Park Joong-Wook, Crowley David E
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA.
Appl Microbiol Biotechnol. 2006 Oct;72(6):1322-9. doi: 10.1007/s00253-006-0423-5. Epub 2006 Jun 28.
Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5 x 10(3) copy numbers per gram of soil, but increased by over a thousand fold to 10(7) copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.
许多降解多环芳烃（PAHs）的细菌都含有nahAc基因，该基因编码多聚萘双加氧酶的一个组分。由于nahAc基因高度保守，因此该基因可作为PAH降解活性的潜在生物标志物。本研究的目的是使用常规PCR和实时PCR检测土壤中萘降解速率与nahAc基因拷贝数之间的关系。基于33种细菌的nahAc DNA序列设计了四组用于实时PCR的简并引物。在添加萘之前，nahAc基因的拷贝数低于检测限，为每克土壤5×10³个拷贝数，但在接触萘蒸气（土壤水浓度约为30 ppm）6天后增加了一千多倍，达到10⁷个拷贝。通过对来自nahAc引物的PCR产物进行克隆和测序，在添加萘的土壤中发现了两个未报道的萘双加氧酶同源物。这些实验结果证明了土壤微生物群落接触萘后发生的高度动态变化，并表明在PAH污染土壤中，萘的基因拷贝数与降解速率之间存在直接关系。
