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Appl. Environ. Microbiol. doi:10.1128/AEM.02835-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of indole derivatives as a self-growth inhibitor of Symbiobacterium thermophilum, a unique bacterium whose growth depends on coculture with Bacillus sp

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

^* To whom correspondence should be addressed. Email: ueda{at}brs.nihon-u.ac.jp.

	Abstract

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. EI-MS and NMR 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 minimal inhibitory concentrations (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 micro-organisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MIC values (<0.1 µg/ml) while a marked amount of BIE (6–7 µg/ml) equivalent to the MIC value had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivity 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 micro-organisms.