Cultivation-dependent and -independent approaches for determining bacterial diversity in fleavy-metal-contaminated soil
Cultivation-dependent and -independent approaches for determining bacterial diversity in fleavy-metal-contaminated soil
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DOI：
10.1128/aem.69.6.3223-3230.2003
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发表时间：
2003-06-01
影响因子：
4.4
通讯作者：
Fry, JC
中科院分区：
文献类型：
--
作者：
Ellis, RJ;Morgan, P;Fry, JC
近年来，非培养方法已优先于传统的分离技术用于微生物群落分析。然而，来自特定样本的未培养生物体对于确定人为压力对土著社区的影响是否重要，这是值得怀疑的。为了调查这一点，土壤样品取自一个网站与片状金属污染，和细菌群落结构进行了评估与各种方法。显微镜下荧光细菌计数存在微小差异。直接从土壤样品中扩增的16 S rRNA基因片段（16 S-DGGE）的变性梯度凝胶电泳（DGGE）图谱高度相似。从污染最严重的样本中产生的克隆文库揭示了一个多样化的细菌群落，这与其他研究中的原始土壤群落相似。然而，从土壤样品中的细菌的比例，可培养的标准板计数介质之间的0.08%和2.2%的变化，这些值与金属浓度呈负相关。通过平板洗涤液的16 S-DGGE和单个分离株的脂肪酸谱比较来自每个样品的可培养群落。每种方法都表明，每个样本的可培养社区组成之间存在相当大的差异。对来自基于培养和不依赖培养的方法的DGGE条带进行测序和比较。这些数据表明，金属污染并没有显着影响的总遗传多样性，但影响生理状态，使能够响应实验室培养和它们的分类分布的细菌的数量被改变。因此，平板计数可能是一个更合适的方法来确定重金属对土壤细菌的影响比培养独立的方法。
In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.