Figure 3: EF-P is required for MgtB production in a manner dependent on Pro555 and Pro556 codons.

(A) Schematic diagram of the MgtB protein and location of proline codons used in this work. (B–D) Western blot analysis of crude extracts prepared from a strain with the wild-type mgtB gene (14028s), the efp mutant (DN337), an mgtB derivative with the Pro 73, 74 codons substituted by Ala codons (EN793), a mutant with both the Pro 73, 74 substitution and the efp insertion (EN794), an mgtB derivative with the Pro 555, 556 codons substituted by Ala codons (EN873), or a mutant with both the Pro 555, 556 substitution and the efp insertion (EN877). Blots were probed with anti-MgtB (B), anti-MgtC (C), anti-EF-P (D) or anti-Fur (E) antibodies to detect MgtB, MgtC, EF-P and Fur proteins respectively. Bacteria were grown for 5 h in N-minimal media containing 0.01 mM Mg2+ as described in Methods.