Figure 11. MetE Is Oxidized When E. coli Experience Oxidative Stress.

(A) Growth of a trxB gor strain (WP778) containing a plasmid expressing MetE was monitored by measurement of the OD₆₀₀ during growth in LB medium supplemented with 4 mM DTT (filled symbols). At an OD₆₀₀ of approximately 0.4, cells were filtered to remove the DTT, resuspended in fresh LB medium, and growth was monitored (open circles).

(B) In vivo thiol-trapping experiments were performed before and after removal of DTT from the medium. Lanes 1 and 7 contain thiol-trapped samples of purified reduced MetE, and lanes 2 and 8 contain thiol-trapped samples of glutathionylated MetE prepared in vitro by treatment of MetE with GSSG.

(C) Growth of wild-type E. coli (strain W3110) in glucose-minimal medium was monitored by the OD₆₀₀ with (filled circles) and without (open circles) the addition of diamide (at OD₆₀₀ approximately 0.5) to a final concentration of 0.9 mM.

(D) In vivo thiol-trapping experiments were carried out to assess the oxidation state of MetE at various times following diamide addition. Lanes 1, 2, and 8 contain thiol-trapped samples of purified reduced and/or diamide-oxidized MetE.