Metabolism of inositol stereoisomers in a thermophile,Geobacillus kaustophilusHTA426

嗜热土芽孢杆菌（GeobacilluskaustophilusHTA 426）是一种嗜热芽孢杆菌属的近缘种，以肌醇、D-手性肌醇和鲨肌醇等肌醇立体异构体为唯一碳源。在其基因组中有三个旁系同源基因，可能编码肌醇脱氢酶。这些基因gk 1897、gk 1898和gk 1899串联在一个大的基因簇内，该基因簇包含与B中参与肌醇催化的基因同源的几乎完整的iol基因组。枯草杆菌。将三种合理的肌醇六磷酸酶中的每一种纯化为His 6-标签融合体。酶表现出嗜热活性，每一个肌醇立体异构体和辅因子具有其自身的特征特异性。北方印迹和引物延伸分析表明，这三种酶由相同的5-kb多顺反子转录本编码，并在肌醇的存在下同时诱导。对HTA 426进行EMS诱变，以分离出不能利用肌球蛋白的突变株PS 8， 关于我们 - 肌醇。在PS 8中，肌醇脱氢酶活性被废除沿着5-kb的转录，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。对在肌醇存在下生长的HTA 426细胞中的代谢物的分析表明，在稳定期细胞内出现大量的D-手性肌醇和鲨肌醇，而在PS 8细胞中仅存在肌醇，这表明肌醇立体异构体的相互转化可能涉及这三种酶。嗜碱菌属（kaustophilusto）分别具有三种肌醇脱氢酶基因。gk 1897基因是该菌生长所必需的，它依赖于木糖醇作为唯一碳源。有趣的是，发现缺乏gk 1897的突变体在较低温度下积累高水平的胞内scyllo-inositol以显示出CFU的显著增加。另一方面，gk 1899对肌肌醇和D-手性肌醇的生长都是必需的。PS 8的抑制突变体被分离出来，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本的表达。对10个突变体进行鸟枪测序，发现9个突变体在核糖体结合位点或推定基因的翻译起始密码子上与B直向同源。枯草芽孢杆菌结果提示，PS 8中iol转录丢失的原因可能与Crh的过度阻遏有关。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