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 λ 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 λ lysogens carrying prophages with either the λ 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 GFPΦRz 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.

Fig 1

Topology and localization of λ and phage 21 lysis genes. The λ holin (S105) and endolysin (R) are localized to the IM and cytoplasm, respectively. The holin protein oligomerizes in the IM to form large lesions that enable the R protein to diffuse into the periplasmic space, where its substrate, the PG, is located. The lipopolysaccharide leaflet of the OM is indicated by a darker shade of gray. For simplicity, the PG layer and IM holes are not illustrated here. The phage 21 pinholin (S²¹) and SAR endolysin (R²¹) are both initially localized to the IM. Both S²¹ and R²¹ exhibit dynamic membrane topology, as indicated by an arrowhead and dotted line. The first transmembrane domain of S²¹ is a SAR domain, and it exits the bilayer as a result of S²¹ hole formation. Irreversible hole formation by S²¹ collapses the IM energy gradient, thus triggering global release of the single N-terminal SAR domain of R²¹. Release of the R²¹ SAR domain from the IM results in a periplasmic localization of the R²¹ enzyme. For simplicity, the spanin subunits of both λ and phage 21 are represented by the same illustration, as the respective IM and OM subunit proteins sequences are 97% and 95% identical. The spanin is illustrated here as a dimer based on unpublished results and reference . The IM spanin (λRz or Rz²¹) has a single N-terminal transmembrane domain with an N_in/C_out topology and an extended and primarily alpha-helical periplasmic domain. The approximate position of a predicted hinge region for the periplasmic domain of the IM spanin is indicated by a gray shaded box. The OM spanin (λRz1 or Rz1²¹) is a small (40-aa) OM lipoprotein that interacts directly with the C-terminal end of the IM spanin via its own periplasmic domain. The OM spanin subunit is tethered to the inner leaflet of the OM following the posttranslational addition of 3 N-terminal fatty acid moieties (solid black lines). The function of the spanin is OM disruption.

Fig 2

The spanin complex is required for rapid lysis. (A to H) MC4100 lysogens were thermally induced, and 1 min prior to the onset of lysis, a field of cells was observed with phase-contrast microscopy in order to document the morphology of phage lysis. Time lapse series of individual cells beginning immediately prior to and continuing through completion of morphological conversion are shown. Prophages and relevant genotypes are indicated above each group of time series. Time (in seconds) is displayed above each individual frame, with time zero representing the first available frame prior to an observable morphological change or lysis. Bars, 5 μm. (I) Proposed models for holin-endolysin (left) and pinholin-SAR endolysin (right) action prior to spanin function and lysis. The holin accumulates and forms near-micrometer-scale lesions in the IM (irregular white circle with black border), resulting in a localized release of R (notched circles). The pinholin forms a heptameric pore (small white circle) approximately 2 nm in diameter. Pore formation collapses the membrane potential and causes release of the IM-tethered SAR endolysin (notched circle with black bar) to the periplasm. See the text for further details. Note that the PG peptide cross-links and OM are omitted for simplicity.

Fig 3

Saltatory λRz_am/Rz1_am lysis is an artifact. The effect of various physiological parameters on cell lysis was assessed following induction of λ prophages. (A) Shearing forces attendant on flask shaking complement the λRz_am/Rz1_am lysis defect. The following prophage inductions were conducted with constant shaking: λ (open circles), λRz_am/Rz1_am in LB plus 10 mM Mg²⁺ (closed diamonds), and λRz_am/Rz1_am (open diamonds). For the following prophage inductions, culture shaking was ceased at 45 min after induction: λ (closed circles) and λRz_am/Rz1_am (closed triangles). (B) The Mg²⁺-dependent lysis defect is complemented by growth in a baffled flask. For the λ (circles) and λRz_am/Rz1_am (squares) inductions, shaking was ceased and the temperature decreased to 30°C 45 min postinduction. The λRz_am/Rz1_am (diamonds) prophage was induced in a baffled flask in the presence of 10 mM Mg²⁺. (C) Late-log-phase induction leads to a Mg²⁺-independent lysis defect. For the following prophage inductions, culture shaking was ceased at 45 min after induction: λ (closed circles) and λRz_am/Rz1_am (closed squares). The following prophage inductions were subjected to constant shaking: λ (open circles), λRz_am/Rz1_am in LB plus 10 mM Mg²⁺ (closed diamonds) and λRz_am/Rz1_am (open squares).

Fig 4

Deconvolution fluorescence imaging of GFPΦRz reveals the oligomeric state prior to PG degradation. Rows A and B represent samples collected 60 min after induction of GFPΦRz and λS_am7Rz_am/Rz1_am or λS_am7Rz_am/Rz1, respectively. Row C is a deconvoluted z-stack series (step size, 0.2 μm) of GFPΦRz fluorescence following λRz_am/Rz1 induction 60 min earlier. Bar (inset), 5 μm.