Journal
METHODS
Volume 48, Issue 1, Pages 8-13Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2009.02.010
Keywords
Flow cytometry; Stringent response; Bacteria; Serine hydroxamate; ppGpp; EdU; PicoGreen; FM4-64; Click labeling; DNA synthesis
Funding
- NIGMS NIH HHS [GM79510, R01 GM079510, R01 GM051753-14, R01 GM079510-02, R01 GM51753, R01 GM051753] Funding Source: Medline
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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. (C) 2009 Elsevier Inc. All rights reserved.
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