Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate
Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate
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DOI：
10.1111/j.1462-2920.2007.01325.x
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发表时间：
2007-09-01
影响因子：
5.1
通讯作者：
Szewzyk, Ulrich
中科院分区：
文献类型：
--
作者：
Boeckelmann, Uta;Luensdorf, Heinrich;Szewzyk, Ulrich
发现从“河雪”中分离的细菌菌株F8产生丝状网络形式的细胞外纤维。这些细胞外丝此前被证明是由DNA组成的，在本工作中首次通过超微结构和电子能量损失谱进行了研究。菌株F8的“整体"制备表明这些聚合物在超微结构上是均匀的，并形成了宽度为1.8-2.0 nm的基本细丝网络。当在pH 3.5下与胶体阳离子ThO 2示踪剂一起孵育时，它们变得强烈染色（电子致密），提供直接证据表明纤维带负电荷，因此化学上呈酸性。通过能量过滤透射电子显微镜对细胞外细丝进行元素分析，发现磷是存在的主要元素，并且由于用DNA酶预处理F8细胞防止了钍标记，因此纤维必须由细胞外DNA（eDNA）组成。常规细胞裂解既不能导致切片DNA释放，也不能导致“整体封片阴性染色”。此外，在病毒切片中从未观察到感染有ESTs的细胞，并且在整个样品中从未检测到噬菌体颗粒，这排除了ESTs直接参与eDNA释放的可能性。
Strain F8, a bacterial isolate from 'river snow', was found to produce extracellular fibres in the form of a filamentous network. These extracellular filaments, which were previously shown to be composed of DNA, have been studied for the first time by ultrastructural and electron energy-loss spectroscopy in the present work. 'Whole mount' preparations of strain F8 indicate these polymers are ultrastructurally homogeneous and form a network of elemental filaments, which have a width of 1.8-2.0 nm. When incubated at pH 3.5 with colloidal cationic ThO2 tracers they become intensely stained (electron dense), affording direct evidence that the fibres are negatively charged and thus acidic chemically. Elemental analysis of the extracellular filaments by Energy-filtered Transmission Electron Microscopy revealed phosphorus to be the main element present and, because pretreatment of F8 cells with DNase prevented thorium labelling, the fibres must be composed of extracellular DNA (eDNA). Neither ultrathin sections nor 'whole mount negative stain' caused DNA release by general cell lysis. Additionally, cells infected with phages were never observed in ultrathin sections and phage particles were never detected in whole mount samples, which rules out the possibility of phages being directly involved in eDNA release.