Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa, 237-0061, Japan.
Appl Microbiol Biotechnol. 2014 Dec;98(24):10177-86. doi: 10.1007/s00253-014-6053-4. Epub 2014 Sep 10.
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, σ(54). The evidence suggests that the transcription of some genes involved in amino acid metabolism is σ(54)-dependent, and that a bacterial enhancer-binding protein containing a PAS domain controls the transcription under the presence of high levels of bicarbonate.
色氨酸酶阳性的嗜热栖热菌是一种自由生活的共生细菌，它可以在与嗜热脂肪地芽孢杆菌的共培养中有效地生长。我们的研究表明，嗜热栖热菌的生长取决于嗜热脂肪地芽孢杆菌预先建立的高 CO2 和低 O2 条件。使用含有高 CO2 的无氧气氛可以使嗜热栖热菌在不依赖嗜热脂肪地芽孢杆菌的情况下生长，但细胞产量比共培养时低十倍。在这项研究中，我们研究了色氨酸酶在嗜热栖热菌中的共培养依赖性表达和活性。在与嗜热脂肪地芽孢杆菌的共培养中，嗜热栖热菌细胞积累了大量的吲哚，但在细菌的纯培养中，无论是否添加色氨酸，都没有积累吲哚。嗜热栖热菌细胞在由条件培养基（由嗜热脂肪地芽孢杆菌的培养上清液供应的培养基）组成的纯培养中积累吲哚。蛋白质组学分析鉴定了在嗜热栖热菌细胞中特异性产生的蛋白质，该蛋白质是由 tna2（STH439）编码的色氨酸酶。试图从嗜热脂肪地芽孢杆菌的培养上清液中分离出诱导色氨酸酶的成分，但没有成功，但我们确实发现，当向培养基中添加 10 mM 碳酸氢盐时，会发生吲哚积累。RT-PCR 分析表明，添加碳酸氢盐刺激了 tna2 的转录。在 tna2 启动子内鉴定的转录起始位点之前，有一个由替代σ因子σ(54)指定的-24 和-12 共识序列。这一证据表明，一些参与氨基酸代谢的基因的转录依赖于σ(54)，并且含有 PAS 结构域的细菌增强子结合蛋白在存在高浓度碳酸氢盐的情况下控制转录。
