Lactococcus lactis stringent response. Lactococcus lactis

The stringent response was defined in Lactococcus lactis through transcript profiling after the addition of a chemical inductor, the norvaline. Gene expression was measured in the exponential growth phase (reference sample) and at 1.6 h after norvaline addition. Four hundred and sixty one differentially expressed genes were identified and constituted the stringent response regulon. Keywords: stress response, time course Overall design: Stringent response was imposed through norvaline addition during the growth of Lactococcus lactis IL1403 under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested in exponential phase and 1.6 h after norvaline addition. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 2053 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. The 2 time-points were analyzed simultaneously and 3 independent repetitions were performed.严谨应答（stringent response）首次通过添加化学诱导物正缬氨酸（norvaline）后的转录谱分析，在乳酸乳球菌（Lactococcus lactis）中得以定义。本研究在指数生长阶段（参照样本）以及添加正缬氨酸1.6小时后检测了基因表达水平。共鉴定得到461个差异表达基因，这些基因构成了严谨应答调控子（stringent response regulon）。 关键词：应激响应（stress response）、时间进程（time course） 实验设计：在可控条件（30℃、pH 6.6、氮气环境）下培养乳酸乳球菌IL1403（Lactococcus lactis IL1403），通过添加正缬氨酸诱导严谨应答。分别在指数生长期以及添加正缬氨酸1.6小时后收集细胞样本。从上述样本中提取总RNA，制备放射性标记的互补DNA（cDNA），并将其与尼龙膜芯片进行杂交。芯片上共点样了2053个针对乳酸乳球菌IL1403基因的扩增子（amplicon），每个均重复点样一次。同时对这两个时间点的样本进行同步分析，并完成了3次独立生物学重复。