Journal: Antimicrobial Agents and Chemotherapy

Article Title: Peptide Antibiotic Sensing and Detoxification Modules of Bacillus subtilis ▿

doi: 10.1128/AAC.00352-10

Figure Lengend Snippet: Organization of the psdRS-AB and bceRS-AB loci and induction by peptide antibiotics. (A) Graphic representation of the psdRSAB and bceRSAB loci. Genes belonging to the psd and bce loci are shown in blue (two-component system) and green (ABC transporter); the genes flanking both operons are white. Promoters are marked with bent arrows, and putative terminators are represented by vertical bars and a circle. (B) Regulatory principle and genetic setup of the Psd and Bce biosensor strains. The response regulator (PsdR or BceR), activated by the sensor kinase (PsdS or BceS), binds to its target promoter and induces the expression of the ABC transporter encoding the psdAB or bceAB operon (detoxification) and lacZ (production of β-galactosidase). (C) Example of a qualitative β-galactosidase assay with nisin, actagardine, mersacidin, and gramicidin (disk diffusion assay). The reporter strain carrying a chromosomal PpsdA-lacZ fusion was used in soft-agar overlays on LB plates containing X-Gal. Bactericidal activity is visualized as the presence of a growth inhibition zone around the filter disk, and PpsdA-dependent induction is visualized as a blue ring around the inhibition zone. (D) Qualitative β-galactosidase assay with the subtilin producer strain B. subtilis ATCC 6633. PpsdA-lacZ reporter strain TMB299 was streaked out onto LB-X-Gal plates directly next to the B. subtilis ATCC 6633 cultures. The appearance of a blue zone in the reporter strain next to the subtilin-producing strain shows the induction of PpsdA.

Article Snippet: Moreover, we are grateful to H.-G. Sahl for samples of Pep5 and mersacidin and Antony Appleyard (Novacta Biosystems) for the generous gift of purified actagardine.

Techniques: Expressing, Diffusion-based Assay, Activity Assay, Inhibition

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Peptide Antibiotic Sensing and Detoxification Modules of Bacillus subtilis ▿

doi: 10.1128/AAC.00352-10

Figure Lengend Snippet: Inducers of P psdA , P bceA , P yxdL , and P liaI expression a

Article Snippet: Moreover, we are grateful to H.-G. Sahl for samples of Pep5 and mersacidin and Antony Appleyard (Novacta Biosystems) for the generous gift of purified actagardine.

Techniques: Expressing, Binding Assay, Glycoproteomics

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Peptide Antibiotic Sensing and Detoxification Modules of Bacillus subtilis ▿

doi: 10.1128/AAC.00352-10

Figure Lengend Snippet: Concentration-dependent induction of PpsdA, lysis curves, and MICs of B. subtilis cultures treated with actagardine, enduracidin, gallidermin, nisin, or subtilin (supernatant of B. subtilis strain ATCC 6633). (A) β-Galactosidase activities, expressed as Miller units, of PpsdA-lacZ reporter strain TMB299 induced with the above-mentioned compounds. A log scale is applied on the y axis for reasons of clarity, due to the high dynamic range of β-galactosidase activities. (B) Concentration-dependent killing of TMB299. The times of antibiotic addition are indicated by arrows. The concentrations of actagardine, gallidermin, nisin (in μg/ml), enduracidin (in ng/ml), and subtilin (percent B. subtilis ATCC 6633 supernatant) that affect the growth of B. subtilis are indicated. (C) MIC assays for B. subtilis cultures treated with actagardine, enduracidin, gallidermin, nisin, and subtilin. Wild-type (•) and psdAB deletion mutant (○) strains were inoculated to an OD600 of 0.05 in Mueller-Hinton medium with different concentrations of antibiotics. Cultures were incubated with agitation at 37°C for 6 h before the determination of the cell density (OD600). The MIC was defined as the lowest concentration of antibiotic that fully inhibited growth.

Article Snippet: Moreover, we are grateful to H.-G. Sahl for samples of Pep5 and mersacidin and Antony Appleyard (Novacta Biosystems) for the generous gift of purified actagardine.

Techniques: Concentration Assay, Lysis, Mutagenesis, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Peptide Antibiotic Sensing and Detoxification Modules of Bacillus subtilis ▿

doi: 10.1128/AAC.00352-10

Figure Lengend Snippet: Concentration-dependent induction of PbceA, lysis curves, and MICs of B. subtilis cultures treated with actagardine, bacitracin, and mersacidin. (A) β-Galactosidase activities, expressed as Miller units, of reporter strain TMB279 induced by actagardine, bacitracin, or mersacidin. A log scale is applied on the y axis for reasons of clarity, due to the high dynamic range of β-galactosidase activities. (B) Concentration-dependent killing of B. subtilis. The times of antibiotic addition are indicated by arrows. The concentrations of actagardine, bacitracin, or mersacidin (all in μg/ml) that affect the growth of B. subtilis are indicated. (C) MIC assay for the wild type (•) and isogenic bceAB deletion mutant (○). See the legend of Fig. ​Fig.3C3C for experimental details.

Article Snippet: Moreover, we are grateful to H.-G. Sahl for samples of Pep5 and mersacidin and Antony Appleyard (Novacta Biosystems) for the generous gift of purified actagardine.

Techniques: Concentration Assay, Lysis, Mutagenesis