Boch J, Kempf B, Schmid R, Bremer E
Max-Planck Institute for Terrestrial Microbiology, Federal Republic of Germany.
J Bacteriol. 1996 Sep;178(17):5121-9. doi: 10.1128/jb.178.17.5121-5129.1996.
Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the choline-glycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline- or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to sigma A-dependent promoters and was stimulated by the presence of choline in the growth medium.
从外源提供的前体胆碱或甘氨酸甜菜碱醛合成渗透保护剂甘氨酸甜菜碱，可使枯草芽孢杆菌在高渗培养基中具有相当强的渗透胁迫耐受性。利用一株在甘氨酸甜菜碱合成酶方面存在缺陷的大肠杆菌突变体（betBA），我们通过功能互补克隆了枯草芽孢杆菌中合成渗透保护剂甘氨酸甜菜碱所需的基因。确定了克隆的枯草芽孢杆菌染色体DNA中一个4.1kb片段的DNA序列，并鉴定出两个基因（gbsA和gbsB），其产物对于甘氨酸甜菜碱生物合成和渗透保护至关重要。gbsA和gbsB基因同向转录，由一个短的基因间隔区隔开，可能形成一个操纵子。推导的gbsA基因产物与一类特殊和非特殊醛脱氢酶超家族的成员具有很强的序列同一性。这个超家族包括来自细菌和植物的甘氨酸甜菜碱醛脱氢酶，已知它们参与细胞对高渗胁迫和干旱的适应。推导的gbsB基因产物与III型醇脱氢酶家族具有显著相似性。通过标记替换构建了染色体gbsAB基因存在缺陷的枯草芽孢杆菌突变体，并分析了这些突变菌株在高渗培养基中的生长特性。gbsAB基因的缺失破坏了胆碱 - 甘氨酸甜菜碱合成途径，并消除了枯草芽孢杆菌在含胆碱或甘氨酸甜菜碱醛的培养基中有效应对高渗胁迫 的能力。在gbsAB突变菌株中，放射性标记胆碱的摄取未发生改变。在缺乏甘氨酸甜菜碱生物合成酶的菌株中，胆碱或甘氨酸甜菜碱醛在细胞内的持续积累严重干扰了枯草芽孢杆菌的生长，即使在中等渗透压的培养基中也是如此。通过高分辨率引物延伸分析检测到gbsAB的单个转录起始位点。gbsAB转录从一个与依赖于σA的启动子具有高度同源性的启动子开始，并受到生长培养基中胆碱的刺激。
