Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning

Abstract

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37 degrees C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.

FIG. 1

Characterization of exponentially growing cells in CHG (A), CH (B), TS (C), and S (D) media by digital image analysis. The cells were categorized according to cell length (frequency within the population) and to nucleoid conformation and number (lightest to darkest bars, monucluceate, mononucleate bilobed, binucleate, binucleate bilobed, and tri- or tetranucleate, respectively). The solid and dotted lines show ideal cell length distributions calculated as described in the Appendix, using values for the normalized variance of the birth probability distributions, (Δl/l̄)², of 0.03 and 0.06, respectively. Data are from >400 individual cells from each medium.

FIG. 2

Relationship between DNA content and cell length for populations of cells grown in CHG (A), TS (B), and S (C) media. DNA content (expressed as chromosome equivalents per cell) was measured and plotted against cell length. The standard regression line for each plot is shown. Data are from about 200 individual cells from each medium.

FIG. 3

Models for cell cycle events progression in B. subtilis growing at four different rates. Shown are the major events during the cell cycle: birth (b), initiation (i), termination (t), binucleation (bi), and septation (s). Dots indicate the initiation of DNA replication. The cell length is shown on the scale below the models.

FIG. 4

Relationship between nuclear length and cell length over a range of growth rates for cells growing in CHG (A), CH (B), TS (C), and S (D) media. For each plot standard regression lines are shown. For binucleate cells, nuclear length corresponds to the sum of the two nuclei. Data are from 200 cells from each medium.

FIG. 5

Compilation of representative images of the different cell types found in four different growth media S (A), TS (B), CH (C), and CHG (D). DAPI fluorescence images are overlaid on phase-contrast images. The nucleoids are the brightly stained objects within the dark cell bodies. All of the images are at the same magnification. Scale bar, 2 μm.