J. Microbiol. Biotechnol. (2003), 13(6), 1008–1012 Application of Denaturing Gradient Gel Electrophoresis to Estimate the Diversity of Commensal Thermophiles BAE, JIN-WOO 1 , JOONG-JAE KIM 2 , CHE OK JEON 1 , KWANG KIM 2 , JAE JUN SONG 1 , SEUNG-GOO LEE 1 , HARYOUNG POO 1 , CHANG-MIN JUNG 2 , YONG-HA PARK, AND MOON-HEE SUNG 2,3 1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-600, Korea 2 BioLeaders Corp., 408-1 Sajeung-Dong, Jung-Gu, Daejeon 301-212, Korea 3 Department of Techno Science, College of Natural Sciences, Kookmin University, 861-1, Chongnung-Dong, Songbuk-Gu, Seoul 136-072, Korea Received: April 14, 2003 Accepted: June 4, 2003 Abstract Symbiobacterium toebii has been reported as a thermophile exhibiting a commensal interaction with Geobacillus toebii. The distribution of the commensal thermophiles in various soils was investigated using a denaturing gradient gel electrophoresis (DGGE). Based on the DGGE analysis, the enrichment condition for the growth of Symbiobacterium sp. was found to also enrich populations of several other microbial spp. as well as Symbiobacterium sp. In the enrichment experiment, several different 16S rDNA sequences of commensal thermophiles were detected in all of the soil samples tested, indicating that commensal thermophiles are widely distributed in various soils. Key words: DGGE, diversity, Symbiobacterium toebii It is estimated that there are large numbers of unculturable microorganisms in natural environments, which can only be detected by molecular ecological methods [1, 4, 8, 9, 19]. Among the reasons that many microorganisms can not be cultured in laboratory conditions is the fact that a biological interaction is an essential factor in the growth of these unculturable microorganisms [2]. Symbiobacterium sp. is a thermophile exhibiting a commensal interaction with Geobacillus sp. [14, 15, 16, 17]. The commensal interaction is a unique example of microbe-microbe interaction. Previous studies have revealed the presence of novel lineages of heterotrophic commensal thermophilic organisms, but only two strains of commensal thermophiles, i.e. S. toebii SC-1 and S. thermophilum strain T, have been grown in culture and the factors involved in the commensal interaction are still unclear [11, 12, 18, 20]. Among the molecular ecological methods, DGGE has been the most widely used to investigate distribution patterns of soil bacterial assemblages [3, 6, 7, 21]. However, this technique has not been applied to the estimation of commensal bacterial diversity, because of lack of enough information to understand and reproduce real micro-environmental niches. Among recent molecular ecological studies, there are reports on competitive quantitative PCR (CQ-PCR) and terminal restriction fragment length polymorphism (TRFLP) which focused on the whole Symbiobacterium assemblage by using Symbiobacterium-specific primers [16, 20] and these studies analyzed the diversity of Symbiobacterium from various environment sources, but did not find any populations that existed together with the commensal thermophiles in the natural environmental niche. In the present study, we examined the appropriateness of the enrichment condition of commensal thermophiles with DGGE by comparing enriched samples with non-enriched soil samples. We optimized the conditions for universal primer sets and tested their performance with enriched cultures and environmental samples. The relative effectiveness of each set was evaluated by comparing community profiles obtained from different compost samples from Korea. Finally, the DGGE results were compared with the results obtained by using two other molecular analyses, namely, CQ-PCR and TRFLP analysis. Since knowledge about the ecology of the microorganism is required to understand the microbial interaction, we also investigated possible abundance of the commensal thermophiles in soils using a PCR-DGGE analysis. The results obtained may provide evidence for the presence of unknown novel microbial interactions among uncultivated microorganisms in the ecosystem. *Corresponding author Phone: 82-42-583-7671; Fax: 82-42-583-7670; E-mail: smoonhee@bioleaders.co.kr