Fig. 3.

Molecular mechanism of metal sensing and gene regulation by AdcR and its contribution to GAS pathogenesis. a) Ribbon representation of the crystal structure of AdcR dimer; individual subunits of a AdcR dimer are color-coded. The zinc atom bound to each AdcR subunit are shown as spheres and colored in yellow. The water molecules that participate in zinc coordination are shown as cyan spheres. The secondary structural elements of one subunit are labeled and the amino- and carboxy-terminus of the molecule are labeled as N and C, respectively. The DNA-binding and dimerization domains of AdcR are marked and labeled. b) A close up view of the metal-binding site (boxed in panel A) with the metal binding ligands labeled. Zinc bound to the primary metal sensing site in the structure of AdcR is shown as yellow sphere and the side chains of metal coordinating amino acids are depicted in stick representation. Water molecules are shown as cyan spheres. c) Metal content analysis of AdcR by ICP-MS. Measurements are reported as average from triplicate studies. Data shown are mean ± standard deviation. d) The indicated strains were grown to late exponential phase (A₆₀₀ ~ 1.0) in zinc-replete chemically defined growth medium supplemented with 15 μM ZnSO₄ and transcript levels of adcA were measured by qRT-PCR. Three biological replicates were used. Data graphed are mean ± standard deviation. Average values for wild type strain grown in zinc-rich condition were used as reference and the fold changes in the transcript levels in the indicated strains relative to reference sample are shown above the bars. Statistical significance of P < 0.0001 between the reference and indicated mutant strains is indicated by **. P values were derived from two-sample t-test. e) Fifteen CD-1 mice were infected with each indicated strain intraperitoneally and the near mortality was presented on a Kaplan-Meier survival curve with P-values derived by log rank test. Statistical significance of P < 0.0001 is indicated by **.