Spanin function requires subunit homodimerization through intermolecular disulfide bonds

Berry, Joel D.; Rajaure, Manoj; Young, Ry

doi:10.1111/mmi.12167

Cited by 29 publications

(46 citation statements)

References 26 publications

(57 reference statements)

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“…S7, Inset). The periplasmic domain of gp11 lacks the characteristic features of the two-component spanins, including intermolecular disulfide bonds and predicted coiled-coil helical structure (12,13), and is instead predicted to be dominated by β-strand structures (14). In work to be described elsewhere, gp11 has also been shown to promote spheroplast fusion, which should allow comparative genetic and biochemical studies to focus on the molecular features essential for membrane fusion.…”

Section: Discussion Membrane Fusion As the Lastmentioning

confidence: 99%

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Membrane fusion during phage lysis

Rajaure

Berry

Kongari

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussion Membrane Fusion As the Lastmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

Membrane fusion during phage lysis

Rajaure

Berry

Kongari

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Lysis was monitored as Sample collection for Western blotting. A 1-ml aliquot of whole-cell sample was taken 45 min after induction, precipitated by trichloroacetic acid (TCA), and prepared for Western blotting as described previously (8,10). Briefly, TCA pellets were resuspended in 1ϫ SDS-PAGE sample loading buffer in a volume normalized to the A 550 units recorded at the time of collection.…”

Section: Synthesis Of Disembedded Rz and Rz1 Genesmentioning

confidence: 99%

“…By phasecontrast video microscopy, striking kinetic defects can be readily observed at the single-cell level for spanin alleles that are normal in terms of bulk lysis and plaque formation (8). In the wild-type context, lysis is complete within a few seconds, whereas cells expressing spanin mutant alleles form a spherical intermediate prior to lysis (Fig.…”

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Suppressor Analysis of the Fusogenic Lambda Spanins

Cahill

Rajaure

Holt

et al. 2017

J Virol

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The final step of lysis in phage infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiledcoil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo. Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-ornothing functionality.IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre-and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system.

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The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Roszniowski

Łątka

Maciejewska

et al. 2016

Appl Microbiol Biotechnol

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Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7–49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29–98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4–10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7924-7) contains supplementary material, which is available to authorized users.

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Spanin function requires subunit homodimerization through intermolecular disulfide bonds

Cited by 29 publications

References 26 publications

Membrane fusion during phage lysis

Membrane fusion during phage lysis

Suppressor Analysis of the Fusogenic Lambda Spanins

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

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