Fig. 1.

Carbon starvation causes the E. coli cytoplasm to shrink in a manner distinct from plasmolysis. (A) Experimental setup for inducing cytoplasmic shrinkage. Log-phase cells were pelleted and washed in M9 salts, then incubated in M9 salts without carbon source. The resulting cells were placed onto agarose pads containing M9 salts and imaged. (B) (Left) Log-phase cells grown in LB were washed in M9 salts three times and immediately placed on an agarose pad. Shrinkage of the cytoplasm was observed in most cells (white arrow). (Right) Washing in M9 glucose did not cause shrinkage. (C) Shrinkage was also observed in K. pneumoniae cells after transitioning from LB to M9 salts. The white arrows highlight the pole with shrinkage. (D) Staining of carbon-starved MG1655 cells revealed clear separation between the outer membrane dye FM4-64 and the inner membrane dye MitoTracker green at one pole. (E) After 4 h of starvation, the relative cytoplasmic areas of suddenly starved cells as quantified by segmenting the MitoTracker signal were ∼17% smaller than those of log-phase cells. (F) Representative E. coli MG1655 cells 4 h after rapid starvation induced by transition from LB into M9 salts (Top) or after a hyperosmotic shock with 20% sucrose (Bottom). After a hyperosmotic shock, plasmolysis bays along the cell body (arrows) were visible in phase-contrast and from MitoTracker staining, and the cytoplasm was visibly smaller at both ends. By contrast, the cytoplasm of the suddenly starved cell had shrunk only at one pole. FM4-64 stains the outer membrane, which remained largely unaffected in both cases. (G and H) The percentage of cells that exhibited midcell periplasmic spaces (G) or shrinkage at both poles (H) was significantly higher for osmotic shock–induced plasmolysis than for starvation (Fisher’s exact test).