Figure 1.

Quantitative chromosome organization assay. (A) Fluorescent images of sporulating cells containing the YFP forespore reporter (false-colored green) at -7° and the CFP forespore reporter (false-colored red) at -61°. Cells harboring the wild-type DNA translocase (SpoIIIE+) efficiently pump the chromosome and all forespores contain YFP and CFP fluorescence. In the pumping deficient mutant (SpoIIIE36), the fluorescent image provides a “snap-shot” of the organization of the chromosome at the time of polar division. In many cells the CFP reporter at -61° is not polarly localized and these forespores only contain the -7° YFP reporter (carets). Scale bar, 1 μm. (B) Quantitative analysis of the CFP reporter inserted at positions in the chromosome (red circles) relative to the -7° YFP reporter (green circle). Schematics of the three possible outcomes are shown. For simplicity, only the forespore chromosome is diagramed. Two fields of >400 sporangia each were scored for each strain. (C) Schematic representation of the results in B. The closer a reporter is to the origin, the more likely it is present in the head of the axial filament. The dark gray bar marks the “forespore region” identified by Wu and Errington (Wu and Errington, 1998) and the light gray bar and the gray pie wedges in B and D show the region present in the forespore in >50% of sporulating cells based on the data presented here. This region is off-centered from the oriC and symmetrical around the RacA binding sites (ram sites). (D) Chromosome organization in the absence of Soj and Spo0J. The CFP reporter inserted at three positions (-61°, -35°, +28°) was analyzed relative to the -7° YFP reporter. A fourth class of cells was included in the analysis: forespores that fail to trap either reporter. This class is defined as 0% in wild-type cells (see Experimental Procedures).