Metabolism of inositol stereoisomers in a thermophile,Geobacillus kaustophilusHTA426

KaustophilusHTA426是一种嗜热芽孢杆菌属细菌，它利用一些肌醇立体异构体，包括肌醇、D-甲氧基和肌醇作为唯一的碳源。在它的基因组中有三个相似的基因，可能编码肌醇脱氢酶。这些基因gk1897、gk1898和gk1899位于一个大的基因簇中，该基因簇包含一套几乎完整的与枯草杆菌肌醇分解代谢相关的基因。三个看似合理的肌醇脱氢酶均以His6-tag融合形式纯化。这些酶表现出嗜热活性，每个酶对肌醇立体异构体和辅因子都有自己特有的专一性。Northern杂交和引物延伸分析表明，这三种酶由相同的5kb多顺反子转录本编码，并且在肌醇存在的情况下同时诱导。对HTA426进行电磁诱变，获得不能利用Myo…的突变株pS8。更多-肌醇。在PS8中，肌醇脱氢酶活性与5-kb转录本一起被取消，这表明这三种酶中的任何一种都是肌醇依赖生长所必需的。对HTA426细胞在肌醇存在下的代谢产物的分析表明，在稳定期细胞内出现了大量的D-肌醇和肌醇，而在PS8细胞中只有肌醇存在，这表明肌醇立体异构体的相互转化可能涉及这三种酶。以肌醇为唯一碳源的gk1897基因是该菌生长所必需的。有趣的是，缺乏gk1897的突变体被发现在较低的温度下积累了高水平的胞内肌醇，表现出显著的CFU增加。另一方面，gk1899对肌醇和D-环肌醇的生长都是必需的。分离到了PS8的抑制突变体，使其在肌醇上有较好的生长，恢复了编码三种肌醇脱氢酶的转录本的表达。对10个突变株进行鸟枪测序，发现9个突变株在核糖体结合部位或与枯草杆菌CRH同源的基因翻译起始密码子上存在共同突变。结果提示，PS8基因转录缺失的原因可能与CRH过度抑制有关。较少

Geobacillus kaustophilusHTA426, a thermophilic Bacillus-related species, utilizes someinositol stereoisomers, including myo-, D-chiro-, and scyllo-inositols, as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes, gk1897, gk1898, and gk1899, are located in tandem within a large gene cluster containing analmost complete set of iolgenes homologous to genes involved in inositol catabolism in B. subtilis. Each of the three plausible inositol dehydrogenases was purified as a His6-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5-kb polycistronic transcript and were induced simultaneously in the presence of myo-inositol. HTA426 was subjected to EMS mutagenesis to isolate amutant strain, PS8, which was not able to utilize myo … More -inositol. In PS8, inositol dehydrogenase activity was abolished along with the 5-kb transcript, suggesting that any of the three enzymes is required for myo-inositol-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of myo-inositol revealed that substantial amounts of D-chiro-inositol and scyllo-inositol appeared intracellularly during the stationary phase, while only myo-inositol was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.We have successfully established transformation of G. kaustophilusto inactivate each of the three inositol dehydrogenase genes. The gk1897gene was essential for the growth depending on scyllo-inositol as the sole carbon source. Intriguingly, the mutant lacking gk1897was found to accumulate high level of intracellular scyllo-inositol to exhibited significant increase in CFU at lower temperatures. On the other hand, gk1899was essential for the growth on both myo- and D-chiro-inositols. Suppressor mutants of PS8 were isolated to exhibit better growth on myo-inositol, which regained expression of the transcript encodingthe three inositol dehydrogenases. Shotgun sequencing the 10 individual mutants revealed that 9 were found to carry the common mutation either in ribosome-binding site or translation start codon of a putative gene orthologous to B. subtilis crh. The results suggested that the reason for lost ioltranscription in PS8 might have something to do with excessive repression involving Crh. Less