Distinguishing between Living and Nonliving Bacteria in the Marine Environment: Evaluation of a Combined Molecular Probe and Vital Staining Procedure

在水生生态系统中，细菌被认为是仅次于浮游植物的生物有机体对有机碳转化的作用。据报道，在开阔的海洋水域，活细菌的生物量超过了所有其他浮游生物的生物量，甚至在大陆架和沿海水域也是如此。然而，这些估计都是基于所有DAPI染色细胞都是代谢活跃细胞的假设。这种假设似乎不再站得住脚：很大一部分表面上的细菌显然是不活跃的、垂死的或死亡的。这样的结论有可能促成生态系统功能概念模型的另一个范式转变。至少，这意味着相对较少的原位细菌的生长（和呼吸）速度比以前认为的要快得多，而且很大一部分“表面”细菌实际上是碎屑的组成部分。我们开发了一种荧光生命染色和分子探针（VSP）的新组合，可以定量识别那些膜受损的细胞，以及那些含有足够rRNA的细胞，被认为是代谢活跃的细胞。虽然单独使用这两种方法可能都不足以确定非活性细胞、垂死细胞或死亡细胞的活性，但它们可能构成一种强大的工具，可以研究这些细胞类型在原位的相对重要性，以及它们如何响应环境压力而发生变化。我们已经用大肠杆菌证明了VSP程序的有效性，这里我们将检验其对海洋革兰氏阳性、革兰氏阴性和寡亲菌的适用性。然后，我们将测试VSP的准确性，以量化活的、非活的和饥饿的细胞对营养物质和病毒的反应，并开始记录自然群落中活的和非活的细胞的模式。还有望确定非活性但活的细胞的数值重要性。研究结果不仅将为该方法在海洋环境中的应用提供严格的评估，而且活性细胞与受损膜的现场数据将为细菌在DOC、碎屑和细菌之间转化中的作用的修订概念模型提供第一个定量基础。+ n±!？N¼f + _ Ûª？ÑOhª+ª？0 p + Õ - Ã ？0 T x À - Þ， R:\WWUSER\TEMPLATE\NORMAL。点n细菌被认为是仅次于ph值y S u m m r I f n o r m t I o n ( ++++++++++++ 3Ð ++++++++++++ ++++++++++++ ++++++++++++ ytoplankton在生物体中的作用有助于在水生生态系统有机碳的变换威廉詹姆斯安默曼Joanne MCcreary @ ?Nk + ?* * * * * * * *——¹?L i @ d - Microsoft Word 6.0 7 Õ - e =- e f f f f f f f f ？1 z z z z z z z z Á t t t t ？= ?= + ？t3 Ó ？zz Z Z Z ？z z z z z z z z z Á P X z Á z nz

Bacteria are thought to be second only to phytoplankton in the role living organisms contribute to the transformation of organic carbon in aquatic ecosystems. In open ocean waters, the biomass of living bacteria has been reported to exceed that of all other living plankton, and even in continental shelf and coastal waters it is significant. However, these estimates are all derived from the assumption that all DAPI stained cells are metabolically active cells. This hypothesis no longer appears tenable: a large fraction of apparent bacteria are apparently inactive, moribund, or dead. Such a conclusion threatens to precipitate another paradigm shift in conceptual models of ecosystem function. At the very least, it would imply that a comparatively few bacteria in situ are growing (and respiring) much faster than previously thought, and that a large fraction of `apparent` bacteria is really a component of detritus. We have developed a novel combination of a fluorescent Vital Stain and molecular Probes (VSP) which quantitatively identifies those cells with compromised membranes, and those cells containing sufficient rRNA to be considered metabolically active. While neither approach alone might be considered adequate to specify active from inactive, moribund, or dead cells, together they potentially comprise a powerful tool to investigate the relative importance of these cell types in situ, and how they change in response to environmental pressures. We have already demonstrated the validity of the VSP procedure using E. coli, here we will examine its applicability to marine Gram positive, Gram negative, and oligophiles. We will then test the accuracy of the VSP to quantify living, non living, and starved cells in response to nutrients and viruses, and begin to document patterns of living and non living cells in natural communities. There is also promise of determining the numerical importance of inactive but living cells. The results will not only provide a stringent evaluation of the method for use in the marine environment, but the field data on active cells vs. those with compromised membranes will provide the first quantitative basis for a revised conceptual model of the role of bacteria in transformations between DOC, detritus, and bacteria. +n ±! ?n ¼f + _ Ûª? ÑOh ª' +'ª?0 P + Õ - Ã ? 0 T x À - Þ , R:\WWUSER\TEMPLATE\NORMAL.DOT n Bacteria are thought to be second only to ph S u m m a r y I n f o r m a t i o n ( ++++++++++++ 3 Ð ++++++++++++ ++++++++++++ ++++++++++++ ytoplankton in the role living organisms contribute to the transformation of organic carbon in aquatic ecosystems James William Ammerman Joanne MCcreary @ ?Nk+ ? @ -+Y+ª) @ ? -¹ ? L i @ dª Microsoft Word 6.0 7 Õªe =- e f f f f f f p f ? 1 z z z z z z z z Á t t t ? = ? = + ? T 3 Ó ? z z z z z ? z z z z z z z z z Á P X z Á z N z