Leys Natalie M E J, Ryngaert Annemie, Bastiaens Leen, Verstraete Willy, Top Eva M, Springael Dirk
Environmental Technology, Flemish Institute for Technological Research, 2400 Mol, Belgium.
Appl Environ Microbiol. 2004 Apr;70(4):1944-55. doi: 10.1128/AEM.70.4.1944-1955.2004.
Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.
鞘氨醇单胞菌属的细菌菌株常从受污染土壤中分离得到，因为它们能够将多环芳烃（PAH）作为唯一的碳源和能源。因此，直接检测受污染土壤中本地或接种的鞘氨醇单胞菌菌株对于生物修复目的具有重要意义。在本研究中，开发了一种基于PCR的非培养检测方法，该方法使用靶向鞘氨醇单胞菌16S rRNA基因的特异性引物，并结合变性梯度凝胶电泳（DGGE）来评估PAH污染土壤中鞘氨醇单胞菌的多样性。使用新引物对在一组不同细菌属的模板DNA上进行PCR，结果表明该方法对鞘氨醇单胞菌科的细菌具有选择性。对大多数测试的鞘氨醇单胞菌菌株获得了单带DGGE图谱。属于同一物种的菌株具有相同的DGGE指纹图谱，并且在大多数情况下，这些指纹图谱是一个物种所特有的。接种菌株在土壤细胞浓度为10(4) CFU g(-1)时即可被检测到。通过新的PCR-DGGE方法对几种PAH污染土壤的鞘氨醇单胞菌种群结构进行分析，结果表明，菲浓度最高的土壤中鞘氨醇单胞菌的多样性最低。从土壤DNA扩增的克隆PCR产物的序列分析揭示了新的16S rRNA基因鞘氨醇单胞菌序列，这些序列与已知培养菌株的序列有显著差异（即来自环境克隆的序列在系统发育上与网上其他可用的环境克隆序列分组，可能源自几个潜在的新物种）。总之，新设计的鞘氨醇单胞菌特异性PCR-DGGE检测技术成功地在物种水平上分析了污染土壤中的鞘氨醇单胞菌群落，并揭示了以前通过依赖培养的检测技术未检测到的不同鞘氨醇单胞菌成员。
