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Applied and Environmental Microbiology, October 2007, p. 6159-6165, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.02835-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Indole Derivatives as Self-Growth Inhibitors of Symbiobacterium thermophilum, a Unique Bacterium Whose Growth Depends on Coculture with a Bacillus sp.

Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan,¹ Takasago International Corporation, 1-4-11 Nishi-yawata, Hiratsuka 254-0073, Japan²

Received 6 December 2006/ Accepted 2 August 2007

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a Bacillus sp. Recently, we discovered that CO₂ generated by Bacillus is the major inducer for the growth of S. thermophilum; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by S. thermophilum. We succeeded in purifying two substances from an ether extract of the culture supernatant of S. thermophilum by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3'-indolyl)indoxyl (BII) and 1,1-bis(3'-indolyl)ethane (BIE). The pure growth of S. thermophilum was inhibited by authentic BII and BIE with MICs of 12 and 7 µg/ml, respectively; however, its growth in coculture with Bacillus was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 µg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MICs (<0.1 µg/ml) while a marked amount of BIE (6 to 7 µg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that Bacillus benefits S. thermophilum by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.

Published ahead of print on 10 August 2007.