Low-temperature-induced DnaA protein synthesis does not change initiation mass in Escherichia coli K-12

Abstract

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.

FIG. 1

Replication control parameters at different temperatures. Cultures of strain BBC119 were grown under conditions of balanced growth in AB-thiamine-glucose-Casamino Acids supplemented medium at the indicated temperatures. Samples were taken for flow cytometric analysis of DNA, origins, and light scatter (LS), for immunoblot analysis of DnaA protein, and for determination of DnaA–β-galactosidase specific activity. The indicated values are averages of two (30 and 21°C) or three (14°C) independent experiments, and error bars indicate ranges for the different experiments. All values have been normalized to those determined in parallel for the 37°C cultures. The actual values for DNA per cell and origins per cell are shown in Table 1. We found that the cells at 37°C contained approximately 25 ng of DnaA per ml at OD₄₅₀ = 1, which is in accordance with previous determinations (16), and that the specific activity of DnaA–β-galactosidase was 17 to 20 Miller units.

FIG. 2

DnaA protein is stable at different temperatures. Cultures of strain BBC119 were grown under conditions of balanced growth in AB-glucose-Casamino-Acids medium at 14 and 37°C. Rifampin and cephalexin were added, and incubation was continued at the growth temperature. Samples were taken for immunoblot analysis at time zero and after approximately four culture generation times (corresponding to 31 h [14°C] and 2 h [37°C]).

FIG. 3

Runout kinetics after inhibition of initiation of replication and cell division. Cultures of strain BBC119 were grown under conditions of balanced growth in AB-glucose-Casamino-Acids medium at 14 and 37°C (see Table 1 for generation times). Rifampin and cephalexin were added at time zero, and incubation was continued at the growth temperature (A and B), while half of the 14°C culture was shifted to 37°C (C). Samples were taken at the indicated times and fixed, stained, and analyzed by flow cytometry. All distributions were normalized to contain the same number of cells.

FIG. 4

Chromosome replication and dnaA gene expression following a temperature downshift. Strain BBC119 was grown under conditions of balanced growth in AB-glucose-Casamino Acids medium at 37°C, and part of the culture was shifted to 14°C at the time indicated by the arrow. (A) Growth was followed by measurement of OD₄₅₀, and the culture was diluted when the OD₄₅₀ reached 0.5. (B) Samples were taken for measurement of origins per light scatter (LS), DNA per light scatter, and DnaA–β-galactosidase specific activity. Origins per light scatter was determined in samples incubated with rifampin and cephalexin at 37°C. Open symbols, 37°C; black symbols, 14°C; gray symbols, steady-state 14°C values.

FIG. 5

Growth and dnaA gene expression after temperature upshift. Strain BBC119 was grown under conditions of balanced growth in AB-glucose-Casamino Acids medium at 14°C, and part of the culture was shifted to 37°C at the time indicated by the arrow. (A) Growth was followed by measurement of OD₄₅₀ and the culture was diluted when the OD₄₅₀ reached 0.5. Samples were taken for measurement of DnaA–β-galactosidase specific activity. (B) Differential plot of values for DnaA–β-galactosidase activity against OD₄₅₀ from panel A. Open symbols: 14°C; black symbols, 37°C.

FIG. 6

Nucleotide sequence of the dnaA mRNA translational initiation region. At the top is shown the 16S rRNA sequence which is complementary to the downstream box (40). In the dnaA mRNA sequence, the normal ribosome binding site and GUG start codon are shown in bold; at the bottom is shown the DnaA sequence translated from the first GUG start codon.