Department of Biotechnology and Bioinformatics, NIIT University, Neemrana, Rajasthan, India.
J Appl Microbiol. 2010 Apr;108(4):1252-62. doi: 10.1111/j.1365-2672.2009.04523.x. Epub 2009 Aug 19.
In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available.
Coal-tar-contaminated soil was collected, which consisted of 122.5 mg g(-1) total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (alpha-subunit) common to all PAH dioxygenase enzymes and (iii) beta-subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha-, Epsilon- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of alpha-subunit and beta-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe-2S] cluster binding site suggested that these gene fragments encode for alpha-subunit of dioxygenase gene.
Sequencing of the cloned libraries representing alpha-subunit gene fragments (Rf1) and beta-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil.
The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.
为了开发有效的多环芳烃（PAHs）降解生物修复策略，需要更好地了解微生物群落的组成和代谢潜力，特别是在高度多环芳烃污染的地点，那里几乎没有关于非培养群落的信息。
采集了煤焦油污染的土壤，其中含有 122.5mg g(-1)总可萃取多环芳烃化合物。用这种土壤进行的生物降解研究表明，存在能够降解萘、菲和芘等模型 PAH 化合物的微生物群落，浓度均为 50ppm。从煤焦油污染土壤的 DNA 中建立了针对 16S rRNA 的 PCR 克隆文库，以表征（i）微生物群落，（ii）编码所有 PAH 双加氧酶共有的 Rieske 铁硫中心（α-亚基）的部分基因片段，以及（iii）双加氧酶的β-亚基。在 16S rRNA 衍生的克隆文库中检测到与变形菌门（α-、ε-和γ-变形菌门）、酸杆菌门、放线菌门、厚壁菌门、芽单胞菌门和厚壁菌门相关的类群。从各自的克隆文库中获得的α-亚基和β-亚基双加氧酶基因片段的许多基因片段序列属于与参考双加氧酶基因序列不同的进化枝。 Rieske 型[2Fe-2S]簇结合位点的保守序列的存在表明，这些基因片段编码双加氧酶的α-亚基。
代表α-亚基基因片段（Rf1）和β-亚基双加氧酶的克隆文库测序表明，在煤焦油污染土壤中存在以前未知的双加氧酶。
Rieske 引物和细菌群落分析的结合代表了一种强大的工具，可用于评估生物修复潜力和在污染环境中探索新的双加氧酶基因。
