Sequence conservation between ZitR/AdcR proteins.

Multi-alignment of ZitR/AdcR protein sequences was performed using Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/) and ESPript server (http://espript.ibcp.fr/ESPript/ESPript/) [30]. We noticed that several lactococcal proteins are erroneously annotated as ‘AdcR’ in gene/protein databases, as they share a higher homology level with ZitR proteins than to the prototypal AdcR protein from S. pneumoniae [13, 18, 19]. We therefore renamed them as ZitR proteins. The following sequences are shown: i) ZitR from L. lactis subsp. cremoris (Llaccre) strain MG1363 (gb |A2RNS2.1|), our lactococcal protein model [15, 16]; ii) ZitR from L. lactis subsp. lactis (Llaclac) strain IL1403 (gb |NP_268273.1|, gb |AAK06214.1|, gb |Q9CDU5|, PDB: 5YHX [21]), which is identical to ZitR proteins from L. lactis subsp. lactis strain NCDO 2118 (gb |AII13676.1|) and subsp. lactis bv. diacetylactis (gb |KZK11188.1|), not shown; iii) ZitR (‘AdcR’) from L. piscium (Lpis) strains MKFS47 (gb |CEN27435.1|) and iv) CNCM I-4031 (gb |SCA91076.1|); v) ZitR (‘AdcR’) from L. raffinolactis (Lraf) strain 4877 (gb |CCK19333.1|); vi) ZitR (‘AdcR’) from L. garvieae (Lgar) strains DCC43 (gb |EKF52529.1|) and vii) 122061 (gb |BAV02535.1|), which is identical to the protein from strain M14 (gb |CEF51164.1|), not shown; viii) ZitR (‘AdcR’) from Lactococcus sp. (Lsp) strain DD01 (gb |KXT61547.1|); ix) the prototypal AdcR protein [13, 18] from S. pneumoniae (Spne) strain D39 (gb |Q04I02.1|, PDB: 3TGN [13]); and finally x) AdcR protein from S. pyogenes strain MGAS315 (gb |AAM78676.1|), or serotype M3 (PDB: 5JLU [20]). Secondary structure elements of ZitR_MG protein are displayed above its sequence: α-helices as medium squiggles and β-strands as arrows. ZitR_MG residues shown here (see below) to belong to the dimerization interface or to the metal binding pocket (Fig 2) are respectively indicated by ‘d’ and ‘z’ characters below the sequences. Amino acids that appear in white characters in a red background are identical in all aligned proteins, while those in red characters and in blue frames are conserved in the majority of proteins.