Microscopy

Strains were streaked from glycerol stocks onto individual LB plates containing the appropriate antibiotics and incubated overnight at 30°C. The following morning, cells were removed from the plates by washing with 1 mL of S₇₅₀+2% glucose (WT and deletion strains) or S₇₅₀+1% arabinose (overexpression strains). Optical densities were measured, and cell suspensions were normalized to OD₆₀₀ = 1.0 in the same media. 250 μL of culture was subsequently inoculated into 4.75 mL of media (C_f = 0.05) in a 50 mL flask. Cultures were incubated at 30°C with shaking (225 RPM) until OD₆₀₀ reached 0.2 and then 0.1% xylose was added to overexpression strains; 100 ng/mL MMC was added for experiments requiring DNA damage. Once OD₆₀₀ reached 0.5–0.7, 300 μL of culture was removed and FM4-64 was added to 2 μg/mL and incubated at room temperature for 5 minutes. Samples were then transferred to 1% agarose pads made of 1x S₇₅₀ salts. Images were captured using an Olympus BX61 microscope as described [58,59]. Cell length histograms were created using ggplot2 in R studio. Shapiro-Wilk tests were performed to assess whether the data between strains were normally distributed. Wilcoxon rank-sum tests were performed to assess statistical significance.

tagC-GFP microscopy: WT, ccrZ, and overexpression were streaked onto spectinomycin or spectinomycin and chloramphenicol (overexpression) and incubated at 30°C for 16 hours. The following morning, WT and ccrZ were plate washed with S₇₅₀+2%glucose, OD₆₀₀ was normalized to 1.0, and normalized culture was inoculated at OD₆₀₀ = 0.1 into 3 mL minimal medium in 15 mL test tubes. The overexpression plate was washed with S₇₅₀+1%arabinose, normalized, and inoculated in the same fashion as the other strains. 0.1% xylose was added before incubation to induce ccrZ. All cultures were incubated at 30°C in a 225 RPM rolling rack. Once the cultures reached a density between 0.7–0.9, cells were imaged. RFP was captured at ~300 ms exposure and GFP was captured at ~800 ms. Analysis was performed using ImageJ. A Kruskal-Wallis test was used to determine whether means of GFP positive cells differed according to genotype. Subsequently, paired Wilcoxon tests were performed with a Benjamini-Hochberg correction to determine pairwise significance.