Half-life estimation in the cyanobacterium Synechocystis sp. PCC 6803 Transcriptome wide during iron starvation

Cyanobacteria, photoautotrophic prokaryotes, contribute significantly to the global photosynthesis and require large amounts of the essential micronutrient iron in order to maintain their Fe-rich photosynthetic apparatus. Here we use the model organism Synechocystis sp. PCC 6803 (later referred to Synechocystis) in both, standard and iron stress conditions, to study transcription and post-transcription regulation in iron deprivation. Although iron is one of the most abundant metals on earth, it is not soluble under aerobic conditions. Thus Synechocystis had to find ways to overcome iron deficiency. At the same time, however, free intracellular iron needs to be kept at permissive levels, as it becomes toxic under aerobic conditions by producing reactive oxygen species. For these reasons, complex regulatory networks have evolved to tightly control intracellular iron concentrations, ensuring its essential function yet avoiding cellular damage (Pierre Cornelis et al). In a previous study, we investigated iron deprivation in Synechocystis using customised amplification library for the analysis of global gene expression in the unicellular cyanobacterium (Hernández-Prieto et al, 2012; Georg et al, 2017),(Georg et al, 2017) but it is still little known about RNA stability in this organism. We now extend this study through a transcriptome wide half-life analysis in Synechocystis grown under standard and iron-limiting conditions using oligonucleotide microarrays that detect both protein-coding and non-coding transcripts (ncRNA). We used the antibiotic Rifampicin to stop the transcription. Samples were taken at time points 0 min (before the addition of rifampicin) and in a time series of 2 min, 4 min, 8 min, 16 min, 32 min and 64 min after its addition. Three and two biological replicates were used for standard and iron stress condition respectively. Half-life was estimated for each condition.

蓝细菌（Cyanobacteria）作为光合自养型原核生物，对全球光合作用贡献显著，且为维持其富铁光合装置，需要大量必需微量营养素铁。本研究以模式生物集胞藻属PCC 6803（Synechocystis sp. PCC 6803，后文简称集胞藻（Synechocystis））为研究对象，分别在标准培养与铁胁迫条件下，探究铁剥夺过程中的转录及转录后调控机制。尽管铁是地球上丰度最高的金属元素之一，但在有氧环境下其溶解性极差，因此集胞藻必须演化出应对铁缺乏的策略。与此同时，胞内游离铁需维持在安全水平，因为在有氧条件下，游离铁会通过产生活性氧引发细胞毒性。基于上述原因，生物演化出复杂的调控网络以严格控制胞内铁浓度，既保障铁的必需生理功能，又避免细胞受到损伤（Pierre Cornelis等）。在先前的研究中，本团队曾利用定制扩增文库分析单细胞蓝细菌的全局基因表达，探究集胞藻的铁剥夺过程（Hernández-Prieto等，2012；Georg等，2017；Georg等，2017），但该物种的RNA稳定性仍鲜少被研究。本研究在此前工作的基础上进行拓展：在标准培养与限铁条件下培养集胞藻，使用可同时检测蛋白编码转录本与非编码转录本（ncRNA）的寡核苷酸微阵列，开展转录组范围的半衰期分析。实验中采用利福平（Rifampicin）抑制转录过程，采样时间点包括给药前0分钟，以及给药后2、4、8、16、32、64分钟的时间序列。标准培养与铁胁迫条件分别设置3组与2组生物学重复，并对每个条件下的转录本半衰期进行估算。