| dc.description.abstract | Cellular decision-making is a ubiquitous biological process among all organisms, from simple viruses to complex mammals, occurring when living systems process signals received from the environment and then make appropriate responses for survival. Bacteriophage lambda and its bacterial host E. coli have served as a basic paradigmatic system for understanding cellular decision-making. Upon infection by phage λ, an E. coli bacterium undergoes either of two alternative life cycles, lysis or lysogeny. Despite extensive efforts to uncover the underlying decision-making mechanisms, several hidden variables have yet been characterized, rendering cell-fate decisions mysterious and unpredictable. The λ genetic circuit composed of fate-determining genes drives the lysis-lysogeny decision. It has been suggested that the fluctuations in viral gene expression can cause dramatic changes in cell-fate determination. To evaluate the effect of stochastic gene activity on λ decision-making, we quantify the transcriptional level of the λ cII gene at the single-cell level, as CII is the master regulator during the decision-making process. We reveal that the average cII mRNA levels increase and reach a peak around 10 min after infection, and subsequently drop. Next, by labeling individual phage genomes, we investigate the intracellular organization of phage DNA development in single cells.
We observe that infecting phage DNA can organize their own DNA replication and gene expression by assembling separate entities within a single cell. This phage individuality corroborates our previous hypothesis that each phage DNA has the capability of making decisions independently. Furthermore, we provide evidence of heterogeneity in phage subcellular development, most likely as a result of intracellular phage-phage interactions. Additionally, when the effect of the side tail fibers on λ infection is examined, those side tail fibers significantly reduce the ability of λ for successful infection but do not affect the lysis-lysogeny decision-making outcome. Meanwhile, we visualize viral DNA degradation by a bacterial CRISPR-Cas system at the single-cell level. We quantitatively characterize several factors accounting for effective CRISPR defense and hypothesize that phages can combat the CRISPR system through rapid DNA replication and co-infections. These findings enrich our current understanding of the mechanistic basis of CRISPR-Cas immune systems. | en |
