Gentamycin at 15 mg/ml.Figure 5. The blaA gene is induced by

Gentamycin at 15 mg/ml.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 mg/ml), M (2.5 mg/ml), or L (0.125 mg/ml) amounts, and incubated at 30uC in a shaker at 200 rpm (A) PblaA promoter activities were determined by measuring b-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 Conduritol B epoxide cost presence of ampicillin at the H (50 mg/ml) level were also assayed using qRT-PCR (presented as diamonds). (B) b-lactamase activity assay. At the indicated times, samples were taken for b-lactamase activity measurements. In both panels, experiments were performed at least in triplicate and the error bars represent standard deviation (SD). doi:10.1371/journal.pone.0060460.gExpression of blaA in S. oneidensisFigure 6. Impacts of the loss of LMW PBPs on growth in the presence of ampicillin. (A) Susceptibility assay of LMW PBP mutants ndacB (PBP4), ndacA (PBP5), and npbpG (PBP7) to ampicillin. ndacAc represents the ndacA strain complemented in trans. (B) Growth of the ndacA strain in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/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 bgalactosidase measurements. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (B and C). doi:10.1371/journal.pone.0060460.gConstruction and complementation of in-frame deletion mutantsIn-frame deletion mutants were constructed using the fusion PCR method was as previously described [43]. Primers used in this study are listed in Table S1. Each deletion mutation was verified by sequencing of the mutated region. For genetic complementation, either promoterless pHG101 or its derivative pHG102, which contains the S. oneidensis arcA promoter for genes not in proximity to their promoter, was used [29]. Introduction of each verified complementation vector into the corresponding mutant was achieved by mating with E. coli WM3064 containing the vector, and confirmed by plasmid extraction, restriction enzyme mapping and sequencing.were aliquotted into 24-well plates with a volume of 2 ml per well. Antibiotics and natural products were added to each well at three concentrations. The plates were kept at the room temperature for observation. The morphology of cells was examined with a Motic BA310 phase-contrast microscope. Micrographs were captured with a Moticam 2306 charged-coupled-device camera and Motic images advanced 3.2 software. All experiments were conducted at least in triplicate.Antibiotic susceptibility assayAntibiotic susceptibility of S. oneidensis was determined with both liquid and solid cultures. For antibiotics commonly used in genetic manipulation, the highest concentrations were set PF-299804 price according to the molecular biology manual and lower concentrations were prepared by double dilution. Three ml of ISC cultures were spotted onto LB agar plates containing antibiotics of varying concentrations. The plates were incubated for up to 3 days and scored for growth each day. No growth, some growth after 3 days, and full growth after 1 day were co.Gentamycin at 15 mg/ml.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 mg/ml), M (2.5 mg/ml), or L (0.125 mg/ml) amounts, and incubated at 30uC in a shaker at 200 rpm (A) PblaA promoter activities were determined by measuring b-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 mg/ml) level were also assayed using qRT-PCR (presented as diamonds). (B) b-lactamase activity assay. At the indicated times, samples were taken for b-lactamase activity measurements. In both panels, experiments were performed at least in triplicate and the error bars represent standard deviation (SD). doi:10.1371/journal.pone.0060460.gExpression of blaA in S. oneidensisFigure 6. Impacts of the loss of LMW PBPs on growth in the presence of ampicillin. (A) Susceptibility assay of LMW PBP mutants ndacB (PBP4), ndacA (PBP5), and npbpG (PBP7) to ampicillin. ndacAc represents the ndacA strain complemented in trans. (B) Growth of the ndacA strain in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/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 bgalactosidase measurements. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (B and C). doi:10.1371/journal.pone.0060460.gConstruction and complementation of in-frame deletion mutantsIn-frame deletion mutants were constructed using the fusion PCR method was as previously described [43]. Primers used in this study are listed in Table S1. Each deletion mutation was verified by sequencing of the mutated region. For genetic complementation, either promoterless pHG101 or its derivative pHG102, which contains the S. oneidensis arcA promoter for genes not in proximity to their promoter, was used [29]. Introduction of each verified complementation vector into the corresponding mutant was achieved by mating with E. coli WM3064 containing the vector, and confirmed by plasmid extraction, restriction enzyme mapping and sequencing.were aliquotted into 24-well plates with a volume of 2 ml per well. Antibiotics and natural products were added to each well at three concentrations. The plates were kept at the room temperature for observation. The morphology of cells was examined with a Motic BA310 phase-contrast microscope. Micrographs were captured with a Moticam 2306 charged-coupled-device camera and Motic images advanced 3.2 software. All experiments were conducted at least in triplicate.Antibiotic susceptibility assayAntibiotic susceptibility of S. oneidensis was determined with both liquid and solid cultures. For antibiotics commonly used in genetic manipulation, the highest concentrations were set according to the molecular biology manual and lower concentrations were prepared by double dilution. Three ml of ISC cultures were spotted onto LB agar plates containing antibiotics of varying concentrations. The plates were incubated for up to 3 days and scored for growth each day. No growth, some growth after 3 days, and full growth after 1 day were co.