Analysis of a growth factor of a thermophilic symbiotic bacteria

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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.