Response of Lactococcus lactis to isoleucine starvation

For the first time in Lactococcus lactis, amino acid starvation response was characterized. The natural imposition of isoleucine starvation, by its consumption during growth, associated to transcript profiling, allowed defining exhaustively this stress stimulon. It consisted of a general induction of nitrogen metabolism (amino acid biosynthesis and transport, proteolytic system and proteases), a strong repression of genes encoding major physiological activities (translation, transcription, carbon metabolism, purine and pyrimidine biosynthesis and fatty acid metabolism) and the induction of unexpected cross responses to acid, osmotic and oxidative stresses. Keywords: stress response, time course Isoleucine starvation was imposed by the consumption of this amino acid during the growth of Lactococcus lactis IL1403 on ILV0.1 medium (CDM with ten-fold reduced concentrations of isoleucine, leucine and valine) and under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested in exponential phase and after 30 min, 1.7 h and 3.5 h of isoleucine starvation. 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 4 time-points were analyzed simultaneously and 3 independent repetitions were performed.

本研究首次在乳酸乳球菌（Lactococcus lactis）中解析了氨基酸饥饿应答反应。通过菌株生长过程中异亮氨酸的天然消耗实现异亮氨酸饥饿，并结合转录谱分析（transcript profiling），得以全面定义该胁迫刺激响应组（stimulon）。该响应组涵盖氮代谢的整体诱导（氨基酸生物合成与转运、蛋白水解系统及蛋白酶）、对核心生理活动相关基因的强烈抑制（包括翻译、转录、碳代谢、嘌呤与嘧啶生物合成及脂肪酸代谢），以及诱导了对酸胁迫、渗透胁迫与氧化胁迫的意外交叉应答。关键词：胁迫应答，时间进程。本研究以乳酸乳球菌IL1403为研究对象，在ILV0.1培养基（即异亮氨酸、亮氨酸、缬氨酸浓度降至原浓度十分之一的化学限定培养基（CDM，Chemically Defined Medium））中进行培养，并在可控条件（30 ℃、pH 6.6、氮气氛围）下，借助生长过程中该氨基酸的消耗实现异亮氨酸饥饿。分别在指数生长期及异亮氨酸饥饿30 min、1.7 h和3.5 h时收集细胞样本。从上述样本中提取总RNA，制备放射性标记的cDNA并与尼龙膜阵列进行杂交。该阵列上共点样2053个乳酸乳球菌IL1403基因特异性扩增子（amplicon），每个扩增子重复点样两次。同时对4个时间点的样本进行转录谱分析，并开展3次独立重复实验。