Abstract
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.
Highlights
Spanins are phage lysis proteins required to disrupt the outer membrane
Identification of spanins and implications for automated phage annotation We restricted our primary search to RefSeq genomes of phages of Gram-negative hosts
Spanins have only been recently characterized, compared to the long history of studies on other lysis proteins, and essentially all experimental work has been done with the lambda Rz and Rz1 genes only
Summary
Spanins are phage lysis proteins required to disrupt the outer membrane. Phages employ either twocomponent spanins or unimolecular spanins in this final step of Gram-negative host lysis. It has long been thought that holin-endolysin function was necessary and sufficient to effect bacteriophage lysis and achieve liberation of progeny virions [1,2,3,4], except in phages of the mycolata, where a third functional class, the Lysin B esterases, are essential to degrade the waxy outer membrane [5, 6] Another functional class of proteins, the spanins, have been shown to be required for disruption of the outer membrane (OM) in Gram-negative hosts [7,8,9,10]. This breach in the IM allows the endolysins to escape the cytoplasm and attack the PG (Fig. 1a)
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