Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.
ABSTRACT Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. Recently, the holin S gene of phage λ was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D. L. Smith, D. K. Struck, J. M. Scholtz, and R. Young, J. Bacteriol. 180:2531–2540, 1998). Numerous locations within the S gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function. The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized. A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein. Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function. The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required.
Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.
Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein. In an event that defines the end of the vegetative cycle, the lambda holin S acts suddenly to permeabilize the membrane. This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds. A C-terminal fusion of the R endolysin with full-length beta-galactosidase (beta-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced lambda lysogen. Under these conditions, the hybrid R-beta-Gal product, an active tetrameric beta-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin. Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-beta-Gal fusion protein. The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore. Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context. However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane. Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin. A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed.
Abstract In contrast to dsDNA phages where multiple proteins are involved in programmed host lysis, lysis in ssRNA Fiersviridae and ssDNA Microviridae phages requires only a single gene ( sgl for s ingle g ene l ysis ) to meet the size constraints of some of the smallest genomes in the biosphere. To achieve lysis, Sgl proteins exploit evolutionary “weak spots” in bacterial cell wall biogenesis. In several cases, this is done by inhibiting specific steps in Lipid II synthesis. Recently metatranscriptomics has revealed thousands of novel ssRNA phage genomes, each of which must carry at least one sgl gene. Determining the targets of these Sgl proteins could reveal novel vulnerabilities in bacterial envelope biogenesis and may lead to new antibiotics. Here, we employ a high-throughput genetic screen to uncover genome-wide host suppressors of Sgl activity and apply it to a set of diverse Sgls with unknown molecular targets. In addition to validating known molecular mechanisms, we determined that the Sgl of PP7, an ssRNA phage of P. aeruginosa , targets MurJ, the flippase responsible for Lipid II export which was previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. Another set of Sgls which are thought to cause lysis without inhibiting cell wall synthesis elicit a common set of multicopy suppressors, suggesting these Sgls act by the same or similar mechanism.
The λ Rz and Rz1 proteins are the subunits of the spanin complex, required for the disruption of the outer membrane during host lysis. Rz, the inner membrane or i-spanin, has a largely alpha-helical periplasmic domain, whereas Rz1, the outer membrane or o-spanin, has a 25% proline content with no predicted secondary structure. We report that both Rz and Rz1 accumulate as homodimers covalently linked by intermolecular disulfide bonds involving all three Cys residues, two in Rz and one in Rz1. Moreover, of these three intermolecular disulfides, spanin function requires the presence of at least one of the two linkages nearest the Rz-Rz1 C-terminal interaction domains; i.e. either the Rz1-Rz1 disulfide or the distal Rz-Rz disulfide link. In a dsbC host, but not in dsbA or dsbA dsbC hosts, formation of the covalent homodimers of Rz is severely reduced and outer membrane disruption is significantly delayed, suggesting that the spanin pathway normally proceeds through DsbA-mediated formation of an intramolecular disulfide in Rz. In contrast, efficient formation of the Rz1-Rz1 disulfide requires DsbA. Finally, Dsb-independent formation of the covalent homodimer of either subunit requires the presence of the other, presumably as a template for close apposition of the thiols.
