Cayley Scott, Record M Thomas
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Biochemistry. 2003 Nov 4;42(43):12596-609. doi: 10.1021/bi0347297.
To better understand the biophysical basis of osmoprotection by glycine betaine (GB) and the roles of cytoplasmic osmolytes, water, and macromolecular crowding in the growth of osmotically stressed Escherichia coli, we have determined growth rates and amounts of GB, K(+), trehalose, biopolymers, and water in the cytoplasm of E. coli K-12 grown over a wide range of high external osmolalities (1.02-2.17 Osm) in MOPS-buffered minimal medium (MBM) containing 1 mM betaine (MBM+GB). As osmolality increases, we observe that the amount of cytoplasmic GB increases, the amounts of K(+) (the other major cytoplasmic solute) and of biopolymers remain relatively constant, and the growth rate and the amount of cytoplasmic water decrease strongly, so concentrations of biopolymers and all solutes increase with increasing osmolality. We observe the same correlation between the growth rate and the amount of cytoplasmic water for cells grown in MBM+GB as in MBM, supporting our proposal that the amount of cytoplasmic water is a primary determinant of the growth rate of osmotically stressed cells. We also observe the same correlation between cytoplasmic concentrations of biopolymers and K(+) for cells grown in MBM and MBM+GB, consistent with our hypothesis of compensation between the anticipated large perturbing effects on cytoplasmic protein-DNA interactions of increases in cytoplasmic concentrations of K(+) and biopolymers (crowding) with increasing osmolality. For growth conditions where the amount of cytoplasmic water is relatively large, we find that cytoplasmic osmolality is adequately predicted by assuming that contributions of individual solutes to osmolality are additive and using in vitro osmotic data on osmolytes and a local bulk domain model for cytoplasmic water. At moderate growth osmolalities (up to 1 Osm), we conclude that GB is an efficient osmoprotectant because it is almost as excluded from the biopolymer surface in the cytoplasm as it is from native protein surface in vitro. At very high growth osmolalities where cells contain little cytoplasmic water, predicted cytoplasmic osmolalities greatly exceed observed osmolalities, and the efficiency of GB as an osmolality booster decreases as the amount of cytoplasmic water decreases.
为了更好地理解甘氨酸甜菜碱（GB）渗透保护的生物物理基础，以及细胞质渗透溶质、水和大分子拥挤效应在渗透压胁迫下大肠杆菌生长中的作用，我们测定了在含有1 mM甜菜碱的MOPS缓冲基本培养基（MBM + GB）中，于广泛的高外部渗透压范围（1.02 - 2.17 Osm）下生长的大肠杆菌K - 12细胞质中GB、K⁺、海藻糖、生物聚合物和水的生长速率及含量。随着渗透压升高，我们观察到细胞质中GB含量增加，K⁺（另一种主要的细胞质溶质）和生物聚合物的含量保持相对恒定，生长速率和细胞质水量则大幅下降，因此生物聚合物和所有溶质的浓度随渗透压升高而增加。我们观察到在MBM + GB中生长的细胞，其生长速率与细胞质水量之间存在与在MBM中生长的细胞相同的相关性，这支持了我们的观点，即细胞质水量是渗透压胁迫下细胞生长速率的主要决定因素。我们还观察到在MBM和MBM + GB中生长的细胞，其生物聚合物和K⁺的细胞质浓度之间存在相同的相关性，这与我们的假设一致，即随着渗透压升高，细胞质中K⁺和生物聚合物浓度增加（拥挤效应）对细胞质中蛋白质 - DNA相互作用产生的预期大的干扰效应之间存在补偿作用。对于细胞质水量相对较大的生长条件，我们发现通过假设单个溶质对渗透压的贡献是可加性的，并使用渗透溶质的体外渗透数据和细胞质水的局部本体域模型，可以充分预测细胞质渗透压。在中等生长渗透压（高达1 Osm）下，我们得出结论，GB是一种有效的渗透保护剂，因为它在细胞质中几乎与在体外天然蛋白质表面一样被排除在生物聚合物表面之外。在细胞含有很少细胞质水的非常高的生长渗透压下，预测的细胞质渗透压大大超过观察到的渗透压，并且随着细胞质水量减少，GB作为渗透压增强剂的效率降低。
