Tet-ON and Tet-OFF regulation in hypervirulent Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic gram-negative bacterial pathogen responsible for a variety of infections in hospitals and in the community. Hypervirulent K. pneumoniae (hvKp) strains are characterized by their ability to cause liver abscesses and bloodstream infections. This study describes plasmid-based tetracycline-dependent regulation systems for induction or repression of target genes in hvKp. These plasmids encode a wild-type tetracycline repressor (TetR) or a reverse variant (revTetR), and carry tet -sensitive promoters, for anhydrotetracycline-dependent Tet-ON or Tet-OFF regulation, respectively. The dynamic range, determined by gfp under tet control, was up to 19.3-fold with wt-TetR and 5.5-fold with revTetR. As analyzed for up to 48 h, the reporter-gene responses in both systems were controllable in an inducer concentration-dependent manner. When the virulence-associated factor peg-344 was induced by the Tet-ON system, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella infection model. These tet vectors can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae (hvKp) strains are a serious threat in healthcare environments and beyond. Even in healthy individuals, infections often result in liver abscesses, and the bacteria can spread to multiple infection sites. We here describe two kinds of gene expression control in hvKp, dependent on anhydrotetracycline (ATc). The Tet-ON system uses wild-type tetracycline repressor (TetR), and addition of ATc leads to target gene induction. In the Tet-OFF mode, reverse TetR is co-repressed by ATc, resulting in target gene repression. We have quantified both systems by reporter gene assays and also subjected the hypervirulence marker gene peg-344 to Tet-ON control. When induced, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella (greater wax moth) infection model. These systems can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen.