Emerging heterogeneous compartments by viruses in single bacterial cells.

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.

Complete Genome Sequence of Citrobacter freundii Myophage Maroon.

Citrobacter freundii is a nosocomial opportunistic pathogen that can cause urinary and bloodstream infections. Phage therapies against C. freundii may prove useful in treating infections caused by this ubiquitous bacterium. Here, we report the complete genome of a T4-like myophage, Maroon, that infects C. freundii.

High-resolution studies of lysis-lysogeny decision-making in bacteriophage lambda.

Cellular decision-making guides complex development such as cell differentiation and disease progression. Much of our knowledge about decision-making is derived from simple models, such as bacteriophage lambda infection, in which lambda chooses between the vegetative lytic fate and the dormant lysogenic fate. This paradigmatic system is broadly understood but lacking mechanistic details, partly due to limited resolution of past studies. Here, we discuss how modern technologies have enabled high-resolution examination of lambda decision-making to provide new insights and exciting possibilities in studying this classical system. The advent of techniques for labeling specific DNA, RNA, and proteins in cells allows for molecular-level characterization of events in lambda development. These capabilities yield both new answers and new questions regarding how the isolated lambda genetic circuit acts, what biological events transpire among phages in their natural context, and how the synergy of simple phage macromolecules brings about complex behaviors.

Coupling of DNA Replication and Negative Feedback Controls Gene Expression for Cell-Fate Decisions.

Cellular decision-making arises from the expression of genes along a regulatory cascade, which leads to a choice between distinct phenotypic states. DNA dosage variations, often introduced by replication, can significantly affect gene expression to ultimately bias decision outcomes. The bacteriophage lambda system has long served as a paradigm for cell-fate determination, yet the effect of DNA replication remains largely unknown. Here, through single-cell studies and mathematical modeling we show that DNA replication drastically boosts cI expression to allow lysogenic commitment by providing more templates. Conversely, expression of CII, the upstream regulator of cI, is surprisingly robust to DNA replication due to the negative autoregulation of the Cro repressor. Our study exemplifies how living organisms can not only utilize DNA replication for gene expression control but also implement mechanisms such as negative feedback to allow the expression of certain genes to be robust to dosage changes resulting from DNA replication.

Cell fate decisions emerge as phages cooperate or compete inside their host.

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.

Lysis-lysogeny coexistence: prophage integration during lytic development.

The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis-lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, these two distinct pathways can be visualized by following the DNA movements in vivo. Surprisingly, we frequently observed an interesting "lyso-lysis" phenomenon in lytic cells, where phage integrates its DNA into the host, a characteristic event of the lysogenic pathway, followed by cell lysis. Furthermore, the frequency of lyso-lysis increases with the number of infecting phages, and specifically, with CII activity. Moreover, in lytic cells, the integration site attB on the E. coli genome migrates toward the polar region over time, leading to more spatial overlap with the phage DNA and frequent colocalization/collision of attB and phage DNA, possibly contributing to a higher chance for DNA integration.

Phage DNA dynamics in cells with different fates.

Bacteriophage λ begins its infection cycle by ejecting its DNA into its host Escherichia coli cell, after which either a lytic or a lysogenic pathway is followed, resulting in different cell fates. In this study, using a new technique to monitor the spatiotemporal dynamics of the phage DNA in vivo, we found that the phage DNA moves via two distinct modes, localized motion and motion spanning the whole cell. One or the other motion is preferred, depending on where the phage DNA is ejected into the cell. By examining the phage DNA trajectories, we found the motion to be subdiffusive. Moreover, phage DNA motion is the same in the early phase of the infection cycle, irrespective of whether the lytic or lysogenic pathway is followed; hence, cell-fate decision-making appears not to be correlated with the phage DNA motion. However, after the cell commits to one pathway or the other, phage DNA movement slows during the late phase of the lytic cycle, whereas it remains the same during the entire lysogenic cycle. Throughout the infection cycle, phage DNA prefers the regions around the quarter positions of the cell.

Structure of a novel O-linked N-acetyl-D-glucosamine (O-GlcNAc) transferase, GtfA, reveals insights into the glycosylation of pneumococcal serine-rich repeat adhesins.

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a ß-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides.