Identification of two myo-inositol transporter genes of Bacillus subtilis
Identification of two myo-inositol transporter genes of Bacillus subtilis
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DOI：
10.1128/jb.184.4.983-991.2002
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发表时间：
2002-02-01
影响因子：
3.2
通讯作者：
Fujita, Y
中科院分区：
文献类型：
--
作者：
Yoshida, KI;Yamamoto, Y;Fujita, Y
在参与枯草芽孢杆菌基因组计划功能分析的日本小组系统构建的数百个突变体中，我们发现 iolT 失活的突变体（ydjK）表现出以肌醇作为唯一碳源的生长缺陷。 iolT 的假定产物与数据库中的许多细菌糖转运蛋白表现出显着的相似性。在枯草芽孢杆菌中，已知 iolABCDEFGHIJ 和 iolRS 操纵子参与肌醇利用，其转录受 IolR 阻遏物调节并由肌醇诱导。在 iol 基因中，iolF 预计编码肌醇转运蛋白。单独 iolF 失活不会像 iolT 失活那样导致肌醇明显的生长缺陷，而同时失活两个基因会导致比单独 iolT 失活更严重的缺陷。突变体对肌醇摄取的测定表明，iolT 失活几乎完全消除了摄取，但 IolF 本身的摄取是轻微可检测到的。这些结果以及 IolT 和 IolF 肌醇转运蛋白的 K-m 和 V-max 值表明 iolT 和 iolF 分别编码主要和次要肌醇转运蛋白。 iolT 转录的 Northern 和引物延伸分析表明，该基因是从可能被 sigma(A) RNA 聚合酶识别的启动子单顺反子转录的，并且也受到 IolR 的负调控。通过凝胶阻滞和DNase I足迹实验分析了IolR和iolT启动子区之间的相互作用，这表明相互作用的模式与iol divergon启动子区的相互作用模式非常相似。
Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source. The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases. In B. subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the iol genes, iolF was predicted to encode an inositol transporter. Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation. Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the K-m and V-max values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively. Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by sigma(A) RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.