Categorizing single cell infection dynamics in the P22–<i>S</i>. Typhimurium model system.

<p>In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the <i>parS</i> sequence engineered in P22 <i>parS</i>. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 <i>parS</i> (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 <i>parS</i> prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 <i>parS</i> chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 <i>parS</i> chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 <i>parS</i> episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 <i>parS</i>, (C) of growing cells from a P22 <i>parS</i> lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 <i>c2 parS</i> (an obligate lytic derivative of P22 <i>parS</i>). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 <i>parS</i> infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 <i>parS</i> prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 <i>parS</i> chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.