Kappes R M, Kempf B, Kneip S, Boch J, Gade J, Meier-Wagner J, Bremer E
Philipps University Marburg, Department of Biology, Germany.
Mol Microbiol. 1999 Apr;32(1):203-16. doi: 10.1046/j.1365-2958.1999.01354.x.
Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.
枯草芽孢杆菌中相容性溶质甘氨酸甜菜碱的生物合成赋予了相当程度的渗透耐受性，其通过胆碱的两步氧化过程进行，以甘氨酸甜菜碱醛为中间体。我们利用了在甘氨酸甜菜碱生产中存在缺陷的枯草芽孢杆菌菌株对甘氨酸甜菜碱醛的敏感性，来筛选对这种有毒中间体具有抗性的突变体。这些菌株在胆碱摄取方面也存在缺陷，遗传分析证明，这些表型需要两个影响不同基因座（opuB和opuC）的突变。分子分析使我们能够证明opuB和opuC操纵子各自编码一个由四个组分组成的依赖于结合蛋白的ABC转运系统。这两种ABC转运蛋白的假定结合蛋白均显示为脂蛋白。通过OpuB（K(m)=1 microM；Vmax=21 nmol min-1 mg-1蛋白）和OpuC（K(m)=38 microM；Vmax=75 nmol min-1 mg-1蛋白）对[14C]-胆碱摄取的动力学分析表明，这些ABC转运蛋白中的每一个都表现出高亲和力和相当大的转运能力。用与OpuB和OpuC转运蛋白的假定底物结合蛋白发生交叉反应的多克隆抗血清进行的蛋白质印迹实验表明，opuB和opuC操纵子的表达受生长培养基渗透压升高的调节。引物延伸分析证实了opuB的渗透控制，并确定了该操纵子的启动子。opuB和opuC操纵子在枯草芽孢杆菌染色体上彼此相邻，它们的高度序列同一性强烈表明这些系统是从一个原始基因簇的复制事件进化而来的。尽管OpuB和OpuC密切相关，但这些系统在渗透保护剂的底物特异性方面表现出显著差异，而对于这种密切相关的ABC转运蛋白来说，这种差异是不容易预测到的。
