The continuous risk of antibiotic resistance development underscores the demand for new agents with mechanisms distinct from existing antibacterial drugs. Here, we investigated HSI#6, a small-molecule antibacterial previously identified as a SecA activator, using integrated omics and functional assays. HSI#6 exhibits a rapid, broad-spectrum bacteriostatic activity, and induces a distinct cell envelope-homeostasis stress signature accompanied by global stress reprogramming. Time-resolved transcriptomics and proteomics revealed early activation of envelope stress regulons and oxidative stress pathways, followed by suppression of ribosome biogenesis and central metabolism. Comparative analysis and biomarker-based principal component analysis (PCA) positioned HSI#6 within the envelope stress mechanistic space, closely aligned with membrane-active antibiotics yet displaying a distinct signature. Adaptive laboratory evolution (ALE) combined with whole-genome sequencing (WGS) revealed compensatory mutations in topoisomerase 1A gene (topA) and transcriptional regulators, without adaptive resistance emerged even under prolonged selection pressure. These findings establish HSI#6 as a mechanistically unique antibacterial agent with low resistance potential.

he Sec pathway is an essential protein secretion route for all organisms. In bacteria, the SecA ATPase peripherally associates with the SecYEG channel to form the translocase that mediates preprotein export. Activation of the translocase depends strictly on the synergy of signal peptide and mature domain binding. Thus, client selectivity, translocase activation and protein secretion are coupled by one mechanism. We show here that a previously identified small molecule (HSI#6) binds SecA, modulates its intrinsic dynamics and allosterically activates the translocase in the absence of clients. By uncoupling translocase activation from preprotein binding, HSI#6 transformed the translocase into a promiscuous nanomachine that lost client selectivity and secreted unfolded pre- mature- and cytoplasmic- proteins with high efficiency in vivo or in vitro. To our knowledge, HSI#6 is the first activator of the Sec pathway and might offer unique opportunities for the discovery of new antibacterials.