Entrations (Fig. S1). The phenotype of S. oneidensis with ampicillin at

Entrations (Fig. S1). The phenotype of S. oneidensis with ampicillin at 2.5 mg/ml resembles that of ampicillin-treated E. coli cells except for full recovery of growth by the former [27,28], implying that the antibiotic may cause cell lysis by the same mechanism in these two species. As cells treated with ampicillin at 2.5 mg/ml but not 0.125 mg/ml lysed (cell density at inoculation #0.01 of OD600), we hypothesized that cells with 2.5 mg/ml ampicillin may not be able to promptly Autophagy remove the antibiotic from the culture. If so, larger inocula should allow a faster removal of the antibiotic and thereby alleviate cell lysis. To test this, cells were allowed to grow to an OD600 of ,0.2 without ampicillin, and this culture was then diluted by 1:2, 1:4, 1:8, 1:16 with fresh ampicillin-containing media. As shown in Fig. 3C, ampicillin at 2.5 mg/ml was able to induce cell lysis in 1:4, 1:8, and 1:16 diluted cultures but not in either undiluted or 1:2 diluted cultures, thus supporting our hypothesis. Notably, lysis occurred at the same time, 4 h after inoculation despite the difference in optical densities of these cultures. We then asked whether removal of ampicillin can explain the phenotype of S. oneidensis in the presence of 50 mg/ml. Cells were grown in the presence of 2.5 and 50 mg/ml ampicillin and the Epigenetics amount of the remaining ampicillin was monitored over time (Fig. 3D). At 50 mg/ml of ampicillin the concentration was rapidly reduced, reaching the detection limit (,0.5 mg/ml) within 6 h. In cultures with ampicillin at lysing concentrations, however, ampicillin remained above the threshold for 8 h. These data indicate that cell lysis is due to the slow removal of the agent from the cultures.b-lactamase BlaA dominates ampicillin hydrolysis in S. oneidensisTo address why cells failed to remove ampicillin when supplied at 2.5 mg/ml, we examined the genome for genes predicted to encode putative b-lactamases. In total, S. oneidensis possesses seven such genes, of which six reside on the chromosome (SO0541, blaA(SO0837), SO0914, ampC(SO2388), SO3054 and SO3474) and one on the megaplasmid (SOA0149). SO0541, SO3054, SO3474 and SOA0149 belong to metallo-b-lactamases, requiring a metal ion for enzymatic activity, while AmpC and BlaA are annotated to be serine b-lactamases with substrate specificity for cephalosporins and a progenitor of carbapenem-hydrolyzing oxacillinase, respectively. The function of SO0914 is currently unknown.Ampicillin of sub-MIC induces cell lysisIn the pellicle formation assay, we noticed that growth of S. oneidensis was delayed significantly with ampicillin at 0.49?.25 mg/Expression of blaA in S. oneidensisFigure 1. Pellicle formation of S. oneidensis in the presence of commonly used antibiotics (8 of 10 tested were shown). Lateexponential 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 30uC. The wells were photographed 20 h after inoculation. Concentrations (H, M, L mg/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. doi:10.1371/journal.pone.0060460.gWe deleted each of these candidate genes individually and measured g.Entrations (Fig. S1). The phenotype of S. oneidensis with ampicillin at 2.5 mg/ml resembles that of ampicillin-treated E. coli cells except for full recovery of growth by the former [27,28], implying that the antibiotic may cause cell lysis by the same mechanism in these two species. As cells treated with ampicillin at 2.5 mg/ml but not 0.125 mg/ml lysed (cell density at inoculation #0.01 of OD600), we hypothesized that cells with 2.5 mg/ml ampicillin may not be able to promptly remove the antibiotic from the culture. If so, larger inocula should allow a faster removal of the antibiotic and thereby alleviate cell lysis. To test this, cells were allowed to grow to an OD600 of ,0.2 without ampicillin, and this culture was then diluted by 1:2, 1:4, 1:8, 1:16 with fresh ampicillin-containing media. As shown in Fig. 3C, ampicillin at 2.5 mg/ml was able to induce cell lysis in 1:4, 1:8, and 1:16 diluted cultures but not in either undiluted or 1:2 diluted cultures, thus supporting our hypothesis. Notably, lysis occurred at the same time, 4 h after inoculation despite the difference in optical densities of these cultures. We then asked whether removal of ampicillin can explain the phenotype of S. oneidensis in the presence of 50 mg/ml. Cells were grown in the presence of 2.5 and 50 mg/ml ampicillin and the amount of the remaining ampicillin was monitored over time (Fig. 3D). At 50 mg/ml of ampicillin the concentration was rapidly reduced, reaching the detection limit (,0.5 mg/ml) within 6 h. In cultures with ampicillin at lysing concentrations, however, ampicillin remained above the threshold for 8 h. These data indicate that cell lysis is due to the slow removal of the agent from the cultures.b-lactamase BlaA dominates ampicillin hydrolysis in S. oneidensisTo address why cells failed to remove ampicillin when supplied at 2.5 mg/ml, we examined the genome for genes predicted to encode putative b-lactamases. In total, S. oneidensis possesses seven such genes, of which six reside on the chromosome (SO0541, blaA(SO0837), SO0914, ampC(SO2388), SO3054 and SO3474) and one on the megaplasmid (SOA0149). SO0541, SO3054, SO3474 and SOA0149 belong to metallo-b-lactamases, requiring a metal ion for enzymatic activity, while AmpC and BlaA are annotated to be serine b-lactamases with substrate specificity for cephalosporins and a progenitor of carbapenem-hydrolyzing oxacillinase, respectively. The function of SO0914 is currently unknown.Ampicillin of sub-MIC induces cell lysisIn the pellicle formation assay, we noticed that growth of S. oneidensis was delayed significantly with ampicillin at 0.49?.25 mg/Expression of blaA in S. oneidensisFigure 1. Pellicle formation of S. oneidensis in the presence of commonly used antibiotics (8 of 10 tested were shown). Lateexponential 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 30uC. The wells were photographed 20 h after inoculation. Concentrations (H, M, L mg/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. doi:10.1371/journal.pone.0060460.gWe deleted each of these candidate genes individually and measured g.