Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics

Abstract

Antibiotics such as erythromycin and rifampicin, at low concentrations, alter global bacterial transcription patterns as measured by the stimulation or inhibition of a variety of promoter-lux reporter constructs in a Salmonella typhimurium library. Analysis of a 6,500-clone library indicated that as many as 5% of the promoters may be affected, comprising genes for a variety of functions, as well as a significant fraction of genes with no known function. Studies of a selection of the reporter clones showed that stimulation varied depending on the nature of the antibiotic, the promoter, and what culture medium was used; the response differed on solid as compared with liquid media. Transcription was markedly reduced in antibiotic-resistant hosts, but the presence of mutations deficient in stress responses such as SOS or universal stress did not prevent antibiotic-induced modulation. The results show that small molecules may have contrasting effects on bacteria depending on their concentration: either the modulation of bacterial metabolism by altering transcription patterns or the inhibition of growth by the inhibition of specific target functions. Both activities could play important roles in the regulation of microbial communities. These studies indicate that the detection of pharmaceutically useful natural product inhibitors could be effectively achieved by measuring activation of transcription at low concentrations in high-throughput assays using appropriate bacterial promoter-reporter constructs.

Fig 1.

Comparison between growth inhibition and promoter activation (light production) by erythromycin (A and B) and rifampicin (C and D) over a range of different concentrations as measured with Etest strips placed on bacterial cell overlays. (B and D) Luminescence. The lux–reporter constructions used are listed in Table 1. The bottom of the inhibition zone (where it intersects with the Etest strip) gives a measure of the MICs, which are 32 μg/ml (erythromycin) and 12 μg/ml (rifampicin).

Fig 2.

Combined scatter plot of the actions of rifampicin at 1 μg/ml and erythromycin at 5 μg/ml determined by using a 6,500-clone S. typhimurium random promoter–lux library. RLU, relative light units. Incubation in microtiter plate liquid cultures was for 24 h at 37°C. Points above the diagonal indicate promoter-activated strains and points below the diagonal indicate clones in which promoter activity was repressed. (-, erythromycin; ♦, rifampicin.)

Fig 3.

(Left) Scatter plots of the reassay of selected clones activated by erythromycin at 1 μg/ml (-) and 30 μg/ml (•). (Right) Reassay of selected clones activated by rifampicin at 0.2 μg/ml (×) and 2.5 μg/ml (•). CPS, counts per second. OD is at 620 nm; 1.E+01 = 10¹, etc.

Fig 4.

The effect of mutations to resistance to erythromycin (rplV) and to rifampicin (rpoB) in S. typhimurium on antibiotic inhibition and promoter activation on solid media. (A) Rifampicin-sensitive. (B) Rifampicin-resistant. (C) Erythromycin-sensitive. (D) Erythromycin-resistant.

Fig 5.

The antibiotic specificity of the transcription activation of different promoters. Overlays of S. typhimurium promoter–lux fusions on LB agar were exposed to discs containing antibiotics (10 μg of imipenem, 10 μg of polymixin B, 10 μg of rifampicin, or 15 μg of erythromycin). Light production was measured as described in Materials and Methods. RLU, relative light units. pheA* is a presumptive identification.

Fig 6.

Concentration dependence of promoter activation (plasI′:luxCDABE) in a rifampicin-sensitive (K802; MIC 12 μg/ml) and a rifampicin-resistant (K802NR; MIC >256 μg/ml) E. coli host.