Molecular mechanism of the intermicrobial symbiosis of an obligately symbiotic thermophilic bacterium and its ecological analyses

耐热色氨酸酶产生菌嗜热共生杆菌(ST)是一株在与嗜热芽孢杆菌S(BS)共培养条件下生长的嗜温共生菌。在本研究中，我们阐明了以下几点：(1)对于ST(GF)的生长因子，我们首先建立了定量PCR方法，使ST的特异性定量成为可能。通过基于该技术的精确分析，我们发现GF不仅存在于BS的培养上清液中，而且存在于所有被测试的其他细菌的培养上清液中。分离ST和BS的透析培养使ST能够纯生长，因此推测一个小的可透析分子对GF活性起作用。对低分子物质进行纯化的尝试使我们发现，GF活性存在于蒸汽组分中。BS培养上清液中还含有一种携带GF活性的高分子量物质，推测该物质参与了低分子量GF的产生。其次，我们根据16S rDNA序列进行了系统发育分析，发现ST属于革兰氏阳性细菌群中最外层的一个新分支，与迄今已知的任何其他细菌群都没有聚在一起。第三，我们研究了ST及其相关微生物的环境分布，发现这些细菌非常普遍，不仅分布在土壤环境中，主要包括堆肥，还分布在动物的消化器官中。动物饲料也经常携带这种细菌。16S rDNA系统发育分析表明，在革兰氏阳性菌群中，属于由ST在最外层创建的新分支，进一步的多样性形成了新的分支。此外，对新获得的16S rDNA片段的DGGE分析表明，存在多种共生细菌。

The thermostable-tryptophanase producing microbe, Symbiobacterium thermophilum (ST) is a themophilic symbiotic bacterium which growth in the coculture with thermophilic Bacillus strain S (BS). In the present study, we elucidated following ; (1) as for the growth factor for ST (GF), we first established quantitative PCR procedure, which enabled specific quantification of ST.By the precise analyses based on the technique, we revealed that GF is a general metabolite existing in the culture supernatant not only of BS but of all the other bacteria tested. Dialyzing culture separating ST and BS enabled pure growth of ST, thus it was suggested that a small dialyzable molecule is responsible for the GF activity. Attempt to purify the low molecular weight substance led us to find that the GF activity is present in vapor fraction. BS culture supernatant also contained a high-molecular weight substance carrying the GF activity, which was assumed to be involved in the production of the GF of low-molecular mass.Second, we performed phylogenetic analysis based on the 16S rDNA sequence and revealed that ST belongs to a novel branch created at the outermost position in the Gram-positive bacterial group, which did not cluster to any other bacterial group hitherto known.Third, we examined environmental distribution of ST and its related organisms, and found that these bacteria are quite general, distributing not only in soil environment mainly including compost, but also in digestive organs of animals. Feeds for animals also frequently carried the bacteria. 16S rDNA-based phylogeny of the isolates showed that those fell into the novel branch created by ST at the outermost position in Gram-positive group, and further diversity forming new branches in the cluster. Furthermore, the DGGE analysis of the newly obtained 16S rDNA fragments implied the presence of multiple species belonging to Symbiobacterium.