Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.
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.
Ferullo
Daniel J
DJ
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.
Cooper
Deani L
DL
Moore
Hayley R
HR
Lovett
Susan T
ST
D002453:Cell Cycle / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000378:metabolism; D048168:Escherichia coli K12 / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C092350:FM 4-64; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C028002:serine hydroxamate; D012694:Serine; D003857:Deoxyuridine