Wilson M S, Bakermans C, Madsen E L
Section of Microbiology, Division of Biological Sciences, Cornell University, Ithaca New York 14853, USA.
Appl Environ Microbiol. 1999 Jan;65(1):80-7. doi: 10.1128/AEM.65.1.80-87.1999.
We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.
我们开发了从一个受煤焦油废物污染场地中分解萘的地下水微生物原位转录mRNA进行分离和表征的程序。将地下水抽过孔径为0.22微米的过滤器，然后在干冰 - 乙醇中冷冻。通过煮沸十二烷基硫酸钠裂解和酸性酚 - 氯仿萃取从冷冻的过滤器中提取RNA。使用一系列设计用于扩增nahAc同源物的PCR引物进行转录本表征。发现几对引物可扩增代表萘降解基因全部多样性的nahAc同源物。对环境RNA提取物进行逆转录，然后通过PCR扩增所得的cDNA混合物。通过对地下水cDNA进行PCR扩增产生地高辛标记的探针混合物。该探针混合物在严格条件下与携带各种nahAc同源物的萘降解细菌的相应PCR产物杂交，表明已从地下水中检索到多种双加氧酶转录本。对稀释和未稀释的cDNA制剂进行独立扩增，将所得的28个PCR产物克隆并测序。序列比较揭示了与双加氧酶基因ndoB和dntAc相关的两个主要组，它们先前分别从恶臭假单胞菌NCIB 9816 - 4和伯克霍尔德氏菌菌株DNT中克隆得到。仅在使用未稀释的cDNA制剂进行的实验中发现了一个独特的序列亚组。据我们所知，这些结果首次直接证明了在受污染场地中本地微生物对编码萘分解代谢的基因进行原位转录。检索到的序列比基于培养的方法在研究场地检测到的具有更高的多样性。
