Cell cycle analysis of Escherichia coli cells

Cell cycle analysis of Escherichia coli cells

C period = the time for a round of chromosome replication

D period = the time between the end of a round of chromosome replication and cell division

Determination of initiation age （a_i） and C+D:

From flow cytometry analysis of cells treated with rifampicin and cephalexin （run-out histogram） the proportions of cells that had not initiated replication at the time of drug action （4-origin-cells, streaked） and cells that had initiated （8-origin-cells） can be estimated.The initiation age （a_i） can be found from the theoretical age distribution described by this formula,

where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution （streaked portion）.

This gives:

If you have for example a generation time τ=84 minutes and the portion of cells with 4 origins is 66% the formula gives:

The C+D period is estimated from the initiation age （a_i）, the generation time （τ） and the number of generations spanned per cell cycle.

Example:

4-origin-cells: 23 %

Generation time （τ）: 27 min

Initiation age （a_i）: 5 min

Determination of the C and D periods:

The C period is found from the oriC/terC ratio obtained by Southern blot or qPCR analysis （oriC/ter ratio determination） and the generation time （τ）:

The D period is found from the C+D and C period:

D = （C + D） − C

Example （continues）:

C period calculated from the oriC/terC ratio: 49 min

D period = （C+D） – C

D period = 76 min – 49 min = 27 min

The theoretical exponential DNA histogram:

A theoretical exponential DNA histogram can be drawn to check whether the obtained values fit with the experimental data. From the C+D period the DNA content of the cells at different time points in the cell cycle can be calculated.

Example:

The individual values of C and D can be varied

to obtain a shape of the theoretical histogram

that gives the best fit to the experimental histogram.

Calculation of the average number of replication forks when D=τ:

In the example given above, 23% of the cells contain 4 replication forks （4-origin peak in run-out histogram） and 77% contain 12 replication forks （8-origin peak）, hence the average number of replication forks in the cell population will be:

（4 x 0.23） + （12 x 0.77） = 10.2 forks

Calculation of the average number of replication forks when D≠τ:

Example:

4-origin-cells: 23%

8-origin-cells: 77%

τ = 27 min

a_i = 5 min

C = 51 min

D = 25 min

C+D = 76 min

12 forks → 8-origin peak in run-out histogram = 77% of the cells

6 and 4 forks → 4-origin peak in run-out histogram = 23% of the cells

The fraction of cells containing 6 forks: F = 2 - 2^{（（τ-a_t）/τ）} = 2 – 2^{（（27-2）/27）} = 0.10

The fraction of cells containing 4 forks: 0.23 – 0.10 = 0.13

The average number of replication forks: （6 x 0.10） + （4 x 0.13） + （12 x 0.77） = 10.4 forks