Modular, inducible, and titratable expression systems for Escherichia coli and Acinetobacter baumannii

Abstract

ABSTRACT Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii. We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, PabstBR, that induces to a high level but is less leaky than the commonly used trc promoter. We show that PabstBR is titratable at both the population and single-cell levels, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress σ factor, RpoE, and deploy the reporter in E. coli and A. baumannii, finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers who study the fundamental biology of E. coli and A. baumannii.IMPORTANCEAcinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. In this study, we provide expression tools that can be used to study the gene function in A. baumannii, including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli, enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology.

Keywords

• synthetic biology
• gene expression
• cloning
• shuttle vector
• Tn7 vector