Novel whole-cell antibiotic biosensors for compound discovery

Abstract

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of </=25 mug/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

FIG. 1.

Typical time course of yheI biomarker induction by linezolid between 1 and 4 h and concentration-induction patterns with different translation inhibitors. (A) Linezolid induces the biosensor cells at the earliest after a 3-h exposure time, leading to a maximum signal of 1,300%. (B) The concentration-induction pattern reveals that linezolid, doxycycline, and lead to an approximately 10-fold signal induction, while fusidic acid doubles the signal after the optimal assay incubation time of 4 h. Using 200% signal induction as a significance cutoff, low concentrations of antibiotics (0.02 μM doxycycline, 0.1 μM linezolid, 0.6 μM fusidic acid, and 0.3 μM chloramphenicol) can be detected. These concentrations are 1 to 2 orders of magnitude below the corresponding MICs (MICs, 3.13 μM for linezolid, fusidic acid, and doxycycline; 12.4 μM for chloramphenicol). The fourfold-increased MIC of chloramphenicol is reflected by the maximal signal-inducing concentration of 5 μM in comparison to the other reference antibiotics, which reach the induction maximum around 1 μM. The growth-inhibitory effects of the antibiotics at and above their MICs lead to reduced numbers of viable cells and therefore to a reduction of the signal intensities.

FIG. 2.

Subset of known antibiotics identified in screening of 14,000 pure natural products using the yheI biomarker strain. Structurally diverse chemotypes of antibiotics reported to target the ribosomal function were identified as inducers of the yheI promoter (see Table 5). Some representative structures are presented.

FIG. 3.

MOA studies for ferrimycin A1 by the incorporation of radiolabeled metabolic precursors into whole cells of S. aureus 133. Labeling was performed at different compound concentrations (as indicated).