Growth phase-coupled alterations in cell structure and function of Escherichia coli

Abstract

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells.

FIG. 1.

Percoll gradient centrifugation of E. coli mutants lacking sigma factors. (A) Wild-type KP7600 (W3100 derivative) and mutants lacking RNA polymerase sigma subunits (RpoH, RpoF, and RpoS) were grown on LB medium at 37°C. The cultures were subjected to centrifugation on a Percoll gradient (80%) by the established procedure (26). (B) The rpoS disruptant carrying the rpoS expression plasmid, pRpoS01, was subjected to Percoll gradient centrifugation. As controls, wild-type E. coli KP7600 with the vector pUC18 and the rpoS disruptant with pUC18 were centrifuged in parallel. Log, logarithmic growth; Sta, stationary phase.

FIG. 2.

Electron micrograph of wild-type E. coli. Wild-type E. coli W3110 was grown in LB medium for 2.5 h (A), 4 h (B), 8 h (C), 12 h (D), and 18 h (E). A cell suspension was directly placed on a Collodion-coated grid. The specimens were negatively stained with 2% sodium phosphotungstic acid and examined under a transmission electron microscope. Bar, 2 μm in all the micrographs.

FIG. 3.

Electron micrograph of E. coli rpoS and rpoF disruptants. Wild-type E. coli rpoS (A and B) and rpoF (C and D) disruptants were grown in LB medium for 4 h (A and C) and 18 h (B and D). Electron micrographic observation was carried out as in the experiment in Fig. 2. Bars, 2 μm (A and B) or 0.5 μm (C and D).

FIG. 4.

Determination of the RpoF and FliC levels. (A) Wild-type E. coli KP7600 and its rpoS disruptant were grown in LB medium for the times indicated. Crude cell extracts were prepared as described by Jishage et al. (15), and the amounts of RpoA, RpoF, and FliC proteins were determined by quantitative immunoblotting by the method of Makinoshima et al. (26). (B) The intensity of immunostained filters was measured with LAS (Fuji), and the sigma-F (RpoF) and flegellin (FliC) levels are shown as values relative to the level of the RNA polymerase alpha subunit (RpoA).

FIG. 5.

Electron micrograph of thin sections of wild-type and rpoS mutant E. coli cells. Wild-type E. coli KP7600 (A and B) and its rpoS disruptant (C and D) were grown in LB for 4 h (A and C) and 18 h (B and D). Thin sections were prepared and observed with an electron microscope as described in Materials and Methods. (E and F) Expanded micrographs of 18-h cultures of the wild type (B) and rpoS mutant (D). Bars, 1 μm (A to D) or 0.33 μm (E and F). Arrows in panel E show some of the stationary-phase-specific electron-dense areas.

FIG. 6.

Intracellular level of glycogen. Wild-type E. coli 7600 and its rpoS disruptant were grown in LB medium for the indicated times. The glycogen level was determined by the anthrone-sulfuric acid method (8).