FIG. 4.

The presence or absence of manganese in LB medium has little or no effect on chromosome copy number or the synchrony of replication initiation. Cells were grown in LB medium or LB medium plus 100 μM MnCl₂ at 26°C to an OD₆₀₀ of 0.3 to 0.5 for mid-exponential-phase samples or 37°C until 3 h after the onset of stationary phase for T₃ samples. At the indicated times, a 5-ml aliquot was removed and incubated with 200 μg/ml of chloramphenicol for 6 h to inhibit further rounds of replication from initiating but to allow current rounds to finish. Cells were then fixed in ethanol, washed twice with 200 mM Tris-HCl at pH 7.5, and subsequently incubated with 100 μg/ml RNase A for 1 h at 60°C. Cells were then stained with propidium iodide to a final concentration of 5 μg/ml and diluted (if necessary), and 10,000 cells were analyzed for DNA content with a flow cytometer. The numbers under the flow cytometry profiles correspond to chromosomal equivalents. Strains used were BB1185 (sda⁺ flgMΔ80) and SH455 (Δsda flgMΔ80).