Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model system for studying biochemistry, genetics and molecular biology of photobiological processes. Despite its importance in basic and applied research, the genome-wide picture of transcriptional regulation in this bacterium is limited. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs) to study transcriptional activity of the cyanobacterial genome in response to S deprivation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate withdrawal were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S deprivation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful sulfur to no sulfur, Synechocystis undergoes coordinated transcriptional changes, including genes whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient limitation was dominated by decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular sulfur status indicated that the observed repression has a specific regulatory component. The repression of certain sulfur-related genes was paralleled by activation of genes involved in internal and external S scavenging. Keywords: stress response, time course Synechocystis sp. PCC 6803 was grown photoautotrophically in BG-11 medium supplemented with 8mM NaHCO3 and buffered with 10mM HEPES (pH 7.4). The cells were grown in 250ml flasks at 32oC under a light intensity of 25µmol photons m-2 s-1. Cultures were bubbled with sterile air containing 1% (v/v) CO2. Log phase cells (OD730nm=0.6) were harvested by centrifugation (2000×g for 12 min) washed once and then re-suspended in sulfate-free media (MgSO4 replaced by the same molarity of MgCl2). In addition, all S-containing trace metals in BG-11 were replaced by non-S containing metals. Cells were harvested and fixed for microarray analysis by adding 10% (v/v) ice-cold 5% phenol in ethanol stop solution at the following time points: before S-depravation (time 0, control), 1, 3, 6, 12, 24, 48 and 72 hr after S-depravation. S-deprivation with HEPES buffering control experiment was performed as described above, except that HEPES buffer was used upon sulfate removal. Bacterial samples for a time course were taken at time 0, 1, 12 and 24 hrs after sulfate withdrawal. Growth stage control experiment was done in parallel with S deprivation experiments. Samples were taken at 0, 1, 2.5, 4, 7, 11 and 48 hr after OD730nm reached 0.60. All the experiments were done in biological replicates.

单细胞蓝细菌集胞藻（Synechocystis sp. PCC 6803）是研究光生物学过程的生物化学、遗传学与分子生物学的模式系统。尽管该菌株在基础与应用研究中具有重要价值，但目前对该细菌全基因组范围的转录调控图谱仍知之甚少。针对生长环境变化的典型转录应答，可为构建集胞藻的转录调控网络提供框架，同时也为该基因组内基因的功能注释提供高效手段。我们设计、验证并使用了覆盖集胞藻全基因组的70聚体寡核苷酸微阵列（覆盖96.7%的染色体开放阅读框），用于探究该蓝细菌基因组在硫剥夺胁迫下的转录活性。本研究通过定量逆转录聚合酶链反应（qRT-PCR）对微阵列数据进行了验证。本研究得到五项核心结论：1）硫酸盐撤除后的转录变化相对温和，但具有统计学显著性，且与生长动力学特征相符；2）编码高亲和力硫酸盐转运蛋白的硫摄取基因被显著诱导，而藻胆体、光系统I与II、细胞色素b6/f复合物以及ATP合酶相关基因的转录水平下调，表明光捕获与光合活性受到抑制；3）硫剥夺会引发与整体生长停滞及胁迫相关的转录应答；4）大量受硫可用性调控的基因编码假设蛋白或功能未知蛋白；5）尽管观测到氢气释放量增加，但氢化酶的结构基因与成熟辅助基因并未被鉴定为差异表达基因。基于该寡核苷酸微阵列平台获取的表达谱数据显示，在从硫充足状态过渡到硫缺乏状态的过程中，集胞藻会发生协同性转录变化，包括参与营养限制感知与细菌代谢调控的相关基因。营养限制条件下的转录谱以大量转录本丰度下调为主要特征，但此类变化并非源于生长停滞状态下转录的全面非特异性关闭。编码功能依赖于细胞硫状态的蛋白的转录本出现下调，这表明观测到的基因抑制现象具有特异性调控组分。部分硫相关基因的抑制与参与胞内外硫清除的基因激活现象同时发生。
关键词：胁迫应答，时间序列实验
集胞藻（Synechocystis sp. PCC 6803）采用光自养培养方式，在添加8mM碳酸氢钠、以10mM羟乙基哌嗪乙硫磺酸（HEPES）缓冲至pH 7.4的BG-11培养基中培养。细胞于250ml培养瓶中，在32℃、光照强度25μmol光子·m⁻²·s⁻¹的条件下培养，培养体系通入含1%（体积分数）CO₂的无菌空气。取对数生长期细胞（OD730nm=0.6），经2000×g离心12分钟收集后洗涤一次，随后重悬于无硫培养基（将硫酸镁替换为等摩尔浓度的氯化镁）。此外，BG-11培养基中所有含硫微量元素均替换为非硫微量元素。在以下时间点收集细胞并加入10%（体积分数）冰浴的5%苯酚乙醇终止液进行固定，以用于微阵列分析：硫剥夺前（时间0，对照组），以及硫剥夺后1、3、6、12、24、48和72小时。HEPES缓冲硫剥夺对照实验的操作流程如上所述，仅在移除硫酸盐时添加HEPES缓冲液。时间序列实验的细菌样本采集时间为硫酸盐撤除后0、1、12和24小时。生长阶段对照实验与硫剥夺实验同步开展，在OD730nm达到0.60后的0、1、2.5、4、7、11和48小时采集样本。所有实验均设置生物学重复。