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. 2011 Jun 15;14(12):2399–2412. doi: 10.1089/ars.2010.3782

FIG. 2.

FIG. 2.

The C52-C98 or C81-C108 disulfides are the only ones that allow β-lactamase to retain activity. (A) In vivo activity of one disulfide bond containing β-lactamase mutants. The Western blot after acid-AMS-trapping at the far left shows the expression level and redox status (all oxidized) of each variant as measured from whole cells using β-lactamase antibody. The schemes in the middle indicate the names and linkages of the one disulfide bonded β-lactamase variants (engineered cysteines are shown in red; wild-type cysteines are shown in black). The spot titer panel at the right shows their ampicillin (0.2 g/L) resistance. The single disulfide bonded variants showed decreased ampicillin resistance compared to wild-type β-lactamase. (B) A wild-type strain containing wild-type β-lactamase (52-98) (RGP181) grew well in liquid media containing 0.2 g/L ampicillin in the presence or absence of DTT (upper graph), whereas β-lactamase (C81-C108) (RGP655) grew somewhat in 0.2 g/L ampicillin, but only in the absence of DTT (lower graph). (C) 1 mM DTT restores the ability of wild-type E. coli strains containing β-lactamase C52-C81 (RGP685) (upper graph) or β-lactamase C98-C108 (RGP686) (lower graph) to grow on media containing 0.2 g/L ampicillin. (D) 1 mM DTT also restores the ability of wild-type strains containing β-lactamase C52-C108 (RGP719) (upper graph) or β-lactamase C81-C98 (RGP720) (lower graph) to grow in media containing 0.2 g/L ampicillin. The requirement of disulfide bond catalysis machinery for β-lactamase activity. (E) Cell-titer curves for strains expressing PDI detector β-lactamase based on the spot titer assay on ampicillin gradient plates. Shown are wild type (RGP663), dsbA− (RGP664), dsbC− (RGP665), dsbD− (RGP666), dsbA− dsbC− (RGP667), and trxA− (RGP668). (F) Cell-titer curves for strains expressing wild-type β-lactamase based on the spot titer assay on ampicillin gradient plates. Shown are wild type (RGP181), dsbA− (RGP188), dsbC− (RGP195), dsbD− (RGP286), and dsbA− dsbC− (RGP202). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).