CodY regulates the transcription of virulence and metabolic genes under limiting concentrations of BCAAs.

<p>A. Relative luminescence measurements (RLU) indicating activation of <i>hly</i> promoter under growth of WT <i>L. monocytogenes</i> (harboring pPL2-P<i>_hlylux</i> plasmid) in BHI, MDM and MDM with low concentration of isoleucine. B. Optical density measurements of the same cultures of WT <i>L. monocytogenes</i> containing pPL2-P<i>_hlylux</i> plasmid, growing in BHI, MDM and MDM with low concentration of isoleucine. The results represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean. C. Relative luminescence measurements indicating activation of <i>hly</i> promoter in <i>ΔcodY</i> mutant and WT bacteria during growth in MDM and MDM with low concentration of isoleucine. The results represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean. D. RT-qPCR analysis of <i>prfA</i> and <i>actA</i> transcription levels during growth in MDM with low levels of isoleucine in the <i>ΔcodY</i> mutant, WT bacteria and <i>ΔcodY</i> complemented strain harboring pPL2-<i>codY</i> plasmid with and without IPTG. Levels are represented as RQ, relative to BHI and normalized to <i>rpoD</i> mRNA. The data represent 3 independent experiments (N = 3). Error bars indicate a 95% confidence interval. E. RT-qPCR analysis of <i>ilvC</i> and <i>purH</i> transcription levels in <i>ΔcodY</i> mutant and WT bacteria during growth in BHI and in MDM media with reduced concentrations of BCAAs: 100 µg/ml and 10 µg/ml. Represented as RQ, relative to BHI and normalized to <i>rpoD</i> mRNA. The data represent 3 independent experiments (N = 3). Error bars indicate a 95% confidence interval. In all experiments (A–E) overnight precultures were grown in MDM. F. Intracellular growth curve of the <i>ΔcodY</i> mutant, WT bacteria and the <i>ΔcodY</i> complemented strain (with and without IPTG) in primary BMD macrophage cells. The data represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean.