Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain

Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene that as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S, thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.