| Budget Amount *help |
¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
Fiscal Year 2012: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2011: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000)
Fiscal Year 2010: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000)
|
|---|
| Research Abstract |
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
|
