Cell-Size Homeostasis and the Incremental Rule in a Bacterial Pathogen

Abstract

How populations of growing cells achieve cell-size homeostasis remains a major question in cell biology. Recent studies in rod-shaped bacteria support the "incremental rule" where each cell adds a constant length before dividing. Although this rule explains narrow cell-size distributions, its mechanism is still unknown. We show that the opportunistic pathogen Pseudomonas aeruginosa obeys the incremental rule to achieve cell-length homeostasis during exponential growth but shortens its cells when entering the stationary phase. We identify a mutant, called frik, which has increased antibiotic sensitivity, cells that are on average longer, and a fraction of filamentous cells longer than 10 μm. When growth slows due to entry in stationary phase, the distribution of frik cell sizes decreases and approaches wild-type length distribution. The rare filamentous cells have abnormally large nucleoids, suggesting that a deficiency in DNA segregation prevents cell division without slowing the exponential elongation rate.

Figure 1

Cell-size regulation in Pseudomonas aeruginosa and its elongated mutant frik. (A) Correlation between relative growth (log(L_d/L_b)) and time between divisions reveals identical growth rates in both strains. Binned data. (B) Cell width in exponential growth is independent of cell length in both wild-type and frik (Pearson correlation coefficient: 0.061 (wt) and −0.039 (frik)). Data are rescaled by average length and width for each strain. Average cell length is 3.14 μm for wt and 5.60 μm for frik. Average cell width is 0.75 μm for wild-type and 0.74 μm for frik (p = 0.58, N = 117 for wild-type and N = 107 for frik). (C) There is no correlation between growth from birth to division (ΔL = L_d-L_b) and length at birth L_b (Pearson correlation coefficient: −0.010 (wt) and 0.114 (frik)). (D) Cell lengths measured in exponential growth in different liquid media show that wild-type and frik follow the growth law. Media used were (from slowest to fastest) minimal glycerol media, minimal glucose media, casamino acid media, and LB. Error bars are 25 and 75% percentiles. Linear fits (solid lines) are L = 0.89 μ + 2.18 (μm) for wt, L = 4.21 μ + 2.32 (μm) for frik. Exponential fits (dashed lines) are L = 2.22 exp(0.32 μ) (μm) for wt, L = 2.82 exp(0.83 μ) (μm) for frik. (Inset) Micrographs of a frik cell (left) and a wt cell (right) in casamino acid media, imaged by fluorescence microscopy. Scale bar: 2 μm. To see this figure in color, go online.

Figure 2

Cell growth in agar pads is exponential. (A and B) Tracking single cells over several hours reveals exponential growth even holds for single cells in both wild-type (A) and frik (B). (Arrows) Elongation events that lead to filamentous cells in the frik mutant; (dashed lines) when growth arrests. (C) Both wild-type (black) and frik (cyan) have the longest cells in the exponential phase in liquid cultures with a steady decrease as cells transition into the stationary phase. (Straight lines through the various points are guides to the eyes.) (Inset) Representative growth curve from which the cell sizes were determined. (Gray area) What we call the “exponential” phase. (Anything outside of the gray area is considered the “stationary” phase.) (D) Frik continues to divide even after growth arrests in agar pads. Growth stops after 360 min in this example (indicated by the black outline around the picture). After this time, both a long cell and the shorter cells still divide to become shorter. (E) Frik tends to show budding off of two cells on opposite poles in short sequence. (Gray arrowhead) First septation event, followed by a second septation event (pointed out by the black arrowhead). To see this figure in color, go online.

Figure 3

The filamentous cell in frik. (A) Distributions of L_d from experiments (dots) and simulations (lines). The X axis is rescaled with ΔL of each strain. The Y axis is rescaled for consistency. (B) Lineage tree from one colony of frik. To see this figure in color, go online.

Figure 4

Frik has longer nucleoids; filamentous cells have abnormal nucleoids. Wild-type (A) and frik (B) from exponentially growing liquid cultures were stained with FM 4-64 (membrane dye) and Hoechst 33342 (a DNA intercalating dye). (Red arrowheads) Areas where the membrane has septated. To see this figure in color, go online.