Molecular Ecological Approach to Sulfur-Metabolizing Bacteria in Aquatic Ecosystems

对不同水生环境中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层沉积物中发现了一种淡水丝状滑翔硫化物氧化菌（Thioploca），并对其进行了形态、生态和系统发育鉴定。琵琶湖的Thioploca在形态上与丹麦峡湾的微咸水物种Thioploca ingica相似。单株毛状体直径为3 ~ 5.6 μm；完整的硫斑细丝直径在18 ~ 75 μm之间。细胞长度范围为1.2 ~ 3.8 μm。在用醋酸铀酰染色的TEM样品中，发现致密的细胞内颗粒，在x射线分析中未显示任何磷和硫的阳性信号。采用新设计的Thioploca特异性引物（706-Thioploca， Biwa160F和Biwa829R）与普通细菌引物联合扩增biioploca 16S rRNA基因，避免了污染细菌DNA的非特异性扩增。对所有三个重叠PCR产物进行DGGE分析，结果显示DGGE条带单一，表明扩增了单个16S rRNA基因。用同样的方法对康斯坦茨湖的硫菌卡进行了检测。16S rRNA序列经FISH杂交验证，定位于琵琶湖的特定基序。针对琵琶湖的细菌探针EUB-338、γ-蛋白质细菌探针GAM42a和探针Biwa829均获得阳性信号。基于近乎完整的16S rRNA序列和形态相似性，Biwa湖和Constance湖的Thioploca与ingrica Thioploca亲缘关系密切，且彼此亲缘关系密切。

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.