Gene regulation by elevated c-di-AMP levels in Streptococcus pneumoniae

Streptococcus pneumoniae harbors two cyclic di-AMP (c-di-AMP) phosphodiesterases Pde1 and Pde2. Previously, we demonstrated that deletion of one or both of these proteins leads to growth retardation in culture media, defects in the bacterial stress response, and attenuation in mouse models of disease. All of these phenotypes are due to increased levels of c-di-AMP, since the nature and break down products of each protein are different, and mutations that lower c-di-AMP levels partially restore growth and stress tolerance in these mutants. However, how c-di-AMP mediates pneumococcal stress resistance and virulence is unknown. To establish how c-di-AMP affects the transcriptome, RNA-Seq analysis was employed to compare gene expression between wild-type and Δpde1Δpde2 (ST2734) pneumococci. Overall, the competence regulon was upregulated in the Δpde1Δpde2 mutant. The goal of this research was to determine c-di-AMP-responsive genes in Streptococcus pneumoniae. We prepared RNA samples of the wild-type and Δpde1Δpde2 strains with three independent biological replicates. RNA samples were treated with DNase to remove contaminated DNA. rRNA was removed using Ribo-Zero magnetic kit (Epicentre). cDNA library was then generated using ScriptSeq Complete Kit for Bacteria (Epicentre). Samples 1 to 6 were amplified with index PCR primers 1to 6, respectively, from Script Seq Index PCR primers (Epicentre). RNA-Seq was performed at the Next Generation Sequencing and Expression Analysis Core of University of Buffalo.