Abstract
Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda ...development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the
E. coli
nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.
The system of the bacterium Escherichia coli and its virus, bacteriophage lambda, is paradigmatic for gene regulation in cell-fate development, yet insight about its mechanisms and complexities are ...limited due to insufficient resolution of study. Here we develop a 4-colour fluorescence reporter system at the single-virus level, combined with computational models to unravel both the interactions between phages and how individual phages determine cellular fates. We find that phages cooperate during lysogenization, compete among each other during lysis, and that confusion between the two pathways occasionally occurs. Additionally, we observe that phage DNAs have fluctuating cellular arrival times and vie for resources to replicate, enabling the interplay during different developmental paths, where each phage genome may make an individual decision. These varied strategies could separate the selection for replication-optimizing beneficial mutations during lysis from sequence diversification during lysogeny, allowing rapid adaptation of phage populations for various environments.
When the process of cell-fate determination is examined at single-cell resolution, it is often observed that individual cells undergo different fates even when subject to identical conditions. This ...“noisy” phenotype is usually attributed to the inherent stochasticity of chemical reactions in the cell. Here we demonstrate how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the subcellular level. We follow the postinfection decision in bacteriophage lambda at single-virus resolution, and show that a choice between lysis and lysogeny is first made at the level of the individual virus. The decisions by all viruses infecting a single cell are then integrated in a precise (noise-free) way, such that only a unanimous vote by all viruses leads to the establishment of lysogeny. By detecting and integrating over the subcellular “hidden variables,” we are able to predict the level of noise measured at the single-cell level.
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► Each infecting λ phage makes an independent choice between lysis and lysogeny ► Cell lysogeny is pursued only if all infecting phages choose that fate ► The observed single-cell phenotype is much noisier than that of the single phage
Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. ...Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics.
We perform direct numerical simulations of a rigid sphere translating parallel to a flat wall in an otherwise quiescent ambient fluid. A spectral element method is employed to perform the simulations ...with high accuracy. For $Re\,{<}\,100$, we observe the lift coefficient to decrease with both Reynolds number and distance from the wall. In this regime the present results are in good agreement with the low-Reynolds-number theory of Vasseur & Cox (1977), with the recent experiments of Takemura & Magnaudet (2003) and with the simulations of Kim et al. (1993). The most surprising result from the present simulations is that the wall-induced lift coefficient increases dramatically with increasing $Re$ above about 100. Detailed analysis of the flow field around the sphere suggests that this increase is due to an imperfect bifurcation resulting in the formation of a double-threaded wake vortical structure. In addition to a non-rotating sphere, we also simulate a freely rotating sphere in order to assess the importance of free rotation on the translational motion of the sphere. We observe the effect of sphere rotation on lift and drag forces to be small. We also explore the effect of the wall on the onset of unsteadiness.
Phage P1 is a temperate phage which makes the lytic or lysogenic decision upon infecting bacteria. During the lytic cycle, progeny phages are produced and the cell lyses, and in the lysogenic cycle, ...P1 DNA exists as a low-copy-number plasmid and replicates autonomously. Previous studies at the bulk level showed that P1 lysogenization was independent of
ultiplicity
f
nfection (MOI; the number of phages infecting a cell), whereas lysogenization probability of the paradigmatic phage λ increases with MOI. However, the mechanism underlying the P1 behavior is unclear. In this work, using a fluorescent reporter system, we demonstrated this P1 MOI-independent lysogenic response at the single-cell level. We further observed that the activity of the major repressor of lytic functions (C1) is a determining factor for the final cell fate. Specifically, the repression activity of P1, which arises from a combination of C1, the anti-repressor Coi, and the corepressor Lxc, remains constant for different MOI, which results in the MOI-independent lysogenic response. Additionally, by increasing the distance between phages that infect a single cell, we were able to engineer a λ-like, MOI-dependent lysogenization upon P1 infection. This suggests that the large separation of coinfecting phages attenuates the effective communication between them, allowing them to make decisions independently of each other. Our work establishes a highly quantitative framework to describe P1 lysogeny establishment. This system plays an important role in disseminating antibiotic resistance by P1-like plasmids and provides an alternative to the lifestyle of phage λ.
Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. In this work, we built a simple genetic model to elucidate this MOI independency based on the gene-regulatory circuitry of P1. We also proposed that the effective communication between coinfecting phages contributes to the lysis-lysogeny decision-making of P1 and highlighted the significance of spatial organization in the process of cell fate determination in a single-cell environment. Finally, our work provides new insights into different strategies acquired by viruses to interact with their bacterial hosts in different scenarios for their optimal survival.
Reliable information on forces on a finite-sized particle in a turbulent boundary layer is lacking, so workers continue to use standard drag and lift correlations developed for a laminar flow to ...predict drag and lift forces. Here we consider direct numerical simulations of a turbulent channel flow over an isolated particle of finite size. The size of the particle and its location within the turbulent channel are systematically varied. All relevant length and time scales of turbulence, attached boundary layers on the particle, and particle wake are faithfully resolved, and thus we consider fully resolved direct numerical simulations. The results from the direct numerical simulation are compared with corresponding predictions based on the standard drag relation with and without the inclusion of added-mass and shear-induced lift forces. The influence of turbulent structures, such as streaks, quasi-streamwise vortices and hairpin packets, on particle force is explored. The effect of vortex shedding is also observed to be important for larger particles, whose Re exceeds a threshold.
Bacteriophage P1 is the premier transducing phage of
. Despite its prominence in advancing
genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we ...report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes:
,
,
,
, and
. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery.
Here we consider the effect of a finite-sized stationary particle in a channel flow of modest turbulence at
Re
τ
=
178.12
. The size of particle is varied such that the particle Reynolds number ...ranges from about 40 to 450. The location of the particle is chosen to be either in the buffer layer
(
y
p
+
=
17.81
)
or at the channel center. Fully resolved direct numerical simulations of the turbulent channel flow around the particles is performed. Here the ambient turbulence intensity relative to the mean velocity seen by the particle is large
(
I
=
23.16
%
)
in the buffer region, while it is substantially lower
(
I
=
4.09
%
)
at the channel center. We present results on turbulence modulation due to the particle in terms of wake dynamics and vortex shedding.
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