Kuhlmann Anne U, Bremer Erhard
Department of Biology, Laboratory for Microbiology, Philipps University Marburg, Karl-von-Frisch Strasse, D-35032 Marburg, Federal Republic of Germany.
Appl Environ Microbiol. 2002 Feb;68(2):772-83. doi: 10.1128/AEM.68.2.772-783.2002.
By using natural-abundance (13)C-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography (HPLC) analysis we have investigated the types of compatible solutes that are synthesized de novo in a variety of Bacillus species under high-osmolality growth conditions. Five different patterns of compatible solute production were found among the 13 Bacillus species we studied. Bacillus subtilis, B. licheniformis, and B. megaterium produced proline; B. cereus, B. circulans, B. thuringiensis, Paenibacillus polymyxa, and Aneurinibacillus aneurinilyticus synthesized glutamate; B. alcalophilus, B. psychrophilus, and B. pasteurii synthesized ectoine; and Salibacillus (formerly Bacillus) salexigens produced both ectoine and hydroxyectoine, whereas Virgibacillus (formerly Bacillus) pantothenticus synthesized both ectoine and proline. Hence, the ability to produce the tetrahydropyrimidine ectoine under hyperosmotic growth conditions is widespread within the genus Bacillus and closely related taxa. To study ectoine biosynthesis within the group of Bacillus species in greater detail, we focused on B. pasteurii. We cloned and sequenced its ectoine biosynthetic genes (ectABC). The ectABC genes encode the diaminobutyric acid acetyltransferase (EctA), the diaminobutyric acid aminotransferase (EctB), and the ectoine synthase (EctC). Together these proteins constitute the ectoine biosynthetic pathway, and their heterologous expression in B. subtilis led to the production of ectoine. Northern blot analysis demonstrated that the ectABC genes are genetically organized as an operon whose expression is strongly enhanced when the osmolality of the growth medium is raised. Primer extension analysis allowed us to pinpoint the osmoregulated promoter of the B. pasteurii ectABC gene cluster. HPLC analysis of osmotically challenged B. pasteurii cells revealed that ectoine production within this bacterium is finely tuned and closely correlated with the osmolality of the growth medium. These observations together with the osmotic control of ectABC transcription suggest that the de novo synthesis of ectoine is an important facet in the cellular adaptation of B. pasteurii to high-osmolarity surroundings.
通过使用天然丰度的（13）C - 核磁共振光谱法和高效液相色谱（HPLC）分析，我们研究了在高渗透压生长条件下多种芽孢杆菌属物种中从头合成的相容性溶质的类型。在我们研究的13种芽孢杆菌中发现了五种不同的相容性溶质产生模式。枯草芽孢杆菌、地衣芽孢杆菌和巨大芽孢杆菌产生脯氨酸；蜡样芽孢杆菌、环状芽孢杆菌、苏云金芽孢杆菌、多粘类芽孢杆菌和嗜解硫胺素芽孢杆菌合成谷氨酸；嗜碱芽孢杆菌、嗜冷芽孢杆菌和巴氏芽孢杆菌合成四氢嘧啶；嗜盐芽孢杆菌（以前称为芽孢杆菌）产生四氢嘧啶和羟基四氢嘧啶，而泛酸芽孢杆菌（以前称为芽孢杆菌）合成四氢嘧啶和脯氨酸。因此，在高渗生长条件下产生四氢嘧啶类四氢嘧啶的能力在芽孢杆菌属和密切相关的分类群中广泛存在。为了更详细地研究芽孢杆菌属物种组内的四氢嘧啶生物合成，我们重点研究了巴氏芽孢杆菌。我们克隆并测序了其四氢嘧啶生物合成基因（ectABC）。ectABC基因编码二氨基丁酸乙酰转移酶（EctA）、二氨基丁酸转氨酶（EctB）和四氢嘧啶合酶（EctC）。这些蛋白质共同构成四氢嘧啶生物合成途径，它们在枯草芽孢杆菌中的异源表达导致了四氢嘧啶的产生。Northern印迹分析表明，ectABC基因在遗传上被组织成一个操纵子，当生长培养基的渗透压升高时，其表达会强烈增强。引物延伸分析使我们能够确定巴氏芽孢杆菌ectABC基因簇的渗透调节启动子。对经渗透压挑战的巴氏芽孢杆菌细胞的HPLC分析表明，该细菌内四氢嘧啶的产生是精细调节的，并且与生长培养基的渗透压密切相关。这些观察结果以及ectABC转录的渗透控制表明，四氢嘧啶的从头合成是巴氏芽孢杆菌细胞适应高渗环境的一个重要方面。
