A constitutive promoter toolbox for tunable gene expression in Acinetobacter baumannii

Constitutive promoters with defined strengths are essential for gene expression studies in bacteria; however, such tools remain limited in the clinically important pathogen Acinetobacter baumannii. In this study, we developed an eGFP-based reporter system to identify and characterize constitutive promoters in A. baumannii. Candidate promoter regions were selected from highly expressed genes based on transcriptomic data. Among 25 tested promoters, 19 exhibited detectable activity spanning a broad range of expression strengths. Quantitative analyses using RT-qPCR, SDS-PAGE, and western blotting confirmed differential transcriptional and translational outputs. Fluorescence-based measurements further revealed distinct temporal activity patterns of these promoters across growth phases. Based on these results, a set of 19 constitutive expression vectors was constructed, providing a platform for tunable gene expression in A. baumannii. As a proof-of-concept, overexpression of the endogenous gene cluster strAB, encoding aminoglycoside phosphotransferases (APHs), resulted in reduced susceptibility to streptomycin, demonstrating the applicability of this system for functional studies. This work establishes a well-characterized promoter library and a practical expression platform for A. baumannii. KEY POINTS: • A reporter system that enables systematic identification of promoters in A. baumannii was established. • A diverse promoter library spanning a wide range of expression strengths was identified. • A tunable vector toolkit that enables gene expression in A. baumannii was developed.