Figure 5. Analysis of SOS at a single cell level in the absence of anionic PLs.

(A) Measurement of RecA bundle lengths 1.5 h after treatment with 1X MIC of norfloxacin shows that in WT (MG1655), >20% of bundles are >2 μm. These RecA bundles are 15% longer than in BKT29 in which ~85% RecA bundles are typically <1.5 μm. (B) ~60% of bundles display a straight morphology (eccentricity = 1) in WT compared to ~20% in BKT29. (C) Representative SIM images showing bundles in WT, and punctate, ellipsoidal RecA- GFP structures in BKT29. (D) Using a sulA-promoter fusion to GFP, we observed that BKT29 has a reduced SOS response compared to WT when treated with 1X MIC of norfloxacin. This difference is also observed at 3X MIC. Shaded area represents SD of three biological replicates, with line and mean. (E) The length of RecA bundles within the cell is positively correlated to the degree of SOS induced as observed by a recN-mCherry promoter fusion expressed with RecA-GFP. Shaded area indicates 95% confidence intervals of a linear regression fit. (F) Representative SIM image showing the overlay of SOS intensity with RecA bundles for WT and BKT29. White arrows indicate RecA bundles. Most cells show increased SOS (orange-white) when long bundles are present. The image displays the maximum intensity within one frame, which is a subset of the population of cells analyzed. Some outliers exist for BKT29 (seen in the accompanying images) in which short filaments have high levels of SOS and are visible in the plot. Because cells are also normalized by total area, longer cells with SOS intensities equal to shorter cells will have an overall higher SOS response. Cells that are blue indicate a lower level of SOS. Also see Fig S6 and Fig S7.