Generation and comparison of broad-host range inducible expression vectors for use in Gram-negative bacteria including ESKAPE pathogens.

Abstract

The objective of this study is a controlled comparison of several carbohydrate inducible promoter in important bacterial species. Inducible promoter systems are invaluable for biotechnology and basic science applications. However, few inducible promoters are available on plasmids that replicate in Saccharomyces cerevisiae, which enables gap-repair recombination, or on broad host-range vectors, which allows replication in many Gram-negative genera. In this study we generated shuttle vectors with S. cerevisiae and the broad host range pBBR1 bacterial replicon. These contained a variety of inducible promoters and used a highly sensitive luxCDABE reporter in Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. Tested carbohydrate-inducible promoters were P _BAD , P _rhaB , P _T5 , and P _xut , isolated from A. baumannii. In the Enterobacterales the P _BAD and P _rhaB promoters demonstrated the highest levels of inducibility at 100- and 5,600-fold, respectively. For P. aeruginosa P _rhaB and P _xut were the most inducible at 40- and 10-fold, respectively. For P. fluorescens all of the carbohydrate-inducible promoters had similar inducibility, under 10-fold. None of the carbohydrate-inducible promoters were effective for A. baumannii; however, they could be used as constitutive promoters. Data indicated that the rhamnose-inducible promoter excelled among the carbohydrate-inducible promoters for most tested organisms, and this study highlights the need for better inducible promoters for A. baumannii.