The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress [microarray]. The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress [microarray]

To understand the role of CdhR and its adjacent gene PG1236 in nitric oxide (NO) stress resistance, isogenic mutants P. gingivalis FLL457 (ΔPG1237::ermF), FLL458 (ΔPG1236::ermF) and FLL459 (ΔPG1236-37::ermF) were made by allelic exchange mutagenesis and their gene expression was studied under control and NO stress conditions. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were up regulated and over 1% of the genes down regulated. Transcriptome analysis of FLL458 and FLL459 under NO stress showed similar modulation patterns in both mutants for a few genes. The PG1236-7 gene cluster seemed to be part of the same transcriptional unit that showed increased expression under NO stress. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495. Overall design: Fresh cultures of P. gingivalis strains (mutants and wild type) were grown from overnight cell cultures under anaerobic conditions at 37ºC in BHI broth. P. gingivalis strains were grown to early exponential phase (OD600:~0.3) in BHI broth under anaerobic conditions at 37ºC. At that time point, the bacteria cultures were stressed with Diethylamine (DEA) NONOate (15µl, 24mM stock concentration) for the NO stress studies and culture samples were taken 15min post-treatment with DEA NONOate. P. gingivalis W83 cultures and total RNA extracted from them using a total RNA isolation kit (Promega, WI). Additional DNase treatment was carried out using DNase kit (Ambion, Austin, TX). DNA microarray gene expression was carried out using Roche NimbleGen customer arrays (100910_CW_P_ging_W83_expr_HX12, Roche, IN) according to the standard NimbleGen procedure (NimbleGen Arrays User’s Guide: Gene Expression Analysis v5.1). cDNA was synthesized from the RNA samples using Transcriptor High Fidelity cDNA Synthesis kit (Roche, IN). Both RNA and cDNA quality were checked using Agilent Bioanalyzer and Agilent RNA 6000 Nano and DNA1000 chips. Differentially expressed genes were determined using fold-change (≥1.25) plus p (≤ 0.05) with FDR = 0.05. ***Please note that [1] raw data files have been lost and thus are not provided, [2] only the list of differentially-expressed genes is available and thus included as processed data*****

为明确CdhR及其相邻基因PG1236在一氧化氮（nitric oxide, NO）应激抗性中的功能，研究人员通过等位基因交换诱变（allelic exchange mutagenesis）构建了牙龈卟啉单胞菌（Porphyromonas gingivalis, P. gingivalis）的同基因缺失突变株FLL457（ΔPG1237::ermF）、FLL458（ΔPG1236::ermF）与FLL459（ΔPG1236-37::ermF），并在对照及NO应激条件下对其基因表达特征展开研究。
对FLL457的DNA微阵列分析结果显示，约2%的基因呈现上调表达，超过1%的基因呈现下调表达。对NO应激下的FLL458与FLL459进行转录组分析发现，两个突变株在部分基因上呈现相似的表达调控模式。PG1236-7基因簇似乎属于同一转录单元，在NO应激条件下表达水平显著升高。重组CdhR蛋白可与PG1459及PG0495的预测启动子区域结合。

### 总体实验设计
将牙龈卟啉单胞菌突变株与野生型菌株的新鲜培养物由过夜培养物转接，于37℃厌氧条件下在脑心浸液（Brain Heart Infusion, BHI）肉汤中培养。将菌株在37℃厌氧条件下培养至早期指数生长期（OD600≈0.3），此时向细菌培养液中加入二乙胺（diethylamine, DEA）NONOate（15μL，24mM储备液）以构建NO应激模型，于处理15分钟后采集培养样本。
提取牙龈卟啉单胞菌W83菌株的总RNA，使用总RNA提取试剂盒（Promega，威斯康星州）完成提取，并通过脱氧核糖核酸酶（deoxyribonuclease, DNase）试剂盒（Ambion，德克萨斯州奥斯汀）进行额外的DNase处理以去除基因组DNA污染。
采用罗氏NimbleGen定制芯片（100910_CW_P_ging_W83_expr_HX12，罗氏，印第安纳州）按照"NimbleGen Arrays User’s Guide: Gene Expression Analysis v5.1"的标准流程开展DNA微阵列基因表达检测。使用Transcriptor高保真互补脱氧核糖核酸（complementary DNA, cDNA）合成试剂盒（罗氏，印第安纳州）以RNA样本为模板合成cDNA。通过安捷伦（Agilent）生物分析仪及Agilent RNA 6000 Nano、DNA1000芯片对RNA和cDNA的质量进行质检。
采用倍数变化（fold-change）≥1.25、p值≤0.05且错误发现率（False Discovery Rate, FDR）=0.05的标准筛选差异表达基因。

***请注意：[1] 原始测序数据文件已丢失，无法提供；[2] 仅可获取差异表达基因列表，故将其作为已处理数据纳入本数据集***