Analysis of the tryptophanase expression in Symbiobacterium thermophilum in a coculture with Geobacillus stearothermophilus

Abstract

The tryptophanase-positive Symbiobacterium thermophilum is a free-living syntrophic bacterium that grows effectively in a coculture with Geobacillus stearothermophilus. Our studies have shown that S. thermophilum growth depends on the high CO₂ and low O₂ condition established by the precedent growth of G. stearothermophilus. The use of an anoxic atmosphere containing high CO₂ allows S. thermophilum to grow independently of G. stearothermophilus, but the cellular yield is ten times lower than that achieved in the coculture. In this study, we characterized the coculture-dependent expression and activity of tryptophanase in S. thermophilum. S. thermophilum cells accumulated a marked amount of indole in a coculture with G. stearothermophilus, but not in the bacterium’s pure culture irrespective of the addition of tryptophan. S. thermophilum cells accumulated indole in its pure culture consisting of conditioned medium (medium supplied with culture supernatant of G. stearothermophilus). Proteomic analysis identified the protein specifically produced in the S. thermophilum cells grown in conditioned medium, which was a tryptophanase encoded by tna2 (STH439). An attempt to isolate the tryptophanase-inducing component from the culture supernatant of G. stearothermophilus was unsuccessful, but we did discover that the indole accumulation occurs when 10 mM bicarbonate is added to the medium. RT-PCR analysis showed that the addition of bicarbonate stimulated transcription of tna2. The transcriptional start site, identified within the tna2 promoter, was preceded by the −24 and −12 consensus sequences specified by an alternative sigma factor, σ⁵⁴. The evidence suggests that the transcription of some genes involved in amino acid metabolism is σ⁵⁴-dependent, and that a bacterial enhancer-binding protein containing a PAS domain controls the transcription under the presence of high levels of bicarbonate.