 528 two-component spanins and 58 unimolecular spanins identified in this analysis. Primary analysis revealed significant differences in the secondary structure predictions for the periplasmic domains of the two-component and unimolecular spanin types, as well as within the three different genetic architectures of the two-component spanins. Using a threshold of 40% sequence identity over 40% sequence length, we were able to group the spanins into 143 i-spanin, 125 o-spanin and 13 u-spanin families. More than 40% of these families from each type were singletons, underlining the extreme diversity of this class of lysis proteins. Multiple sequence alignments of periplasmic domains demonstrated conserved secondary structure patterns and domain organization within family members. Furthermore, analysis of families with members from different architecture allowed us to interpret the evolutionary dynamics of spanin gene arrangement. Also, the potential universal role of intermolecular disulfide bonds in two-component spanin function was substantiated through bioinformatic and genetic approaches. Additionally, a novel lipobox motif, AWAC, was identified and experimentally verified.ConclusionsThe findings from this bioinformatic approach gave us instructive insights into spanin function, evolution, domain organization and provide a platform for future spanin annotation, as well as biochemical and genetic experiments. They also establish that spanins, like viral membrane fusion proteins, adopt different strategies to achieve fusion of the inner and outer membranes.Electronic supplementary materialThe online version of this article (10.1186/s12859-018-2342-8) contains supplementary material, which is available to authorized users.

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.spanin | membrane fusion | spheroplast | holin | endolysin P hage lysis, the most common cytolytic event in the biosphere, has been extensively studied in phage λ, where four genes encoding five proteins (Fig. 1A) effect a three-step lytic process that releases the progeny virions (1, 2). The infection cycle suddenly terminates when the S105 holins, small membrane proteins encoded by gene S, are redistributed into large 2D foci, resulting in the formation of μm-scale holes in the cytoplasmic membrane (3). This event, called holin "triggering," occurs at a time specific to the allelic state of S and is temporally regulated by the proportion of a second S product, the antiholin S107 (4, 5). The R endolysin is then able to escape through the holes to attack the peptidoglycan (PG). Because the PG layer confers shape and mechanical integrity to the cell, holin and endolysin function was long thought to be necessary and sufficient for lysis (6, 7). However, recent genetic and physiological studies revealed that two other λ proteins, Rz and Rz1, are also required (Fig. 1B) (8). Rz and Rz1 are a type II integral membrane protein (N-in, C-out) and an outer membrane (OM) lipoprotein, respectively (9-11). Interacting by the C-termini of their periplasmic domains, Rz and Rz1 form a complex spanning the entire periplasm, designated as the spanin complex to denote its topology in the envelope. Accordingly, Rz and Rz1 are designated as the inner membrane (IM) (i-spanin) and OM (o-spanin) subunits of the spanin complex ( Fig. 1 A and B) (12). Experiments with GFP-Rz chimeras and biochemical analysis of envelope proteins indicate that the spanin complexes accumulate in the envelope throughout the morphogenesis period of the infection cycle (8, 13). The available data indicate that, after destruction of the PG by the endolysin, the spanin complex functions to disrupt the OM. In the absence of spanin function, the infection cycle termin...

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.

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