Metabolism of inositol stereoisomers in a thermophile,Geobacillus kaustophilusHTA426

Geobacillus kaustophilusHTA426是一种与嗜热杆菌相关的物种，它利用一些肌醇立体异构体，包括肌醇，d -基和三基肌醇，作为唯一的碳源。在其基因组中有三个同源基因可能编码肌醇脱氢酶。gk1897、gk1898和gk1899这三个基因串联在一个大的基因簇中，该基因簇包含几乎完整的与枯草芽孢杆菌肌醇分解代谢相关的基因同源的基因。三种可能的肌醇脱氢酶中的每一种都被纯化为his6标签融合。这些酶表现出嗜热活性，每种酶对肌醇立体异构体和辅因子都有自己的特征特异性。Northern blot和引物延伸分析表明，这三种酶由相同的5kb多顺反电子转录物编码，并且在肌醇存在下同时被诱导。对HTA426进行EMS诱变，分离出不能利用肌醇的突变株PS8。在PS8中，肌醇脱氢酶活性与5kb转录本一起被消除，这表明这三种酶中的任何一种都是肌醇依赖性生长所必需的。对HTA426细胞在肌醇存在下生长的代谢物的分析显示，在固定期细胞内出现了大量的d -chiro-肌醇和scylo -肌醇，而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。我们已经成功地建立了G. kaustophilus3个肌醇脱氢酶基因灭活的转化。gk1897基因对以三基肌醇为唯一碳源的生长至关重要。有趣的是，缺乏gk1897的突变体被发现在较低温度下细胞内积累大量的肌醇，从而显着增加CFU。另一方面，gk1899对于肌醇和d -肌醇的生长都是必不可少的。分离出的PS8抑制突变体在肌醇上表现出更好的生长，恢复了编码三种肌醇脱氢酶的转录物的表达。对10个突变体进行霰弹枪测序，发现9个突变体在核糖体结合位点或翻译起始密码子上携带共同突变，这些基因被认为是与枯草芽孢杆菌crh同源的基因。结果表明，PS8缺失的原因可能与涉及Crh的过度抑制有关。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