The Spanin Complex Is Essential for Lambda Lysis

Abstract

Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced lysogens carrying prophages with either the canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFP⌽Rz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.

Membrane fusion during phage lysis

“…Instead, the spanin complexes can cause fusion upon partial depletion of the PG imposed by starvation for D-Ala, even when the PG is sufficiently intact to preserve the rod-shaped cell morphology. These results support our interpretation of the dramatic "local blowout" morphology characteristic of λ lysis (8). We have suggested that endolysin molecules escaping through the μm-scale IM holes formed by the triggered holin population initially degrade the patch of the PG over the hole.…”

“…Cultures induced for the fusion assay (see Spheroplast Fusion Assay and Fluorescence Microscopy below) were chemically induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Phase-contrast microscopy was conducted as described previously (8), except that an EC Plan-Neofluor 100×/1.3 oil Ph3/∞/0.17 objective was used to capture the images. For imaging, a 1 mL sample of growing culture was harvested by centrifugation at 9,500 × g for 1 min and immediately resuspended in 100 μL of LB, of which 1 μL was spotted on the ethanol-washed glass slide and covered with the 22 × 22 mm coverslip before mounting on the stage for the inverted microscope objective.…”

“…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.…”

“…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).…”

Stochastic holin expression can account for lysis time variation in the bacteriophageλ

“…Holin accumulates evenly in the Escherichia coli plasma membrane until a genetically encoded time when they spontaneously aggregate to form large holes [31]. These holes allow endolysin and the spanins to access and disrupt their targets beyond the periplasmic space [32]. Subsequently, the cell ruptures and phage progeny are released into the surrounding medium.…”