Changes in relative transcript amounts caused by the addition of mitomycin C in Streptococcus pneumoniae. Streptococcus pneumoniae

Bacteriophages (phages) are widespread in Streptococcus pneumoniae, with most strains carrying phage genomes integrated into the chromosome. RNA sequencing was utilised to explore whether phage gene expression could be detected. The pneumococcal reference strain PMEN3 (Spain9V-3), which contained two full-length phages and one partial phage, was grown in broth culture and mitomycin C was added to facilitate phage induction. PMEN3 culture samples were taken at sequential time points and RNA was extracted and sequenced. Overall design: Seven 10 ml tubes of brain-heart infusion broth were inoculated with pneumococcal reference strain PMEN3 and incubated at 37°C+5% CO2. An aliquot of 0.5 ml broth was removed and the absorbance at OD600 was measured at each time point from 0-6 h to measure increased bacterial growth. Mitomycin C (Sigma-Aldrich) was added to the broth culture tubes after 3 h of incubation (OD600 ≥0.5). Just prior to the addition of mitomycin C at 3 h and after 4, 5 and 6h of incubation, respectively, broth cultures were removed from the incubator for processing. A 0.5 ml aliquot was used to measure the absorbance and the RNA was stabilised in the remaining 9.5 ml of broth culture by the addition of 19 ml of RNAprotect Bacteria Reagent (Qiagen). RNA was immediately extracted from the samples using the Promega Maxwell® 16 Instrument and LEV simplyRNA Cells purification kit, following the manufacturer’s protocol. RNA extracts were sent to the Oxford Genomics Centre where library preps were made using RNA-Seq Ribozero kits (Illumina, Inc) and sequencing was performed on the MiSeq (Illumina, Inc). The sequenced forward and reverse reads were paired and mapped to reference genomes using Bowtie2 with the highest sensitivity option. Differential gene expression was assessed in Geneious using the DESeq method. Genes with an adjusted P value <0.05 were deemed to be differentially expressed.

噬菌体（Bacteriophages，简称phages）广泛存在于肺炎链球菌（Streptococcus pneumoniae）中，多数菌株的染色体上整合有噬菌体基因组。本研究采用RNA测序技术，探究是否可检测到噬菌体基因的表达情况。

本研究选取携带2个完整噬菌体基因组与1个部分噬菌体基因组的肺炎链球菌参考菌株PMEN3（Spain9V-3），将其接种于肉汤培养基中培养，并添加丝裂霉素C（mitomycin C）以诱导噬菌体裂解。按预设时间点采集PMEN3的培养样本，提取RNA并进行测序。

整体实验设计：取7支装有10 ml脑心浸液肉汤（brain-heart infusion broth）的试管，接种肺炎链球菌参考菌株PMEN3，置于37℃、5% CO₂环境中培养。在0至6小时的各时间点，各取0.5 ml肉汤培养液，测定其OD600吸光度以监测细菌生长增殖情况。培养3小时（此时OD600≥0.5）后，向各肉汤培养管中加入丝裂霉素C（Sigma-Aldrich）。分别于添加丝裂霉素C前（培养3小时时）、培养4小时、5小时及6小时后，将培养管从培养箱中取出进行后续处理。各取0.5 ml培养液用于测定吸光度，剩余9.5 ml培养液中加入19 ml RNAprotect细菌稳定剂（RNAprotect Bacteria Reagent，Qiagen）以稳定RNA。按照制造商的操作流程，使用Promega Maxwell® 16全自动核酸提取仪（Promega Maxwell® 16 Instrument）及LEV simplyRNA细胞纯化试剂盒（LEV simplyRNA Cells purification kit），即刻从样本中提取RNA。将提取得到的RNA样本送至牛津基因组学中心（Oxford Genomics Centre），使用Illumina公司的RNA-Seq Ribozero试剂盒（RNA-Seq Ribozero kits，Illumina, Inc）构建文库，并在Illumina MiSeq测序平台（Illumina, Inc）上完成测序。将测序得到的正向与反向读段进行配对拼接后，采用Bowtie2软件的最高灵敏度模式将其比对至参考基因组。在Geneious软件中采用DESeq方法评估基因的差异表达情况。校正后P值<0.05的基因被认定为差异表达基因。