Construction of a novel reporter strain indicating inhibition of cell wall biosynthesis

The WalRK two-component system monitors cell wall biosynthesis, and its activity decreases when biosynthesis is inhibited. As WalRK is essential for many Gram-positive bacteria, including Staphylococcus aureus and Bacillus subtilis, directly inhibiting WalK would disrupt cell wall biosynthesis and result in cell death. This study presents the construction of new reporter strains with two WalRK-dependent promoters: PiseA and PssaA. These reporter strains enable indirect in vivo measurement of WalRK activity based on luminescence. We evaluated these reporters in combination with the established PliaI reporter system, which is regulated by LiaRS, in order to improve the identification of cell wall inhibitors during the screening of natural products. The WalRK-dependent PiseA reporter yielded specific responses to almost all known cell wall biosynthesis inhibitors, including β-lactams, tunicamycin, glycopeptides, daptomycin, and fosfomycin, with no discernible response to other antibiotic classes. Furthermore, combining the PliaI reporter system with the PiseA reporter enhanced the range of potential cell wall inhibitors that could be detected. Deleting the gene of the eukaryotic-like Ser/Thr kinase PrkC increased the PiseA signal obtained with certain antibiotics, as well as after a longer incubation period. Additionally, deletion of prkC lowered PliaI expression in the presence of lipid II-targeting antibiotics. Screening natural products with an unknown mode of action identified several compounds that elicited vancomycin-like patterns with the reporter systems, indicating potential cell wall inhibitory activity. Overall, combining WalRK and LiaRS reporters enables a rapid, complementary evaluation of cell wall stress responses, providing a powerful method for discovering new cell wall-targeting antibiotics. KEY POINTS: • The PiseA/PssaA reporters enable continuous in vivo observation of WalRK activity • Cell envelope stress responses are differentially sensed by WalRK and LiaRS • The PiseA reporter strain detects nearly all cell wall biosynthesis inhibitors.