Table 1.
Attributes of Cys-pair mutants of FepA
| Cys paira | Locationb | Exprc, −βME | S-Sd, −βME | Siderophore nutritione |
[59Fe]Ent uptakef |
FD sensor uptakeg |
|||
|---|---|---|---|---|---|---|---|---|---|
| −βME | +βME | −βME | +βME | −βME | +βME | ||||
| None (++) | NAh | 1.0 | − | 15 | 15 | 1.0 | 1.03 | +++ | ++++ |
| Intra-N-domain Cys pairs | |||||||||
| G27C:R126C | Nβ1–Nβ5 | 0.97 | + | 21 | 18 | 0.23 | 1.08 | − | ++ |
| A33C:E120C | Nα2–Nβ5 | 0.90 | + | 28* | 15 | 0.31 | 1.24 | − | +++ |
| D44C:T111C | Nα3–NL2 | 1.31 | - | 20 | 16 | 1.05 | 1.26 | +++ | +++ |
| N56C:D73C | Nβ2–Nβ3 | 0.85 | + | 33* | 20 | 0.19 | 0.99 | − | +++ |
| I84C:V142C | Nβ4–Nβ6 | 1.2 | + | 24 | 17 | 0.13 | 0.95 | − | ++ |
| L125C:V141C | Nβ5–Nβ6 | 1.44 | + | 23 | 16 | 0.12 | 0.88 | − | ++ |
| Inter-N-domain–C-domain Cys pairs | |||||||||
| I14C:G300C | TonB-box–β7 | 0.86 | + | 17* | 15 | 0.03 | 1.03 | − | − |
| A42C:N162C | Nα3–β1 | 0.83 | − | 15 | 15 | NDi | ND | +++ | ND |
| T51C:N608C | Nβ2–β18 | 0.88 | + | 15 | 16 | 0.57 | 0.74 | +++ | ND |
| G54C:T585C | Nβ2–β17 | 1.17 | + | 22 | 23 | 0.37 | 0.31 | − | − |
| M77C:E511C | Nβ3–β14 | 1.67 | + | 21* | 18* | 0.57 | 0.48 | +++ | ND |
| P128C:A463C | Nβ5–β13 | 0.71 | + | 13 | 14 | ND | ND | +++ | ND |
| A137C:A465C | Nβ6–β13 | 1.57 | + | 14 | 15 | ND | ND | +++ | ND |
| A138C:A445C | Nβ6–β12 | 1.58 | + | 19 | 20 | 0.94 | 0.70 | +++ | ND |
| G149C:T180C | Nβ6–β2 | 0.71 | − | 15 | 15 | ND | ND | +++ | ND |
a Cys-pair mutants were engineered in wildtype FepA and carried on the pHSG575 (66) derivative pITS23 (39). Mutants identified in bold type showed severely impaired FeEnt uptake, unless exposed to βME.
b We designed Cys pairs using the Modeller algorithm of CHIMERA (UCSF) and PDB file 1FEP (8).
c Using ImageJ (73), we quantified the immunoreactive bands of each Cys-pair mutant in Fig. 2A, relative to the amount of wildtype FepA. Mean expression among 15 mutants was 0.96 of wildtype; the range of expression values was 0.71–1.67 of wildtype expression.
d Evidence of disulfide formation, from observation of altered mobility in anti-FepA immunoblots of SDS-polyacrylamide gels of OM fractions, or reversible (i.e. relieved by βME) inhibition of FeEnt uptake.
e The tabulated values are the diameters of the growth halo (41, 42) around a paper disk containing 50 μm FeEnt, after incubation at 37 °C overnight. Results marked with an asterisk (*) denote samples with a faint halo.
f Vmax values of [59Fe]Ent uptake from Vmax screening experiments were calculated relative to the rate of wildtype FepA (96 pmol/109 cells/min; Fig. 3, C and D).
g FeEnt uptake was measured as the rate of fluorescence recovery in FD sensor experiments, relative to wildtype FepA.
h NA means not applicable.
i ND means no data.