Daims H, Nielsen J L, Nielsen P H, Schleifer K H, Wagner M
Lehrstuhl für Mikrobiologie, Technische Universität München, 85350 Freising, Germany.
Appl Environ Microbiol. 2001 Nov;67(11):5273-84. doi: 10.1128/AEM.67.11.5273-5284.2001.
Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO(3)(-) or as CO(2)) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.
通过非培养分子方法研究了不同生物膜和活性污泥样品中未培养的硝化螺旋菌属类细菌。首先，通过16S rRNA基因序列分析确定了硝化生物膜中硝化螺旋菌属类细菌的系统发育关系。随后，构建了包含所有公开可用序列的硝化螺旋菌门系统发育共识树。该分析表明，硝化螺旋菌属至少由四个不同的亚谱系组成。基于这些数据，分别开发了两种针对硝化螺旋菌门和属的16S rRNA导向寡核苷酸探针，并评估了其用于荧光原位杂交（FISH）的适用性。通过FISH、共聚焦激光扫描显微镜和计算机辅助三维可视化，使用这些探针研究了污水处理厂中硝化螺旋菌属类亚硝酸盐氧化菌细胞聚集体的原位结构。通过荧光素染色证明，在硝化螺旋菌小菌落内部检测到了可透水的空洞和无细胞通道网络。通过结合FISH和微放射自显影技术，研究了不同培养条件下自然生境中硝化螺旋菌属类细菌对不同碳源的摄取情况。在有氧条件下，生物反应器样品中的硝化螺旋菌属类细菌摄取无机碳（以HCO₃⁻或CO₂形式）和丙酮酸，但不摄取乙酸盐、丁酸盐和丙酸盐，这表明这些细菌在丙酮酸存在下可以进行兼性营养生长。相比之下，在缺氧或厌氧条件下未观察到硝化螺旋菌属类细菌对任何测试碳源的摄取。
