Heterogeneously Expressed GadAB Identified by Single-Cell RNA Sequencing Mediates Antibiotic Tolerance Under Acid Stress

Bacterial population heterogeneity serves as the intrinsic basis for persister formation. Single-cell RNA sequencing is a powerful tool for investigating such heterogeneity. In this study, we analyzed single-cell transcriptomic data, previously generated via RiboD-PETRI-seq, from exponential-phase Escherichia coli MG1655 populations. We identified subpopulations with heterogeneous expression of gadA and gadB. Dual-fluorescent reporter strains were subsequently utilized to verify the heterogeneous protein abundance of GadA and GadB across the bacterial community. The expression levels of these two proteins were positively correlated within individual cells. We further observed that the naturally occurring GadA/GadB-low subpopulation exhibited a higher persister ratio than the GadA/GadB-high subpopulation under acid stress, and a similar phenotype was recapitulated in the ΔgadAB mutant. Integrated transcriptomic and metabolomic analyses collectively demonstrated that the gadAB double mutant exhibits a dormant-like state upon acid exposure. This study confirms heterogeneous gadA and gadB expression within bacterial populations. The GadA/GadB-low subpopulation exhibits enhanced antibiotic tolerance under acid stress. This work offers theoretical insights into the acid and antibiotic tolerance mechanisms of enteric pathogens in the acidic gastrointestinal niche.