Juretschko S, Timmermann G, Schmid M, Schleifer K H, Pommerening-Röser A, Koops H P, Wagner M
Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich, Germany.
Appl Environ Microbiol. 1998 Aug;64(8):3042-51. doi: 10.1128/AEM.64.8.3042-3051.1998.
The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.
采用多相方法研究了一家接收高氨浓度污水的工业废水处理厂硝化活性污泥中存在的氨氧化细菌和亚硝酸盐氧化细菌种群。使用一组针对氨氧化细菌的分级16S rRNA靶向探针进行原位杂交，结果显示类似运动亚硝化球菌的细菌占主导地位。通过分离运动亚硝化球菌作为数量上占优势的氨氧化菌，并随后进行比较16S rRNA基因（rDNA）序列和DNA-DNA杂交分析，证实了荧光原位杂交（FISH）所表明的系统发育关系。为了对氨氧化菌群进行分子精细尺度分析，从氨氧化菌分离株和活性污泥中提取的总DNA中扩增了编码氨单加氧酶（amoA）活性位点多肽的基因的部分片段。然而，对来自活性污泥的13个amoA克隆序列的比较序列分析表明，这些序列彼此之间以及与欧洲亚硝化单胞菌Nm50和运动亚硝化球菌分离株的相应amoA基因片段高度相似。运动亚硝化球菌分离株和欧洲亚硝化单胞菌的amoA基因片段之间意外的高序列相似性表明可能发生了横向基因转移事件。虽然分离出了一株硝化杆菌菌株，但通过原位杂交在活性污泥中未检测到亚硝酸盐氧化菌属硝化杆菌属成员。因此，我们使用rRNA方法研究其他知名亚硝酸盐氧化细菌属的丰度。使用三种不同的方法从活性污泥中提取DNA。对于每种DNA制备物，分别扩增编码小亚基rRNA的几乎全长基因，并用于构建三个16S rDNA文库。通过比较序列分析，60个随机选择的克隆中有2个可归为硝化螺菌属的亚硝酸盐氧化细菌。基于这些克隆序列，开发了一种特异性的16S rRNA靶向探针。用该探针对活性污泥进行FISH分析表明，类似硝化螺菌的细菌大量存在（占细菌总数的9%），并且经常与运动亚硝化球菌一起出现在共聚集的微菌落中。
