Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using 4′-6-Diamidino-2-Phenylindole (DAPI)
Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using 4′-6-Diamidino-2-Phenylindole (DAPI)
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DOI：
10.1021/es201123r
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发表时间：
2011-09-15
影响因子：
11.4
通讯作者：
McGrath, John W.
中科院分区：
文献类型：
--
作者：
Kulakova, Anna N.;Hobbs, Darragh;McGrath, John W.
无机聚磷酸盐（polyP）作为一种重要的磷素汇，在磷素交换和磷素合成中发挥着重要作用。然而，尽管聚磷酰胺研究取得了重大进展，但缺乏快速有效的分析方法来量化微生物培养物和环境样品中聚磷酰胺的积累。一个主要的缺点是需要在分析之前从细胞中提取polyP。由于提取效率低，这可能导致低估细胞内polyP水平及其环境池大小：我们使用标准溶液和当前方案观察到23-58%的polyP损失。在这里，我们报告了一种基于直接荧光的DAPI测定系统，该系统消除了在定量之前对先前polyP提取的要求。这将微生物培养物中polyP检测的效率提高了28-55%，表明细胞内polyP库的定量测量。它提供了一种直接的聚P测定，其将定量能力与技术简单性相结合。这是我们探索polyP在细胞生物学和生物地球化学营养循环中的作用的重要一步。
Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.