Response of Lactococcus lactis to different growth rates. Lactococcus lactis

The development of transcriptomic tools has allowed exhaustive description of stress responses. These responses always superimpose a general response associated to growth rate decrease and a specific one corresponding to the stress. The exclusive growth rate response can be achieved through chemostat cultivation, enabling all parameters to remain constant except the growth rate. We analysed metabolic and transcriptomic responses of Lactococcus lactis in continuous cultures at different growth rates ranging from 0.09 to 0.47 h-1. Growth rate was conditioned by isoleucine supply. Although the metabolism was constant and homolactic, a widespread transcriptomic response involving 30 % of the genome was observed. The expression of genes encoding physiological functions associated with biogenesis increased with growth rate (transcription, translation, fatty acid and phospholipids metabolism). Many phages, prophages and transposons related genes were down regulated by growth rate suggesting genome plasticity to be involved in the adaptation to slow growth. The growth rate response was compared to carbon and amino-acid starvation transcriptomic responses, revealing constant and significant involvement of growth rate regulations in these two stressful conditions (overlap 26%). Although stringent response mechanism is considered as the one governing growth deceleration in bacteria, the rigorous comparison of the two transcriptomic responses clearly indicated the mechanisms are distinct. Moreover it was established that genes positively regulated by growth rate are preferentially located in the vicinity of replication origin while those negatively regulated are mainly encountered at the opposite. This result demonstrates the often neglected relationship between genes expression and their location on chromosome. Keywords: growth rate impact, continuous cultures Overall design: Continuous cultivation of Lactococcus lactis IL1403 were carried out on a chemically defined medium and under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested at steady state. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. Samples corresponding to various growth rates were analyzed simultaneously and 3 independent repetitions were performed.

转录组学工具（transcriptomic tools）的发展使得对胁迫响应的全面描述成为可能。这些响应始终叠加着与生长速率降低相关的一般性响应，以及对应特定胁迫的特异性响应。仅通过除生长速率外其余所有参数均可保持恒定的恒化器培养（chemostat cultivation），即可获得纯粹的生长速率响应。

我们分析了乳酸乳球菌（Lactococcus lactis）在连续培养（continuous cultures）中，于0.09至0.47 h⁻¹的不同生长速率下的代谢与转录组学响应。生长速率由异亮氨酸（isoleucine）的供给量调控。尽管代谢模式恒定且为同型乳酸发酵，仍观测到涉及基因组30%的广泛转录组学响应。与生物发生相关的生理功能编码基因的表达量随生长速率升高而上升，涵盖转录、翻译、脂肪酸与磷脂代谢相关基因。诸多噬菌体、前噬菌体（prophages）及转座子（transposons）相关基因的表达量随生长速率升高而下调，这表明基因组可塑性参与了对低生长速率的适应过程。

将生长速率响应与碳源及氨基酸饥饿胁迫的转录组学响应进行比较后发现，生长速率调控机制在这两种胁迫条件下均持续且显著地发挥作用，二者重叠度达26%。尽管严谨型响应（stringent response）机制被认为是调控细菌生长减速的核心机制，但对两种转录组学响应的严谨比对清晰表明二者的作用机制存在差异。此外，研究证实受生长速率正向调控的基因优先定位于复制起点（replication origin）附近区域，而受负向调控的基因则主要分布于其相反一侧。该结果揭示了长期以来被忽视的基因表达与其在染色体上的位置之间的关联。

关键词：生长速率影响，连续培养
整体实验设计：将乳酸乳球菌IL1403在化学限定培养基（chemically defined medium）中进行连续培养，实验条件可控（30 ℃、pH 6.6、氮气氛围）。在稳态（steady state）下收集细胞样本，从中提取总RNA并制备放射性标记的cDNA（complementary DNA），随后将其与尼龙膜基因芯片（nylon arrays）进行杂交。本芯片上共点样了1948个针对乳酸乳球菌IL1403基因的扩增子（amplicon），每个重复点样两次。同时分析不同生长速率对应的样本，并设置3次独立生物学重复。