Project name: Streptococcus pneumoniae

Description: 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.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2016-10-26