The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Abstract

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain heart infusion; BHI) under microaerobic conditions reveals upregulation of 13 genes including adcR and adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and a ≈2-fold increase in cell-associated Zn(II). Apo-and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K DNA-Zn = 2.4 ×10 8 M −1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with step-wise Zn(II) affinities K Zn1 and K Zn2 of ≥10 9 M −1 at pH 6.0 and ≥10 12 M −1 at pH 8.0. X-ray absorption spectroscopy (XAS) of the high affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild-type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild-type apo AdcR, but fail to conformationally switch upon Zn(II) binding. These studies clearly identify His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.

The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

“…In addition to genes encoding the Adc/Lmb system, we found a perfect AdcR box in the promoter region (400 bp upstream from the start codon) of 7 supplementary genes or operons (Table 3), in particular, upstream of the adh gene (sak_0087), encoding a potential zinc-containing alcohol dehydrogenase, which has already been identified to be a direct binding target of AdcR in S. pneumoniae (26,42).…”

“…We have shown that the expression of the lmb, adcA, and adcAII genes is zinc dependent. Zn 2ϩ -dependent regulation is mediated in S. pneumoniae and S. pyogenes by the AdcR regulator (26,27,42). S. pneumoniae AdcR has a unique effector in zinc, and its crystal structure revealed the presence of two metal-binding sites (43).…”

“…We thus analyzed their promoter regions to identify AdcR operator sites (TTAACN NGTTAA) (23,42). One conserved 10-bp palindromic motif was present in the promoter region of the adcAII-shtII and adcRCB operons, and two overlapping copies were also present upstream from the lmb-sht and adcA genes (Fig.…”

Stress Physiology of Lactic Acid Bacteria

“…S. pneumoniae encodes a homologous Zn 2ϩ uptake system (AdcABC) that is regulated by a ZitR homolog (AdcR) (76). AdcR also regulates the expression of the four pneumococcal histidine triad (Pht) proteins, PhtA, PhtB, PhtD, and PhtE (77), involved in surface adhesion, as well as the Zn 2ϩ -specific AdcA homolog AdcAII and a Zn 2ϩ -dependent alcohol dehydrogenase (78,79). The adc operon is present in most streptococci and has also been studied in Streptococcus gordonii, where it is involved in Mn 2ϩ homeostasis (80), and in Streptococcus suis, where it regulates the expression of a Zn 2ϩ /Mn 2ϩ uptake system, ribosomal genes, and pht genes (81).…”

Lactococcus lactis ZitR Is a Zinc-Responsive Repressor Active in the Presence of Low, Nontoxic Zinc Concentrations In Vivo

“…S. pneumoniae and Streptococcus suis AdcR proteins were then found to be DNA-binding proteins (1,3), and S. suis AdcR was shown to be a metalloregulator specifically binding the TTAACYRGTTAA palindrome (1). While this work was submitted for publication, the S. pneumoniae AdcR protein was characterized in both apo-and Zn(II)-bound forms, and three of its Zn(II)-binding residues were identified (44).…”