qRT-PCR was performed using a two-step protocol as in (Ramos-Montanez et al., 2008 (link), Kazmierczak et al., 2009 (link)). Specifically, cDNA was synthesized from 100 ng of total RNA and random primers using the qScript Flex cDNA Kit (Quanta BioSciences). RT-PCR was performed using the Brilliant SYBR Green qPCR Master Mix (Stratagene), the Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent), or the FastStart Universal SYBR Green Master Mix (Roche) and appropriate primers (see Table S6) as in (Kazmierczak et al., 2009 (link), Ramos-Montanez etal., 2008). Reactions were performed in duplicate and normalized to 16S rRNA amounts. The 16S rRNA was quantified using the same cDNA samples except that the samples were diluted 100-fold further. Data were collected on an MX3000P thermocycler (Stratagene) or on a CFX96 thermocycler (Bio Rad) and analyzed with the SYBR Green (with dissociation curve) program associated with each machine. Four dilutions of cDNA from S. pneumoniae strains wild-type for tprA and phrA (either IU1781 or Spn049) were used to generate standard curves for each primer set. Normalized transcript amounts were compared as indicated by performing pairwise unpaired two-tailed t tests.