The Spermidine Synthase Gene as a Reporter of Transcription Inhibition in Escherichia coli.

Antimicrobial resistance is a major threat to modern society and healthcare, as it severely compromises the efficacy of standard antibiotic treatments. To meet the ever-increasing demand for novel antimicrobial drugs, it is crucial to develop new strategies for screening antimicrobial compounds and improve existing high-throughput techniques. Reporter systems that employ specific genetic markers are powerful tools not only for detecting antimicrobial activity of the substance being studied, but also for identifying the potential mechanism of its action. Among other metabolic pathways, RNA biosynthesis machinery is considered a promising molecular target as it remains underutilized in current antimicrobial therapy and therefore is rarely exposed to drug pressure. However, there is no suitable biomarker for identifying compounds that inhibit the transcription in Gram-negative bacteria. Combining bioinformatic search and RT-qPCR experimental validation, we have established the overexpression of the spermidine synthase gene (speE) as a biomarker associated with impaired transcription in Escherichia coli. Monitoring the expression level of speE in antibiotic-treated cells enables reliable detection of compounds that inhibit bacterial RNA-polymerase, such as rifampicin and fidaxomicin. Moreover, our screening system was successfully applied in practice to analyze chromatography fractions from fermentation broth of antibiotic producers, with compounds of the rifamycin family being identified as hits and isolated. The proposed method has the potential to be used in sequential screening procedures to reveal active antimicrobial compounds that inhibit bacterial transcription process, giving the world novel antimicrobials with minimal risk of resistance development.