FIG 1.

The AmpC-AmpR operon is responsible for induced ampicillin resistance in Agrobacterium tumefaciens C58. (A) Operon organization and proposed ampicillin resistance mechanism. (Left, No AMP) Briefly, in the absence of β-lactams such as ampicillin (AMP), ampC expression is repressed by AmpR. AmpR-mediated repression is maintained as long as the AmpR-inactivating ligand, UDP-GM-pentapeptide, is bound to AmpR (PampC OFF). (Right, +AMP) In contrast, the presence of ampicillin (AMP) increases the pools of AmpR-activating ligands or the cell wall degradation fragments (anhydro modification is depicted by a ring), which are known to displace AmpR-inactivating ligands. As a result, the increase in AmpR-activating ligands activates AmpR and ampC is transcribed (PampC ON). (B) Growth of A. tumefaciens WT, ΔampC, and ΔampR cells in the absence (No AMP) and presence of ampicillin at 25 or 50 μg/mL (AMP 25 or AMP 50, respectively) for 24 h (n = 1; 2 replicates). (C) Quantitative analysis of phase-contrast microscopy of exponentially growing strains in the absence (No AMP) or presence of ampicillin at 25 μg/mL (AMP 25). The percent phenotype was calculated by counting the number of cells displaying one of the phenotypes indicated (1 cell = 1 phenotype) and dividing it by the total number of cells per strain. (D) Ampicillin susceptibility assay performed by spotting dilutions. Briefly, exponential cultures were serially diluted, spotted on LB solid medium containing no ampicillin (No AMP) or ampicillin at 25 μg/mL (AMP 25), and incubated at 28°C for 36 h before imaging. Plates used to demonstrate complementation (ΔampC + pAmpC or ΔampR + pAmpR) included 1 μM IPTG to induce expression of plasmid-encoded AmpC or AmpR. (E) Disk susceptibility was performed on lawns of the indicated strains grown on LB plates for 24 h at 28°C (n = 2). AMP 10, disk containing 10 μg/mL of ampicillin; AMP 10/SUL 10, disk containing 10 μg/mL of ampicillin and 10 μg/mL of sulbactam, a broad-spectrum β-lactamase inhibitor. Data represent the mean (±standard deviation [SD]) of three independent experiments. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.1; ns, not significant. (F) Determination of β-lactamase production performed by a nitrocefin assay using cell lysates. No AMP or AMP 25 indicates cells untreated or treated with ampicillin at 25 μg/mL, respectively, for 2 h before the generation of cell lysates. The data shown represent one of two biological replicates.