Double-Strand Break Repair and Holliday Junction Processing Are Required for Chromosome Processing in Stationary-Phase Escherichia coli Cells

Abstract

As nutrients are depleted and cell division ceases in batch cultures of bacteria, active processes are required to ensure that each cell has a complete copy of its genome. How chromosome number is manipulated and maintained in nondividing bacterial cells is not fully understood. Using flow cytometric analysis of cells from different growth phases, we show that the Holliday junction-processing enzymes RuvABC and RecG, as well as RecBCD, the enzyme complex that initiates DNA double-strand break repair, are required to establish the normal distribution of fluorescent peaks, which is commonly accepted to reflect the distribution of chromosome numbers. Our results reveal that these proteins are required for the proper processing of chromosomes in stationary phase.

Keywords: DNA repair; double-strand break repair; genome stability; homologous recombination; stationary phase.

Figure 1

Growth parameters and viability of wild-type and recombination-defective strains over time. (A) Optical densities measured at a wavelength of 600 nm (OD₆₀₀) are plotted against the time (in hours) after inoculation. Samples were removed from the cultures at each time point marked. (B) At each time point, samples were collected from cultures started by diluting overnight cultures by a factor of 10⁵ into fresh LB medium; the number of colony-forming units was determined by appropriate dilution and plating on LB agar. The number of cells per milliliter is plotted on a logarithmic scale against the time (in hours) after inoculation. Diamonds = FC36 (wild-type); triangles = FC457 (recG258); circles = FC484 (ruvA60); squares = FC400 (recB21); dashes = FC513 (recG258 ruvA60).

Figure 2

Effects of recombination defects on cell size. The relative FL2-W values (cell size) are plotted against the relative FL2-A values (total propidium iodide fluorescence) in arbitrary units. Increasing values on the Y-axis indicate larger cells, and increasing values on the X-axis indicate greater total propidium iodide fluorescence intensity.

Figure 3

Populations of cells with distinct fluorescence intensities develop during growth and stationary phase of the wild-type strain. Propidium iodide–stained cells of the wild-type strain (strain FC36) were analyzed by flow cytometry at the indicated time points. (A) The numbers of cells (Y-axis) are plotted against their relative fluorescence intensities (X-axis). The histograms from the time points indicated are aligned along the Z-axis. (B) Phase-contrast micrographs of unfixed cells from the time points indicated (1000× magnification).

Figure 4

The Holliday junction–migrating protein RecG is required for the development of normal fluorescent population dynamics during stationary phase. Propidium iodide–stained cells (strain FC457) were analyzed by flow cytometry at the indicated time points. (A) The numbers of cells (Y-axis) are plotted against their relative fluorescence intensities (X-axis). The histograms from the time points indicated are aligned along the Z-axis. (B) Phase-contrast micrographs of unfixed cells from the time points indicated (1000× magnification).

Figure 5

The Holliday junction–processing RuvABC complex is required for normal fluorescent population dynamics during growth and in stationary phase. Propidium iodide–stained cells (strain FC484) were analyzed by flow cytometry at the indicated time points. (A) The numbers of cells (Y-axis) are plotted against their relative fluorescence intensities (X-axis). The histograms from the time points indicated are aligned along the Z-axis. (B) Phase-contrast micrographs of unfixed cells from the time points indicated (1000× magnification).

Figure 6

Loss of both Holliday junction–processing pathways severely alters fluorescent population dynamics during growth and in stationary phase. Propidium iodide–stained cells (strain FC513) were analyzed by flow cytometry at the indicated time points. (A) The numbers of cells (Y-axis) are plotted against their relative fluorescence intensities (X-axis). The histograms from the time points indicated are aligned along the Z-axis. (B) Phase-contrast micrographs of unfixed cells from the time points indicated (1000× magnification).

Figure 7

Loss of RecB alters fluorescent population dynamics during growth and in stationary phase. Propidium iodide–stained cells (strain FC400) were analyzed by flow cytometry at the indicated time points. (A) The numbers of cells (Y-axis) are plotted against their relative fluorescence intensities (X-axis). The histograms from the time points indicated are aligned along the Z-axis. (B) Phase-contrast micrographs of unfixed cells from the time points indicated (1000× magnification).