Figure 6.

Structural model of Alx and functional relevance of its negatively charged residues in Mn²⁺ export. (A) The two-dimensional topological model of Alx structure predicted with DeepTMHMM algorithm and relative positions of negatively charged residues in transmembrane segments (TMS). (B) AlphaFold-predicted structure of Alx and relative positions negatively charged residues in TMS. A hypothetical path for the export of Mn²⁺ is displayed in the predicted structure. (C) Tenfold serial dilutions of overnight-grown cultures of ΔmntP::Kan mutant bearing one of the following plasmids were spotted on the surface of LB agar containing the appropriate concentration of ampicillin, MnCl₂ and IPTG: a vector (pHYD5001), a derivative of pHYD5001 expressing Alx from a P trc promoter (pRA27), a derivative of pHYD5001 expressing Alx^HA from a p_trc promoter (pRA50), a derivative of pRA50 expressing Alx^HA_D24N (pRA61), Alx^HA_D92N (pRA62), Alx^HA_E213Q(pRA63), Alx^HA_D216N (pRA64), and Alx^HA_D284N (pRA58). (D) β-galactosidase activity (Miller units) as a reporter of alx translation (P_alx-PRE-alx’-lacZ, pRA54) was measured in mid-log phase grown cultures of Ao/x::Kan strain (RAS31) bearing vector (pHYD5001) and a derivative of pHYD5001 expressing Alx^HA (pRA27), AIx^HA_D92N (pRA62), Alx^HA_E213Q (pRA63), Alx^HA_D216N (pRA64) from a P_trc promoter. The cultures were grown in LBK media with pH 6.8 and 8.4, supplemented with appropriate concentration of ampicillin and IPTG. The error shown is standard deviation of three repeats of the experiment.