Figure 1.
Pellicle formation of S. oneidensis in the presence of commonly used antibiotics (8 of 10 tested were shown).
Late-exponential phase cultures (∼0.6 of OD600) were diluted 1∶100 with LB broth, aliquotted into 24-well plates (2 ml/well) and incubated statically at 30°C. The wells were photographed 20 h after inoculation. Concentrations (H, M, L µg/ml): ampicillin (Amp, 50, 2.5, 0.125), vancomycin (Van, 50, 2.5, 0.125), and ciprofloxacin (Cipro, 50, 2.5, 0.125), rifampicin (Rif, 50, 2.5, 0.125), tetracycline (Tet, 1.2, 0.06, 0.003), erythromycin (Em, 12.5, 0.625, 0.031), kanamycin (Kan, 5, 0.25, 0.0125), chloramphenicol (Cm, 8.5, 0.42, 0.021). In this and all other figures, Con. represents the antibiotic-free control.
Table 1.
Susceptibility of S. oneidensis to various antibiotics.
Figure 2.
Pellicle formation of S. oneidensis in the presence of β-lactam antibiotics.
(A) Inhibitory effects on pellicle formation were found with ampicillin and penicillin (Pen), but not carbenicillin (Carb). (B) Pellicle formation in LB broth containing ampicillin prepared by double dilution. Pellicle formation was inhibited by ampicillin at concentrations ranging from 0.49 to 6.25 µg/ml.
Figure 3.
Cell lysis caused by ampicillin at 2.5 µg/ml.
Cultures of late-exponential phase cells (∼0.6 of OD600) were diluted 1∶100 with LB broth, and incubated at 30°C in a shaker at 200 rpm. (A) Growth of S. oneidensis in the presence of ampicillin at H (50 µg/ml), M (2.5 µg/ml) or L (0.125 µg/ml) levels. (B) Microscopic images of cells at various times in the presence of ampicillin at 2.5 µg/ml. Arrows point to knobs and branches characterstic of treated cells. (C) Growth of cultures varying in initial cell density in the presence of ampicillin at 2.5 µg/ml. (D) Amounts of ampicillin remaining at the indicated times in cultures supplemented initially with ampicillin at 50 µg/ml or 2.5 µg/ml. In all panels, experiments were performed at least in triplicate and the error bars represent standard deviation (SD).
Figure 4.
Impact of the loss of blaA on growth.
(A) Growth of the ▵blaA strain in the presence of ampicillin at H (50 µg/ml), M (2.5 µg/ml) or L (0.125 µg/ml). Hc and Mc represent the ▵blaA strain complemented in trans. (B) Susceptibility assay of the ▵blaA strain to ampicillin. ▵blaAc represents the ▵blaA strain complemented in trans. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (A).
Table 2.
MICs (µg/ml) of β-lactams for S. oneidensis wild type and derivative strains.
Figure 5.
The blaA gene is induced by ampicillin only at high levels.
Cultures of late-exponential phase cells (∼0.6 of OD600) were diluted 1∶100 with LB broth containing ampicillin at H (50 µg/ml), M (2.5 µg/ml), or L (0.125 µg/ml) amounts, and incubated at 30°C in a shaker at 200 rpm (A) PblaA promoter activities were determined by measuring β-galactosidase (in Miller units) using the PblaA-lacZ reporter system in the wild type. Results are averages of at least three replicates, and the error bars represent standard deviation (SD). Activity of PblaA in the presence of ampicillin at the H (50 µg/ml) level were also assayed using qRT-PCR (presented as diamonds). (B) β-lactamase activity assay. At the indicated times, samples were taken for β-lactamase activity measurements. In both panels, experiments were performed at least in triplicate and the error bars represent standard deviation (SD).
Figure 6.
Impacts of the loss of LMW PBPs on growth in the presence of ampicillin.
(A) Susceptibility assay of LMW PBP mutants ▵dacB (PBP4), ▵dacA (PBP5), and ▵pbpG (PBP7) to ampicillin. ▵dacAc represents the ▵dacA strain complemented in trans. (B) Growth of the ▵dacA strain in the presence of ampicillin at H (50 µg/ml), M (2.5 µg/ml) or L (0.125 µg/ml) levels. H-WT and M-WT represent growth of the wild type strain under the specified conditions. (C) Activities of PblaA-lacZ in strains devoid of one of the LMW PBPs. After growth for two hours, samples were taken for β-galactosidase measurements. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (B and C).
Table 3.
Bacterial strains and plasmids used in this study.