Holin triggering in real time

White, R. W. G.; Chiba, Shinobu; Pang, Ting; Dewey, Jill S.; Savva, Christos G.; Holzenburg, Andreas; Pogliano, Kit; Young, Ry

doi:10.1073/pnas.1011921108

Cited by 138 publications

(159 citation statements)

References 35 publications

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“…Subsequently the cell lysis and phage progeny are released into the surrounding medium. Because hole formation and cell rupture are nearly simultaneous, lysis timing depends on de novo expression and accumulation of holin in the cell membrane up to a critical threshold (45). Data reveal precision in the timing of lysis-individual cells infected by a single virus lyse on average at 65 min, with an SD of 3.5 min, implying a coefficient of variation of ≈ 5% (SI Appendix, section S9).…”

Section: Discussionmentioning

confidence: 95%

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First-passage time approach to controlling noise in the timing of intracellular events

Ghusinga

Dennehy

Singh

2017

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

113

127

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In the noisy cellular environment, gene products are subject to inherent random fluctuations in copy numbers over time. How cells ensure precision in the timing of key intracellular events despite such stochasticity is an intriguing fundamental problem. We formulate event timing as a first-passage time problem, where an event is triggered when the level of a protein crosses a critical threshold for the first time. Analytical calculations are performed for the first-passage time distribution in stochastic models of gene expression. Derivation of these formulas motivates an interesting question: Is there an optimal feedback strategy to regulate the synthesis of a protein to ensure that an event will occur at a precise time, while minimizing deviations or noise about the mean? Counterintuitively, results show that for a stable long-lived protein, the optimal strategy is to express the protein at a constant rate without any feedback regulation, and any form of feedback (positive, negative, or any combination of them) will always amplify noise in event timing. In contrast, a positive feedback mechanism provides the highest precision in timing for an unstable protein. These theoretical results explain recent experimental observations of single-cell lysis times in bacteriophage λ. Here, lysis of an infected bacterial cell is orchestrated by the expression and accumulation of a stable λ protein up to a threshold, and precision in timing is achieved via feedforward rather than feedback control. Our results have broad implications for diverse cellular processes that rely on precise temporal triggering of events.first-passage time | event timing | stochastic gene expression | feedback control | single cell T iming of events in many cellular processes, such as cell-cycle control (1-3), cell differentiation (4, 5), sporulation (6, 7), apoptosis (8, 9), development (10, 11), temporal order of gene expression (12)(13)(14), and so on, depend on regulatory proteins reaching critical threshold levels. Triggering of these events in single cells is influenced by fluctuations in protein levels that arise naturally due to noise in gene expression (15)(16)(17)(18). Increasing evidence shows considerable cell-to-cell variation in timing of intracellular events among isogenic cells (19-21), and it is unclear how noisy expression generates this variation. Characterization of control strategies that buffer stochasticity in event timing are critically needed to understand reliable functioning of diverse intracellular pathways that rely on precision in timing.Mathematically, noise in the timing of events can be investigated via the first-passage time (FPT) framework, where an event is triggered when a stochastic process (single-cell protein level) crosses a critical threshold for the first time. There is already a rich tradition of using such FPT approaches to study timing of events in biological and physical sciences (22-26). Following this tradition, exact analytical expression for the FPT distribution is computed in experimentally valida...

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Section: Discussionmentioning

confidence: 95%

“…After infecting Escherichia coli, λ expresses a protein, holin, which accumulates in the inner membrane. When holin reaches a critical threshold concentration, it undergoes a structural transformation, forming holes in the membrane (45). Subsequently the cell lysis and phage progeny are released into the surrounding medium.…”

Section: Discussionmentioning

confidence: 99%

First-passage time approach to controlling noise in the timing of intracellular events

Ghusinga

Dennehy

Singh

2017

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

113

127

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“…Following the production of the late mRNA transcript, the lysis proteins are translated by host ribosomes. 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].…”

Section: Introductionmentioning

confidence: 99%

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

Singh

Dennehy

2014

J. R. Soc. Interface.

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The inherent stochastic nature of biochemical processes can drive differences in gene expression between otherwise identical cells. While cell-to-cell variability in gene expression has received much attention, randomness in timing of events has been less studied. We investigate event timing at the single-cell level in a simple system, the lytic pathway of the bacterial virus phage l. In individual cells, lysis occurs on average at 65 min, with an s.d. of 3.5 min. Interestingly, mutations in the lysis protein, holin, alter both the lysis time (LT) mean and variance. In our analysis, LT is formulated as the first-passage time (FPT) for cellular holin levels to cross a critical threshold. Exact analytical formulae for the FPT moments are derived for stochastic gene expression models. These formulae reveal how holin transcription and translation efficiencies independently modulate the LT mean and variation. Analytical expressions for the LT moments are used to evaluate previously published single-cell LT data for l phages with mutations in the holin sequence or its promoter. Our results show that stochastic holin expression is sufficient to account for the intercellular LT differences in both wild-type phages, and phage variants where holin transcription and the threshold for lysis have been experimentally altered. Finally, our analysis reveals regulatory motifs that enhance the robustness of lysis timing to cellular noise.

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“…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).…”

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confidence: 99%

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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Holin triggering in real time

Cited by 138 publications

References 35 publications

First-passage time approach to controlling noise in the timing of intracellular events

First-passage time approach to controlling noise in the timing of intracellular events

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

Membrane fusion during phage lysis

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