Stress responses as a tool To detect and characterize the mode of action of antibacterial agents

Abstract

Single-copy gene fusions between the lacZ reporter gene and Escherichia coli strains containing promoters induced by cold shock (cspA), cytoplasmic stress (ibp), or protein misfolding in the cell envelope (P3rpoH) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the cspA::lacZ or ibp::lacZ gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3rpoH::lacZ and ibp::lacZ fusions, while the beta-lactam antibacterial agent carbenicillin activated only the P3rpoH promoter. Nalidixic acid, a compound that causes DNA damage, downregulated beta-galactosidase synthesis from P3rpoH but had little effect on expression of the reporter enzyme from either the cspA or ibp promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of Streptomyces venezuelae cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.

FIG. 1

Induction characteristics and specificity of AB734 λφ[cspA::lacZ] (A, D, and G), AB734 λφ[ibp::lacZ] (B, E, and H), and AB734 λφ[P3rpoH::lacZ] (C, F, and I) lysogens. In the time courses experiments (A through C), mid-exponential-phase cultures were supplemented with 5 μg of chloramphenicol per ml (A), 8 μg of streptomycin per ml (B), or 1 μg of polymyxin B per ml (C), and enzymatic activities were determined at different times (○). Control cultures (●) received an equal volume of 100% ethanol (A) or no additive (B and C). In the concentration dependence experiments (D through F), enzymatic activities were determined at the time of maximal induction, as follows: 3 h after antibiotic was added for AB734 λφ[cspA::lacZ] (D), 1 h after antibiotic was added for AB734 λφ[ibp::lacZ] (E), and 2 h after antibiotic was added for AB734 λφ[P3rpoH::lacZ] (F). The values inside the bars are the percentages of growth inhibition relative to control cultures at the time of sample collection. In the specificity experiments (G through I), mid-exponential-phase cultures were supplemented with no additive (Ctrl), 8 μg of carbenicillin (Car) per ml, 50 μg of nalidixic acid (Nal) per ml, 5 μg of chloramphenicol (Chl) per ml, 8 μg of streptomycin (Str) per ml, 1 μg of polymyxin B (Pol) per ml, 5 μg of tetracycline (Tet) per ml, or 16 μg of neomycin (Neo) per ml, and enzymatic activities were determined at the time of maximal induction. AB734 λφ[cspA::lacZ] cultures were grown at 37°C, while AB734 λφ[ibp::lacZ] and λφ[P3rpoH::lacZ] cultures were grown at 30°C. The typical levels of growth inhibition 1 h after treatment in the experiments whose results are shown in panels G through I were 3% for carbenicillin, 7% for nalidixic acid, 20% for streptomycin and neomycin, 28% for chloramphenicol, 30% for polymyxin B, and 45% for tetracycline.

FIG. 2

Blue halo formation restricted to specific lysogen-antibiotic combinations. Top agar supplemented with AB734 λφ[ibp::lacZ] (A through D), AB734 λφ[P3rpoH::lacZ] (E), or AB734 λφ[cspA::lacZ] (F) cells was poured over LB agar plates spread with X-Gal. Antibiotic disks (Difco) impregnated with 30 μg of chloramphenicol (A), 30 μg of tetracycline (B and F), 300 U of polymyxin B (C and E), or 10 μg of streptomycin (D) were placed on the surfaces of the plates. The plates were incubated overnight at 30°C (A through E) or 37°C (F).

FIG. 3

Detection of chloramphenicol secreted into the growth medium of S. venezuelae cultures. A 1.5-ml aliquot of supernatant from cultures of wild-type S. venezuelae was extracted with ethyl acetate and transferred to a filter paper disk. Top agar supplemented with AB7341λφ[cspA::lacZ] cells was poured over an LB agar plate spread with X-Gal, and the disk was placed on the surface. The plate was incubated overnight at 37°C. The disk contained ∼7.5 μg of chloramphenicol, as estimated by TLC.