Project name: RNA-seq analysis of Streptococcus pneumoniae during intraspecies competition in broth culture

Description: Streptococcus pneumoniae (‘pneumococcus’) is a leading cause of morbidity and mortality worldwide and a frequent coloniser of the nasopharynx. Competition among bacterial members of the nasopharynx is believed to be mediated by bacteriocins: antimicrobial toxins produced by bacteria to inhibit growth of other bacteria. We have identified 14 newly-discovered bacteriocin gene clusters among pneumococci. Here, RNA-seq analysis was performed to investigate whether bacteriocin genes are transcriptionally active when two different strains are competing in broth culture. Pneumococcal reference strains PMEN3 (Spain9V-3) and PMEN6 (Hungary19A-6) were grown together in broth culture. Samples were taken at sequential time points and RNA was extracted and sequenced. The controls were prepared by growing each strain individually in identical experiential conditions and combining the RNA sequencing reads from each individual strain in silico. Differential expression analyses were performed by comparing reads generated when strains were competing in broth culture to those from when strains were grown individually.Overall design: Pneumococcal reference strains PMEN3 and PMEN6 were grown together in brain-heart infusion broth culture at 37°C + 5% CO2 for 6h. Broth cultures at five time points (2, 3, 4, 5 and 6 h of incubation) were sampled and RNA was extracted using the Promega Maxwell® 16 Instrument and LEV simplyRNA Cells purification kit, following the manufacturer’s protocol. Extracted RNA samples were sent to the Oxford Genomics Centre for processing. Library preps were made using RNA-Seq Ribozero kits (Illumina, Inc) and sequencing was performed on the MiSeq (Illumnia, Inc). The controls were prepared by growing each strain individually in identical experimental conditions and combining the RNA sequencing reads from each individual strain in silico. RNA-seq data for PMEN3 grown individually was previously published by us and retrieved from GEO (accession number GSE89200). A pseudo-reference genome was constructed using PMEN3 and PMEN6 reference genomes by sorting genes into three categories: those unique to PMEN3, those unique to PMEN6, and those shared between the two. RNA-seq reads from all time points were combined in silico and mapped to the pseudo-reference genome using Bowtie2 with the highest sensitivity option. Differential expression analyses were performed using the DESeq method by comparing reads generated when strains were competing in broth culture to those from when strains were grown individually. In theory, the control contained twice the number of reads compared to the in vivo competition experiment as it was compiled in silico from two sets of samples. Thus, although the downregulation of genes could not reliably be assessed, it could be assumed with a high level of confidence that upregulated genes are differentially expressed; nevertheless, this approach provides only relative values and actual fold-change ratios for the upregulated genes are likely to be underestimated using this method.This series contains the re-analysis of samples from GSE89200.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2018-02-16