Molecular mechanisms of extreme desiccation tolerance in the terrestrial cyanobacterium

研究了野外分离的褐藻菌自然群落海藻糖水平对干旱的响应。在完全水化的N. commune菌落中未发现可检测到的海藻糖；然而，海藻糖的积累是对干燥过程中水分流失的反应，在风干的菌落中检测到高水平的海藻糖。海藻糖代谢的三个基因，treZ（麦芽糖低聚糖海藻糖水解酶，Mth）， treY（麦芽糖低聚糖海藻糖合成酶，Mts）和treH（海藻酶），被发现为一个基因簇，这些基因的mrna在干燥过程中被检测到相似的水平。点状芽孢杆菌海藻糖酶在大肠杆菌细胞中以活性形式异源表达，分子量为52kDa。海藻糖酶活性在10mM NaCl的作用下受到强烈抑制，而海藻糖合成活性在盐的作用下保持活跃。这些数据表明，海藻糖的生产速度超过了在水分胁迫条件下海藻糖的水解速度，其特征是细胞溶质浓度增加。陆地蓝藻N.公社形成了一个可见的菌落，其中细胞嵌入细胞外多糖（eps），这在这种生物的极端干燥耐受性中起着至关重要的作用。以人工硝基苯连接糖为底物，研究了糖苷键的水解活性。a - β- d -葡萄糖苷酶分子量为20kda，耐沸煮20min，根据其n端氨基酸序列鉴定为蓝细菌的束状丝状蛋白。其中36 kda的主蛋白（WspA）具有耐热β- d -半乳糖苷酶活性。

We investigated changes of trehalose level in field-isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air-dried colonies. The three genes for trehalose metabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY (maltooligosyltrehalose synthase, Mts), and treH (trehalase), were found as a gene cluster, and the mRNAs for these genes were detectable at similar levels during desiccation. Trehalase of N. punctiforme was heterologously expressed in E. coli cells in an active form with a molecular mass of 52kDa. Trehalase activity was strongly inhibited in the presence of 10mM NaCl while trehalose synthesis activity remained active in the presence of salt. These data suggest that the rate of trehalose production exceeds that of trehalose hydrolysis under water-stress conditions characterized by increased cellular solute concentrations. The terrestrial cyanobacterium N. commune forms a visible colony in which the cells are embedded in extracellular polysaccharides (EPSs), which play a crucial role in the extreme desiccation tolerance of this organism. The activities that hydrolyse glycoside bonds were examined using artificial nitrophenyl-linked sugars as substrates. Aβ-D-glucosidase, with a molecular mass of20kDa, was resistant to20min of boiling and was identified as a cyanobacterial fasciclin protein based on its N-terminal amino-acid sequence. The36-kDa major protein (WspA) showed heat-resistantβ-D-galactosidase activity.