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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter
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Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

机译：结合文化独立和非独立方法估算河水溶解有机物的河口浮游生物的丰富度

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Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.
机译：结合了三种不同的分析自然微生物群落多样性的方法，以最大程度地估计浮游生物分解代谢河中溶解性有机物（RDOM）的丰富度。我们还评估了依赖于培养物的定量DNA-DNA杂交，16S rRNA基因克隆文库和变性梯度凝胶电泳（DGGE）检测同一样品中细菌类群的能力。从丰富和贫穷的固体培养基中分离出42种不同的可耕种菌株。另外，通过将通过PCR扩增的细菌16S rDNA基因从群落DNA克隆到大肠杆菌载体中，获得了50个独特的克隆。从12个DGGE图谱中测序了23条独特的条带，不包括噬菌-黄杆菌属的复合模糊条带。不同的方法在属级和更高级别给出了相似的分类单元分布。但是，这些方法之间在物种级别上的匹配性很差，并且通过这三种方法只能识别出一种。因此，所有这三种方法都确定了细菌物种的独特子集，在实验系统中总计具有97个操作分类单元的总丰富度。但是，对结果的信心取决于系统发育确定和物种定义的当前精确度。在所研究的河口，RDOM的细菌消费者主要是食丝藻-黄杆菌属的可耕种和不可耕种的类群，这与所采用的方法一致。不依赖培养物的方法还暗示了一些尚未培养的β-蛋白细菌成为RDOM消费者。

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