Complementation. Actinobacillus pleuropneumoniae

To better understand effects of iron starvation on A. pleuropneumoniae and to identify new potential vaccine targets, we conducted transcript profiling using a DNA microarray containing all 2025 ORFs of the genome of A. pleuropneumoniae serotype 5b strain L20. Upon comparing growth of this pathogen in iron-sufficient versus iron-depleted medium, 210 genes were identified as being differentially expressed. Some genes (92) were identified as being up-regulated; many have confirmed or putative roles in iron acquisition, such as the genes coding for two TonB energy-transducing proteins and the hemoglobin receptor HgbA. Transcript profiling also led to identification of some new iron acquisition systems of A. pleuropneumoniae. Keywords: stress response Overall design: The bacteria was grown in BHI broth with or without 50 ug/ml EDDHA to generate iron restriction. In the iron-restricted medium, 50 ug/ml FeCl3 was added to the medium 5 min. after the addition of EDDHA. RNA extractions were performed on three different days using the QIAGEN RNeasy Kit, and a total of four hybridization were performed. Data were acquired using a Perkin-Elmer ScanArray Express scanner, and were quantified, normalized and analysed using the SpotFinder, MIDAS and TMEV software from TIGR's TM4 Suite of Microarray Analysis softwares.

为深入解析铁饥饿对胸膜肺炎放线杆菌（A. pleuropneumoniae）的影响，并筛选新型潜在疫苗靶点，我们采用包含血清5b型L20菌株胸膜肺炎放线杆菌全基因组2025个开放阅读框（ORF，Open Reading Frame）的DNA微阵列（DNA microarray）开展转录谱分析。通过对比该病原菌在铁充足与铁匮乏培养基中的生长状况，共鉴定出210个差异表达基因。其中92个基因呈上调表达；多数基因已被证实或推测参与铁获取过程，例如编码两种TonB能量转导蛋白以及血红蛋白受体HgbA的基因。本次转录谱分析还鉴定出胸膜肺炎放线杆菌的若干新型铁获取系统。关键词：应激反应（stress response）。整体实验设计：将细菌在添加或不添加50 μg/ml乙二胺二邻羟苯基乙酸（EDDHA）的脑心浸液肉汤（BHI broth）中培养，以构建铁限制模型。在铁限制培养基中，于添加EDDHA 5分钟后加入50 μg/ml氯化铁（FeCl3）。分别于三个不同日期提取RNA，所用试剂盒为QIAGEN RNeasy Kit，共完成四次杂交实验。数据采用Perkin-Elmer ScanArray Express扫描仪采集，并通过TIGR TM4微阵列分析软件套件中的SpotFinder、MIDAS及TMEV软件进行定量、归一化与分析。