Overexpression of AdeIJK RND efflux pump in Acinetobacter baumannii using the mini-Tn 7 T-based single copy induction system can lead to lacI q mutation

Chromosomal gene complementation with inducible expression is a powerful approach for investigating bacterial gene function. The pUC18T-mini-Tn 7 T system enables isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible, site-specific genomic integration of genes in single copy via the P tac-lacI q regulatory system. While widely used, limitations remain underexplored. Here, we report a rare and possibly protein-specific but critical caveat of this system during overexpression of AdeIJK, a resistance-nodulation-division efflux pump, in Acinetobacter baumannii . IPTG exposure led to the emergence of IPTG-unresponsive mutants with the loss of IPTG-dependent gene expression. Sequencing identified a shared R197S mutation in lacI , impairing IPTG binding and derepression. These findings underscore the need for vigilant monitoring of regulatory elements when using inducible systems, particularly during the expression of genes whose overexpression is not well tolerated by bacteria. IMPORTANCE Genetic tools are essential for gaining deeper insights into bacterial systems. Though these tools have been of great value in the study of genotype-phenotype relationships, it is equally important to recognize their limitations. We believe this observation will help raise awareness of a potential constraint associated with the widely used mini-Tn7 gene expression system and enable researchers to avoid misinterpretation of experimental results. Being conscious of this limitation, subtle changes that may have otherwise gone unnoticed can be more effectively identified.