Yamamoto H, Serizawa M, Thompson J, Sekiguchi J
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386-8567, Japan.
J Bacteriol. 2001 Sep;183(17):5110-21. doi: 10.1128/JB.183.17.5110-5121.2001.
Maltose metabolism and the regulation of the glv operon of Bacillus subtilis, comprising three genes, glvA (6-phospho-alpha-glucosidase), yfiA (now designated glvR), and glvC (EIICB transport protein), were investigated. Maltose dissimilation was dependent primarily upon the glv operon, and insertional inactivation of either glvA, glvR, or glvC markedly inhibited growth on the disaccharide. A second system (MalL) contributed to a minor extent to maltose metabolism. Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of glvA and a polycistronic mRNA of glvA-glvR-glvC. Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5' end of glvA. When glvR was placed under control of the spac promoter, expression of the glv operon was dependent upon the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). In regulatory studies, the promoter sequence of the glv operon was fused to lacZ and inserted into the amyE locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled glvR plasmid, pHYCM2VR. When cultured in Difco sporulation medium containing citrate, this transformant [AMGLV(pHYCM2VR)] expressed LacZ activity, but synthesis of LacZ was repressed by glucose. In an isogenic strain, [AMGLVCR(pHYCM2VR)], except for a mutation in the sequence of a catabolite-responsive element (cre), LacZ activity was expressed in the presence of citrate and glucose. Insertion of a citrate-controlled glvR plasmid at the amyE locus of ccpA(+) and ccpA mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively. In the presence of both glucose and citrate, AMCMVR failed to express the glv operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC. Collectively, our findings suggest that GlvR (the product of the glvR gene) is a positive regulator of the glv operon and that glucose exerts its effect via catabolite repression requiring both CcpA and cre.
对枯草芽孢杆菌麦芽糖代谢及glv操纵子的调控进行了研究，该操纵子由三个基因组成，即glvA（6-磷酸-α-葡萄糖苷酶）、yfiA（现命名为glvR）和glvC（EIICB转运蛋白）。麦芽糖异化主要依赖于glv操纵子，glvA、glvR或glvC的插入失活显著抑制了在二糖上的生长。第二个系统（MalL）对麦芽糖代谢的贡献较小。Northern印迹显示有两种转录本，分别对应于glvA的单顺反子mRNA和glvA-glvR-glvC的多顺反子mRNA。引物延伸分析表明，两种转录本均起始于位于glvA 5'端上游26 bp处的同一个碱基（G）。当glvR置于spac启动子控制下时，glv操纵子的表达依赖于异丙基-β-D-硫代半乳糖苷（IPTG）的存在。在调控研究中，将glv操纵子的启动子序列与lacZ融合并插入amyE位点，然后用柠檬酸控制的glvR质粒pHYCM2VR转化所得菌株（AMGLV）。当在含柠檬酸的Difco芽孢形成培养基中培养时，该转化体[AMGLV(pHYCM2VR)]表达LacZ活性，但LacZ的合成受到葡萄糖的抑制。在一个同基因菌株[AMGLVCR(pHYCM2VR)]中，除了分解代谢物反应元件（cre）序列中的一个突变外，在柠檬酸和葡萄糖存在的情况下表达LacZ活性。在ccpA(+)和ccpA突变体的amyE位点插入柠檬酸控制的glvR质粒，分别产生菌株AMCMVR和AMCMVRCC。在葡萄糖和柠檬酸同时存在的情况下，AMCMVR未能表达glv操纵子，而在相同条件下，菌株AMCMVRCC中两种mRNA转录本均高水平表达。总体而言，我们的研究结果表明，GlvR（glvR基因的产物）是glv操纵子的正调控因子，并且葡萄糖通过需要CcpA和cre的分解代谢物阻遏发挥其作用。
