Analysis of a growth factor of a thermophilic symbiotic bacteria

我们已经纯化了一种生长因子，它支持专性共生细菌嗜热共生菌（SM）的生长。从芽孢杆菌S（BS）30 μ l培养物的无细胞提取液出发，经硫酸铵沉淀、DEAE-Toyoprotein层析、丁基纤维素层析等方法纯化，得到两个不同的活性组分。通过Mono Q、羟基磷灰石层析和从非变性PAGE凝胶洗脱进一步纯化每个级分。纯化蛋白经SDS-PAGE电泳均为62 kDa的单一条带，N端氨基酸序列完全一致。根据测定的氨基酸序列，我们用PCR方法成功地克隆了编码纯化生长因子的基因。另外，我们利用PCR技术从BS和SM的染色体上克隆了16 SrDNA，并根据该基因的核苷酸序列，推测了该菌在细菌中的系统发育位置。BS位于嗜热菌的分支，与嗜热脂肪芽孢杆菌亲缘关系最近。SM虽然位于低GC革兰氏阳性菌、梭杆菌和高GC革兰氏阳性菌的聚簇中，但不与任何一种细菌发生杯状反应。这一结果表明，SM属于一个全新的家族，它在遗传学上不同于任何其他已知的细菌群。

We have purified a growth factor wich support the growth of an obligatory symbiotic bacterium, Symbiobacterium thermophilum (SM). Started from cell free extract of 30 1 culture of Bacillus strain S (BS) , the factor protein was purified by ammonium sulfate precipitation, DEAE-Toyopearl, Butyl cellulofine chromatography and two different active fractions were obtained. Each fraction was further purified by Mono Q,Hydroxylapatite chromatography and elution from Native PAGE gel. Both of the purified protein showed 62 kDa single band on SDS-PAGE gel electrophoresis, and the N-terminal amino acid sequences were identical. We have succeeded in cloning the gene encoding purified growth factor by PCR method based on the amino acid squence determined. The nucleotide sequece to the cloned gene fragment suggested that it encodes a homolog of Enzyme I of phosphotransferase system which is distributed among bacteria involved in uptake of sugar into the cell.On the other hand, we have cloned 16s rDNA from the chromosome of BS and SM by PCR.Based on the nucleotide sequence of the cloned fragment, we deduced the phylogenetic location of the organisms among bacteria. BS was located in the branch of thermophilic Basillus spieces and showed most close relationship with B.stearothermophilus. Although SM was located in the cluster including low-GC gram positive bacteria, Fusobacteria and high-GC gram positive bacteria, it did not cuppled with any of them. This result suggests that SM belongs to a completely new family which is phylogeneticaly different from any other known group of bacteria.