An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential
Figure 3
Zinc binding and transport by ZnuD.
(A) Coomassie-stained SDS-PAGE gels showing the protein content of the OMVs (left) and purified ZnuD preparation (right) used in the PAR-binding assay. OMVs were isolated from strain CE1523 carrying pEN11-znuD that was either induced or not with IPTG for znuD expression as indicated. Purified ZnuD from inclusion bodies (IB ZnuD) was refolded in vitro (Folded ZnuD). The folded ZnuD sample was not heated before SDS-PAGE and the positions of folded and unfolded ZnuD are indicated. (B) Binding of zinc to OMVs either or not containing ZnuD and to purified ZnuD that was either folded or not was measured in a PAR competition assay. Shown are the normalized values of the absorption at 500 nm of five independent measurements. (C) Western blot of whole cell lysates of strain HB-1 (lanes 1–3) and the znuA mutant (lanes 4–6) grown in RPMI (lanes 1 and 4), RPMI with 500 nM ZnSO4 (lanes 2 and 5) or TSB (lanes 3 and 6). (D) Representative growth curves (n = 5) of the znuD and tonB knockout strains and their parent strain in response to zinc limitation. Cultures contained either 0 or 0.3 µM TPEN. (E) Western blot of whole cell lysates of strain HB-1 (lanes 1–2) and the tonB mutant (lanes 3–4) grown in RPMI (lanes 1 and 3) or RPMI with 500 nM ZnSO4 (lanes 2 and 4).