Cell division in Escherichia coli cultures monitored at single cell resolution.
Cell division in Escherichia coli cultures monitored at single cell resolution.
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在单细胞分辨率下监测的大肠杆菌培养物中的细胞分裂。
DOI：
10.1186/1471-2180-8-68
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发表时间：
2008-04-23
期刊：
影响因子：
4.2
通讯作者：
Tenson, Tanel
中科院分区：
文献类型：
--
作者：
Roostalu, Johanna;Joers, Arvi;Luidalepp, Hannes;Kaldalu, Niilo;Tenson, Tanel
细胞的一个基本特征是分裂能力。迄今为止，细菌培养物的大多数参数（包括细胞分裂）都是以细胞群体平均值来测量的，假设所有细菌以统一的速率分裂。我们监测了大肠杆菌培养物在不同生长阶段中单个细胞的分裂。我们的实验是基于细胞分裂时绿色荧光蛋白（GFP）的稀释，通过流式细胞术监测。结果表明，绝大多数E.大肠杆菌细胞在指数生长的培养物中均匀分裂。在已处于稳定期长达四天的培养物中，未观察到细胞分裂。然而，当将稳定期培养物稀释到新鲜培养基中时，出现两个细胞亚群：一个开始分裂，另一个没有。这些群体可通过GFP稀释检测，并在流式细胞术中显示不同的侧向散射参数。进一步的分析表明，非生长亚群中的细菌没有死亡，生长能力的差异也不能归结为稳定期特异性基因表达的差异，因为我们观察到几个应力相关启动子的均匀表达。非生长型持留菌（对抗生素耐受的暂时休眠的细菌）的存在先前已在生长的细菌群体中描述过。使用GFP稀释法结合细胞分选，我们表明，氨苄青霉素裂解生长的细菌，而非生长的细菌保留活力，其中一些重新开始生长后，氨苄青霉素被删除。因此，我们的方法使得即使在其中持续存在形成具有低频率的野生型菌株的液体培养物中也能够监测持续存在。原则上，这里开发的方法可以用来检测细胞分裂的差异，以响应不同的环境条件和培养的单细胞生物体以外的大肠杆菌。杆菌
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. 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. 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.
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