Molecular Ecological Approach to Sulfur-Metabolizing Bacteria in Aquatic Ecosystems

对不同水环境中的硫代谢微生物进行了分子生态学研究。其中，在日本淡水中营养湖泊琵琶湖深处的沉积物中发现了硫化硫化细菌属的淡水丝状滑动硫化物氧化细菌，并对其进行了形态、生态和系统发育特征的研究。Biwa Thiopoca湖在形态上类似于Thiopoca ingrica，一种来自丹麦峡湾的咸水物种。单毛直径为3~5.6μm，完整的硫孔菌丝直径为18~75μm，细胞长度为1.2~3.8μm，经醋酸铀酰染色后，可见致密的胞内颗粒，X射线分析未见磷、硫阳性信号。为了避免污染细菌DNA的非特异性扩增，采用新设计的硫酸盐细菌特异引物(706-Thiopoca、Biwa160F和Biwa829R)与普通细菌引物相结合，扩增琵琶湖硫杆菌16S rRNA基因。对三个重叠的PCR产物进行DGGE分析，均得到单一的DGGE条带，表明已扩增出单一的16S rRNA基因。用同样的方法对来自康斯坦茨湖的硫代普罗卡进行了检测。16S rRNA序列与针对琵琶湖硫代普洛卡湖特定基序的FISH杂交进行了验证。细菌探针EUB-338、γ蛋白细菌探针GAM42a和针对琵琶硫代普洛卡湖的探针Biwa829都获得了阳性信号。根据16S rRNA全序列和形态上的相似性，琵琶湖和康斯坦茨湖的Thiopoca与Thiopoca ingrica有密切的亲缘关系。

Molecular ecological studies were done for sulfur-metabolizing microorganisms in various aquatic environments. Among these, freshwater filamentous gliding sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and characterized morphologically, ecologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameter of individual trichomes was 3-5.6 μm ; the diameter of complete Thioploca filaments was in the range of 18-75 μm. Cell lengths ranged from 1.2 to 3.8 μm. In TEM specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signal of phosphorus and sulfur in the X-ray analysis. The 16S rRNA gene of Thioploca from Lake Biwa was amplified using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F and Biwa829R) in combination with general bacterial primers, to avoid unspecific amplification of contaminating bacterial DNA. DGGE analysis of all three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene has been amplified. With the same method, Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified with FISH hybridization targeted at specific motifs of Lake Biwa Thioploca. Positive signals were obtained for the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and the probe Biwa829 targeting Lake Biwa Thioploca. Based on the near-complete 16S rRNA sequence and on morphological similarities, Thioploca from Lake Biwa and Lake Constance are closely related to Thioploca ingrica and to each other.