Transposition mutagenesis of bacteriophage lambda

Young, Ry; Way, Jeffrey C.; Way, Susan Van; Yin, Jie; Syvanen, Michael

doi:10.1016/0022-2836(79)90262-6

Cited by 92 publications

(72 citation statements)

References 17 publications

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“…As shown in Fig. 1B, in the absence of Rz or Rz1 function, lysing cells can be stabilized in this spherical morphology if millimolar concentrations of divalent cations are supplied in the medium (21,22). Samples taken from an induced pSR culture in medium supplemented with 10 mM MgCl 2 were imaged and found to contain a significantly larger number of intact yet spherical cells similar to the rare unlysed cells in the absence of metal ions.…”

Section: Resultsmentioning

confidence: 99%

Micron-scale holes terminate the phage infection cycle

Dewey¹,

Savva

White³

et al. 2010

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

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Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...

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Section: Resultsmentioning

confidence: 99%

Micron-scale holes terminate the phage infection cycle

Dewey¹,

Savva

White³

et al. 2010

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

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“…Both genes are necessary for lysis in liquid culture if the medium is supplemented with millimolar concentrations of divalent cations. 4,5 Under these conditions, defects in either Rz or Rz1 block lysis and lead to the accumulation of spherical, mechanically fragile cells. This morphology and the dependency of the phenotype on divalent cations suggest that the third step is an Rz-Rz1-dependent disruption of the OM.…”

Section: Introductionmentioning

confidence: 99%

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

et al. 2010

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Phage holins and endolysins have long been known to play key roles in lysis of the host cell, disrupting the cytoplasmic membrane and peptidoglycan (PG) layer, respectively. For phages of Gram-negative hosts, a third class of proteins, the spanins, are involved in disrupting the outer membrane (OM). Rz and Rz1, the components of the lambda spanin, are, respectively, a class II inner membrane protein and an OM lipoprotein, are thought to span the entire periplasm by virtue of C-terminal interactions of their soluble domains. Here, the periplasmic domains of Rz and Rz1 have been purified and shown to form dimeric and monomeric species, respectively, in solution. Circular dichroism analysis indicates that Rz has significant alpha-helical character, but much less than predicted, whereas Rz1, which is 25% proline, is unstructured. Mixture of the two proteins leads to complex formation and an increase in secondary structure, especially alpha-helical content. Moreover, transmission electron-microscopy reveals that Rz-Rz1 complexes form large rod-shaped structures which, although heterogeneous, exhibit periodicities that may reflect coiledcoil bundling as well as a long dimension that matches the width of the periplasm. A model is proposed suggesting that the formation of such bundles depends on the removal of the PG and underlies the Rz-Rz1 dependent disruption of the OM.

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“…The R, or endolysin, gene encodes a soluble transglycosylase which accumulates intracellularly throughout the late protein synthesis phase of the infective cycle (4,13). The Rz gene has an unknown function and is not required for lysis under laboratory conditions, except when the outer membrane is stabilized with high concentrations of divalent cations (8,46). The S gene encodes a small (M r ϭ 8,500) inner membrane protein which acts to permeabilize the membrane in some way to allow endolysin access to the periplasm and its substrate, the peptidoglycan (2,3,15,16).…”

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S gene expression and the timing of lysis by bacteriophage lambda

1995

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The S gene of bacteriophage encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2. . . N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

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Transposition mutagenesis of bacteriophage lambda

Cited by 92 publications

References 17 publications

Micron-scale holes terminate the phage infection cycle

Micron-scale holes terminate the phage infection cycle

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

S gene expression and the timing of lysis by bacteriophage lambda

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