The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function
The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function
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DOI：
10.1128/aem.69.11.6875-6887.2003
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发表时间：
2003-11-01
影响因子：
4.4
通讯作者：
Wagner, M
中科院分区：
文献类型：
--
作者：
Adamczyk, J;Hesselsoe, M;Wagner, M
开发了一种新的微阵列方法，同位素阵列方法，用于鉴定在复杂微生物群落中消耗C-14标记底物的微生物。实验进行了一个小的芯片组成的寡核苷酸探针靶向氨氧化细菌（AOB）的16 S rRNA。从在[C-14]碳酸氢盐存在下生长的真养亚硝化单胞菌的纯培养物中提取总RNA。在荧光标记RNA和微阵列杂交后，扫描所有探针点的荧光和放射性显示获得了特异性信号，并且可以明确地检测到C-14掺入rRNA。随后，我们能够证明适合的同位素阵列方法监测社区组成和CO2固定活性的AOB在两个硝化活性污泥样品与[C-14]碳酸氢盐孵育长达26小时。活性污泥样品中的AOB群落结构，预测的微阵列杂交模式，证实了定量荧光原位杂交（FISH）和比较amoA序列分析。CO2固定活动的复杂的活性污泥社区内的AOB种群的微阵列检测C-14掺入，并通过结合FISH和microautoradiography独立确认。在烯丙基硫脲作为AOB活性抑制剂的存在下，与放射性碳酸氢盐孵育的活性污泥中的AOB rRNA显示没有C-14掺入，因此在微阵列的放射性扫描上检测不到。这些结果表明，同位素阵列可以用于在一个PCR-独立的方式，利用高并行性和微阵列的直接识别微生物消耗特定的基板在环境中的歧视权力。
A new microarray method, the isotope array approach, for identifying microorganisms which consume a C-14-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [C-14] bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of C-14 into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [C-14] bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by C-14 incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of C-14 and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.