Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
PLoS One. 2012;7(2):e30981. doi: 10.1371/journal.pone.0030981. Epub 2012 Feb 13.
Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations. Here, a Visual Basic based simulation program was written for the computation of theoretical DNA distributions for different choices of cell cycle parameters (C and D phase durations, doubling time etc). These cell cycle parameters can be iterated until the best fit between the experimental and theoretical DNA histograms is obtained. The Excel file containing the simulation software is attached as supporting information.Cultures of Escherichia coli were grown at twelve different media and temperature conditions, with following measurements by flow cytometry and simulation of the DNA distributions. A good fit was found for each growth condition by use of our simulation program. The resulting cell cycle parameters displayed clear inter-media differences in replication patterns, but indicated a high degree of temperature independence for each medium. The exception was the poorest medium (acetate), where the cells grew with overlapping replication cycles at 42 °C, but without at the lower temperatures.We have developed an easy-to-use tool for determination of bacteria's cell cycle parameters, and consequently the cells' chromosome configurations. The procedure only requires DNA distribution measurements by flow cytometry. Use of this simulation program for E. coli cultures shows that even cells growing quite slowly can have overlapping replication cycles. It is therefore always important not only to assume cells' replication patterns, but to actually determine the cell cycle parameters when changing growth conditions.
许多研究都集中在大肠杆菌和其他能够进行复杂复制的细菌的染色体复制和分离上，这些细菌的 C 期跨越多个世代。对于这些研究，了解复制模式，包括起点和复制叉的拷贝数，对于正确解释结果至关重要。流式细胞术是生成细胞群体实验 DNA 分布的重要工具。在这里，我们编写了一个基于 Visual Basic 的模拟程序，用于计算不同细胞周期参数（C 期和 D 期持续时间、倍增时间等）选择下的理论 DNA 分布。可以迭代这些细胞周期参数，直到获得实验和理论 DNA 直方图之间的最佳拟合。包含模拟软件的 Excel 文件作为支持信息附加。大肠杆菌培养物在 12 种不同的培养基和温度条件下生长，并通过流式细胞术进行测量和 DNA 分布的模拟。我们的模拟程序为每种生长条件找到了很好的拟合。得到的细胞周期参数显示出复制模式的明显介质差异，但表明每种介质的温度依赖性很高。例外是最差的培养基（乙酸盐），在 42°C 下，细胞的复制周期重叠，但在较低温度下没有。我们开发了一种用于确定细菌细胞周期参数的简单易用的工具，从而确定细胞的染色体构型。该程序仅需要通过流式细胞术测量 DNA 分布。我们对大肠杆菌培养物使用此模拟程序表明，即使生长较慢的细胞也可能具有重叠的复制周期。因此，改变生长条件时，不仅要假设细胞的复制模式，还要实际确定细胞周期参数，这一点非常重要。
