Coordinated replication and sequestration of oriC and dnaA are required for maintaining controlled once-per-cell-cycle initiation in Escherichia coli

Abstract

Escherichia coli cells were constructed in which the dnaA gene was moved to a location opposite oriC on the circular chromosome. In these cells the dnaA gene was replicated with significant delay relative to the origin. Consequently, the period where the newly replicated and hemimethylated oriC was sequestered no longer coincided with the period where the dnaA gene promoter was sequestered. DnaA protein synthesis was therefore expected to continue during origin sequestration. Despite a normal length of the sequestration period in such cells, they had increased origin content and also displayed asynchrony of initiation. This indicated that reinitiation occasionally occurred at some origins within the same cell cycle. The extra initiations took place in spite of a reduction in total DnaA protein concentration to about half of the wild-type level. We propose that this more efficient utilization of DnaA protein results from an increased availability at the end of the origin sequestration period. Therefore, coordinated sequestration of oriC and dnaA is required for maintaining controlled once-per-cell-cycle initiation.

FIG. 1.

Construction of strains with dnaA or dnaA46 in the chromosomal attB site. (A) The dnaA/dnaA46 gene was ligated to a nonreplicating attP-carrying cassette (bottom). Lambda Int protein-dependent site-specific recombination with the chromosomal attB site resulted in cells carrying an additional dnaA gene inserted in attB. Subsequently the dnaA gene was removed from its normal location. (B) In wild-type cells dnaA is replicated shortly after oriC. Consequently, both oriC and dnaA are hemimethylated and sequestered by the SeqA protein (•) in the same part of the cell cycle (left). In cells carrying a dnaA gene in attB only, this is replicated with significant delay relative to oriC. Consequently, the dnaA gene is not sequestered for the same part of the cell cycle as the origin (right).

FIG. 2.

Reinitiation in cells carrying dnaA in attB. Cells of strain CM742 (A) and CM742 dnaA850::Tn10 dnaA46::attB (ALO2073) (B) were grown at 30°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan. At time T = −90 the culture was shifted to 42°C, kept at this nonpermissive temperature for 90 min, and shifted back to 30°C at time T = 0. At the times indicated cell samples were collected for treatment with rifampin and cephalexin for 4 h prior to flow cytometry analysis. The median (the value above and below which 50% of the distribution can be found) was used as a robust measure of the central tendency of individual cells (39) and is plotted as origins per cell. The panels on the right-hand side of the figure show selected DNA histograms for rifampin-treated cultures.

FIG. 3.

Initiation of replication is affected by the dnaA gene location. DNA histograms of CM735 (wt) (A) and ALO2465 (dnaA850::Tn10 dnaA::attB) (B). Cells were grown at 37°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan and treated with rifampin and cephalexin prior to flow cytometry analysis. Distinct peaks represent the accumulation of cells with an integral number of chromosomes which correspond to the number of origins at the time of drug addition (40). The scale on the number of cells is arbitrary and represents a minimum of 60,000 cells in all panels.

FIG. 4.

The initiation mass of slow-growing cells carrying dnaA in attB. Flow cytometry histograms of wild-type (CM735) and dnaA850::Tn10 cells carrying dnaA in attB (ALO2465) grown under steady-state conditions at 37°C in minimal medium supplemented with acetate, tryptophan, histidine, and methionine. The parameters measured were number of origins in rifampin- and cephalexin-treated cells (A and D) and DNA content of exponentially growing cells (B and E). Three-parameter contour plots of exponentially growing cells are shown in panels C and F. The isocontour lines are drawn through points with the same number of cells. Thus, the collection of almost concentric rings at the one- or two-chromosome position represents a “mountain” of cells. The dashed line represents the cell mass at which initiation takes place (8). The scale on number of cells and scattered light is arbitrary and represents a minimum of 100,000 cells in all panels.

FIG. 5.

Initiation of replication is affected by the dnaA46 gene location at slow growth. DNA histograms of CM742 (dnaA46) (A and C) and ALO2073 (dnaA850::Tn10 dnaA46::attB) (B and D) cells. Cells were grown at 30°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan (A and B) or with glycerol, tryptophan, histidine, and methionine (C and D) and treated with rifampin and cephalexin prior to flow cytometry analysis. Distinct peaks represent the accumulation of cells with an integral number of chromosomes which correspond to the number of origins at the time of drug addition (40).The scale on number of cells is arbitrary and represents a minimum of 60,000 cells in all panels.

FIG. 6.

The ori/ter ratio is increased in cells carrying dnaA in attB. Three individual cultures of wild-type (CM735) and dnaA850::Tn10 dnaA::attB (ALO2465) cells were grown exponentially at 37°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan. DNA was extracted and digested with HindIII, and Southern hybridization was performed with an oriC probe and a ter probe (Materials and Methods). A sample of plasmid pTAC4511 digested with HindIII was also included on the blot. This sample contains oriC and terC in a 1:1 proportion.

FIG. 7.

Cells carrying dnaA in attB are heterogeneous with respect to cell size and nucleoid positioning. Strains CM735 (wt) (A) and CM735 dnaA850::Tn10 dnaA::attB (ALO2465) (B and C). Panel B shows the morphology of the majority of ALO2465 cells, whereas the less frequent filamentous cells are shown in panel C. Cells were grown at 37°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan. Cells were fixed and stained prior to microscopic analysis as described in Materials and Methods.

FIG. 8.

Cellular DnaA protein content. Strains CM735 (wt) and CM735 dnaA850::Tn10 dnaA::attB (ALO2465) were grown at 37°C in minimal medium supplemented with glucose, Casamino Acids, and tryptophan (A) or in minimal medium supplemented with acetate, tryptophan, histidine, and methionine (B). Samples were taken, and the total protein content was adjusted to the same amount using a Lowry analysis prior to loading on a sodium dodecyl sulfate-polyacrylamide gel. For fast-growing cells 8 μg of total protein was applied to the gel (A) whereas only 5 μg was used for slow-growing cells (B). Following blotting, the filter was probed with a polyclonal DnaA antibody. The control lanes contain the indicated amounts of purified DnaA protein. The relative amount of DnaA protein was quantified for each sample and normalized to wild-type cells. The gene dosage of dnaA was calculated as described in Materials and Methods.