Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication

Abstract

We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by "click" labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions.

Figure 1

Synchronous DNA replication following release from the stringent response. A. Flow cytometry for DNA content in E. coli K-12 cultures treated with serine hydroxamate (SHX) for 90 minute followed by release into medium without SHX for the indicate period of time in minutes. This panel is from our previously published work [17] B. Additional timepoints following release show synchronous replication, as evident by flow cytometric determination of DNA content in the population (black traces). The overlaid red trace is that prior to release.

Figure 2

Cell division after release from the stringent response. A. Fluorescence microscopy images of wild type E. coli treated with serine hydroxamate (SHX) for 90 minutes and subsequently released into M9 minimal medium with casamino acids without SHX for the indicated length of time and stained with the lipophilic dye FM 4-64. Cells have an increase in cell length by 20 minutes with septa first visible in the 30 minute image. Percentage of cells with visible septa, as indicated, increases with time after release. Bar length = 3.20 μm. B. Distribution of cell lengths observed at each time point after release from SHX. Cell lengths increase from 0-45 minutes after release.

Figure 3

Flow cytometric analysis of DNA content for E. coli K-12 cultures in three different growth media (reproduced from [17]). Untreated cells yield a distribution of DNA content, increasing with growth rate. Doubling times (T_d) in each medium are indicated. Cultures treated with serine hydroxamate (SHX) show quantized DNA content, variable with the growth medium, consistent with DNA content in the B period (G1-like) of the cell cycle. Cultures treated with rifampicin and cephalexin (Rif + Ceph) show integer DNA content, with cells arrested in the D period (G2-like).

Figure 4

EdU-Alexa 488 click-labeling during and after serine hydroxamate treatment. Cultures in the exponential phase of growth were divided and either mock-treated (-) or treated with 1 mg/ml serine hydroxamate.(+). Panel A. After 30, 60, or 90 minutes of treatment EdU was added to aliquots from each culture. Following a further incubation of 15 minutes, cells were fixed in methanol and then processed as described in the text. Panel B. After treatment for 90 minutes with serine hydroxamate, cultures were centrifuged and resuspended into fresh medium without serine hydroxamate. At 0, 20, and 40 minutes after release aliquots were incubated with EdU for 15 min, after which they were fixed and click-labeled. Bottom image: control culture with no EdU treatment.