Global transcriptional landscape and promoter mapping of the gut commensal Bifidobacterium breve UCC2003

The recognition specificity and associated affinity of the RNA polymerase sigma subunit towards its cognate promoter sequence is one of the elements contributing to the modulation of gene expression in bacteria. In the present study we identified and assessed vegetative promoters of the bifidobacterial prototype Bifidobacterium breve UCC2003 employing a combination of tiling array analysis and RNA sequencing. By combining and comparing the outputs of these two distinct technologies we were able to identify a number of transcriptional units, including their initiation and termination signals, which correspond to promoters and terminators, respectively. Canonical -10 (TATAAT) and -35 (TTGACA) sequences were identified upstream of transcribed genes or operons, where deviations from this consensus correspond to transcription level variations. Using a Random Forest approach, we were able to define promoter characteristics that most substantially impact on transcription in B. breve. We observed that constitutively transcribed genes frequently encode house keeping functions that are part of the core and essential genome of this species. A comparative analysis between the tiling array-based and RNA-seq technologies allowed us to evaluate their performance and applicability for investigations on (regulation of) bifidobacterial gene expression. Complete genome tiling arrays containing oligonucleotide primers of 60 bp (designed on a 22nt sliding window) across the forward and reverse strand of the genome sequence of B. breve UCC2003 (O'Connell Motherway et al., 2011). Probes were obtained from Agilent Technologies (Palo Alto, Ca., USA). Overnight culture of B. breve UCC2003 was inoculated into 2 % glucose MRS (Difco) medium until reaching mid-log phase (OD600 of 0.5). Following growth, total bifidobacterial DNA and RNA was isolated as described previously (O' Connell Motherway et al., 2011). Labelling was performed using the Kreatech ULS labelling kit according to the manufacturer’s instructions (Kreatech, Amsterdam, The Netherlands). Labelled total gDNA and mRNA was hybridized as described in the Agilent manual Two-Color Microarray-Based Gene Expression Analysis (v4.0) (publication no. G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data sets were processed as previously described (Garcia De La Nava et al., 2003). Differential signal intensity between gDNA (baseline) and mRNA (transcriptome) were analyzed using the Limma package implemented in the statistical environment R (https://cran.r-project.org)

RNA聚合酶σ亚基（RNA polymerase sigma subunit）对其同源启动子序列的识别特异性与结合亲和力，是调控细菌基因表达的关键因素之一。本研究结合瓦片阵列（tiling array）分析与RNA测序（RNA sequencing）技术，对双歧杆菌模式菌株短双歧杆菌（Bifidobacterium breve）UCC2003的营养型启动子进行了鉴定与评估。通过整合并比对这两种不同技术的输出结果，我们成功鉴定出多个转录单元，包括其对应启动子与终止子的转录起始信号与终止信号。我们在转录基因或操纵子的上游区域鉴定到了经典的-10（TATAAT）与-35（TTGACA）保守序列，与该保守序列存在偏差的位点会导致转录水平发生变化。本研究采用随机森林（Random Forest）方法，明确了对短双歧杆菌转录过程影响最为显著的启动子特征。我们发现，组成型转录的基因通常编码持家功能蛋白，而这些功能是该物种核心必需基因组的组成部分。通过对基于瓦片阵列与RNA测序技术的比较分析，我们得以评估这两种技术在双歧杆菌基因表达（调控）研究中的性能与适用性。覆盖短双歧杆菌UCC2003基因组正、负链的全基因组瓦片阵列包含60bp长度的寡核苷酸引物（基于22nt滑动窗口设计）（O'Connell Motherway等，2011）。该阵列探针购自安捷伦科技公司（Agilent Technologies，美国加利福尼亚州帕洛阿尔托市）。将短双歧杆菌UCC2003的过夜培养物接种至2%葡萄糖MRS（Difco）培养基中，培养至对数中期（OD600为0.5）。培养完成后，按照此前报道的方法（O'Connell Motherway等，2011）分离双歧杆菌总DNA与总RNA。参照制造商说明书，使用Kreatech ULS标记试剂盒进行标记（Kreatech，荷兰阿姆斯特丹）。按照安捷伦《基于双色微阵列的基因表达分析（v4.0）》操作手册（产品编号G4140-90050）中的方法，对标记后的总基因组DNA与mRNA进行杂交。杂交完成后，按照安捷伦标准流程对微阵列进行洗涤，并使用安捷伦DNA微阵列扫描仪（型号G2565A）进行扫描。扫描得到的图像文件通过安捷伦特征提取软件（Version 9.5）转换为数据文件。DNA微阵列数据集的处理按照此前报道的方法进行（Garcia De La Nava等，2003）。以基因组DNA（基线）与mRNA（转录组）之间的信号强度差异为研究对象，使用统计环境R中集成的Limma软件包进行分析（https://cran.r-project.org）