Cell division in Escherichia coli cultures monitored at single cell resolution.
BACKGROUND
A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate.


RESULTS
We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency.


CONCLUSIONS
In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli.
Roostalu
Johanna
J
Institute of Technology, University of Tartu, Tartu, Estonia. johanna@roostalu.info
Jõers
Arvi
A
Luidalepp
Hannes
H
Kaldalu
Niilo
N
Tenson
Tanel
T
D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D049408:Luminescent Agents / Q000032:analysis; D008826:Microbial Sensitivity Tests; D011419:Propidium / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D013997:Time Factors
D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; D011419:Propidium; D000667:Ampicillin