Zhang Zhigang, Pendse Ninad D, Phillips Katherine N, Cotner James B, Khodursky Arkady
BioTechnology Institute, 1479 Gortner Avenue, University of Minnesota, St, Paul, MN 55108, USA.
BMC Genomics. 2008 Jul 21;9:344. doi: 10.1186/1471-2164-9-344.
The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model microbe for studying biochemistry, genetics and molecular biology of photobiological processes. Importance of this bacterium in basic and applied research calls for a systematic, genome-wide description of its transcriptional regulatory capacity. 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 annotated at the time of the beginning of this project) to study transcriptional activity of the cyanobacterial genome in response to sulfur (S) starvation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate starvation 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 starvation 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 S to S starvation, Synechocystis undergoes coordinated transcriptional changes, including changes in gene expression whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient starvation was dominated by a 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 S status indicated that the observed repression has a specific regulatory component. The repression of certain S-related genes was paralleled by activation of genes involved in internal and external S scavenging.
单细胞蓝藻聚球藻属6803株(Synechocystis sp. PCC 6803)是用于研究光生物学过程的生物化学、遗传学和分子生物学的模式微生物。这种细菌在基础研究和应用研究中的重要性要求对其转录调控能力进行系统的全基因组描述。预期其对生长环境变化的特征性转录反应将为描述聚球藻转录调控网络提供框架,并为基因组中基因的功能注释提供有效手段。
我们设计、验证并使用了聚球藻全基因组寡核苷酸(70聚体)微阵列(代表本项目开始时注释的所有染色体开放阅读框的96.7%)来研究蓝藻基因组在硫(S)饥饿响应中的转录活性。微阵列数据通过定量逆转录聚合酶链反应(RT-PCR)进行验证。我们有五个主要发现:1) 硫酸盐饥饿时的转录变化相对温和,但显著且与生长动力学一致;2) 编码高亲和力硫酸盐转运蛋白的硫获取基因被显著诱导,而藻胆体、光系统I和II、细胞色素b6/f和ATP合酶基因的转录减少,表明光捕获和光合活性降低;3) 硫饥饿引发了与一般生长停滞和应激相关的转录反应;4) 大量受硫可用性调控的基因编码假设蛋白或功能未知的蛋白;5) 尽管观察到析氢增加,但未发现氢化酶结构和成熟辅助基因有差异表达。
使用基于寡核苷酸的微阵列平台记录的表达谱显示,在从硫充足状态转变为硫饥饿状态的过程中,聚球藻经历了协调的转录变化,包括其产物参与感知营养限制和调节细菌代谢的基因表达变化。营养饥饿的转录谱以许多转录本丰度的降低为主。然而,这些变化不太可能是由于生长停滞状态下转录的全面、非特异性关闭所致。编码功能依赖于细胞硫状态的蛋白质的转录本下调表明,观察到的抑制具有特定的调控成分。某些与硫相关基因的抑制与参与内部和外部硫清除的基因的激活同时发生。