Metabolism of inositol stereoisomers in a thermophile,Geobacillus kaustophilusHTA426

嗜热土芽孢杆菌 HTA426 中肌醇立体异构体的代谢

基本信息

批准号：
22310130
负责人：
YOSHIDA Ken-ichi
金额：
$ 12.06万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22310130/
关键词：
遺伝子資源ゲノム機能 Geobacillus kaustophilus inositol Bacillus subtilis

项目摘要

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

Geobacillus kaustophilushta426是一种与嗜热芽孢杆菌相关的物种，使用某种音析醇立体异构体，包括肌含量为碳源，包括肌酸，d-chiro-和scyllo---肌醇。在其基因组中是三个可能编码肌醇脱氢酶的寄生基因。这些基因GK1897，GK1898和GK1899位于一个大基因簇中，该基因簇包含含有抗肌醇分解代谢的基因同源的肛门完整的iolgenes集合。三种合理的肌醇脱氢酶中的每一个均被纯化为His6-Tag融合。这些酶暴露了嗜热活性，每种活性都具有对肌醇立体异构体和辅因子的特征性特异性。 Northern印迹和引物扩展分析表明，这三种酶是由相同的5-kb多源转录本编码的，并在肌醇存在的情况下仅诱导。 HTA426经历了EMS诱变，以分离出Amtant菌株PS8，该菌株无法利用Myo…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，这表明三种酶中的任何一种是肌醇依赖性生长所必需的。在肌醇存在下生长的HTA426细胞中代谢产物的分析表明，大量的D- chiro-肌醇和scyllo-尼罗醇在固定阶段内细胞内出现，而仅在PS8中存在肌醇，而在PS8细胞中才存在，这表明了这些转换的互感，这些转化为这些转化剂的互感性互动了，这可能是互动的。 G. kaustophilusto使三个肌醇脱氢酶基因中的每一个失活。 GK1897Gene对于生长至关重要，具体取决于Scyllo-肌醇作为唯一的碳源。有趣的是，缺乏GK1897WA的突变体发现在较低温度下会累积高水平的细胞内Scyllo-粘合醇，从而暴露了CFU的显着增加。另一方面，GK18999是肌肌醇和D-Chiro肌醇的生长至关重要的。将PS8的抑制突变体分离出来，以暴露于肌醇上的更好的生长，这仍然是编码三种肌醇脱氢酶的转录本的表达。 shot弹枪测序的10个单个突变体显示，发现9个在核糖体结合位点携带了共同的突变，或者是推定的基因与枯草芽孢杆菌CRH的基因的转换起始密码子。结果表明，在PS8中丢失ioltransscription的原因可能与涉及CRH的过量表达有关。较少的