BACTERIAL-GROWTH CONTROL STUDIED BY FLOW-CYTOMETRY

By employing flow cytometry, the DNA content and cell size of individual bacterial cells may be determined rapidly and with high precision. Also, the number of DNA replication origins in Escherichia coli cells can be measured after treating the cells with rifampicin together with the cell division inhibitor cephalexin. As opposed to wild type cells, certain mutants contain, with high frequency, a number of origins different from 2n, indicating that the mutants do not initiate DNA replication at all origins simultaneously. Here we give evidence that this asynchrony phenotype cannot occur as a consequence of aberrant chromosomal segregation or cell division, but can only be caused by defective coordination of multiple initiation events within one and the same cell. Flow cytometry has been used to perform exact and detailed analyses of the growth and cell cycle of E. coli. While the DNA distribution of a bacterial culture was unchanged as long as steady-state growth was maintained, the cellular DNA content was reduced when the culture approached and entered stationary phase. Only after prolonged incubation in stationary phase did the cells contain fully replicated chromosomes, and rapidly growing cells ended up with either 2 or 4 chromosomes in stationary phase.