Institute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, United Kingdom.
Appl Environ Microbiol. 2010 Apr;76(8):2468-77. doi: 10.1128/AEM.01964-09. Epub 2010 Feb 12.
The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a (13)C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with (13)C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.
可以通过使用指纹技术（如变性梯度凝胶电泳（DNA-DGGE 和 RNA-DGGE）或在 16S rRNA 基因与 13C 标记的底物孵育后进行稳定同位素探测（SIP））来确定 16S rRNA 基因序列的相对丰度的 DNA 或 RNA 靶向变化，从而评估自然微生物群落对环境变化的反应。在含有两个或三个具有不同耐高氨浓度的 Nitrosospira 纯培养或富集培养物的群落分批生长过程中，比较了这三种方法的敏感性。培养物分别用低、中或高初始氨浓度和 13C 标记的二氧化碳供应。DNA-SIP-DGGE 提供了最直接的生长证据，并且最敏感，DGGE 图谱的变化在 DNA 和 RNA-DGGE 图谱的变化以及亚硝酸和硝酸盐产生的可检测增加之前就已经出现。RNA-DGGE 提供了中等的敏感性。此外，这三种分子方法用于跟踪群落中单个菌株的生长。通常，可以根据单培养物生长特性来预测群落中单个菌株相对活性的变化。在所有氨浓度下，耐氨 Nitrosospira 簇 3b 菌株都主导着混合群落，而在中等氨浓度下，氨敏感菌株被淘汰。然而，在最低氨浓度下，耐氨和氨敏感菌株共存，并且在某些条件下，在高氨浓度下在单培养物中被抑制的菌株在混合培养物中在高氨浓度下是活跃的，在那里它们与耐氨菌株共存。因此，结果表明 SIP 对于检测具有相对低产量和低活性的生物的活性具有敏感性，并且能够跟踪相互作用的微生物群落结构的变化。
