Shedding light on Klebsiella pneumoniae virulence: Engineering of broad host range bioluminescence reporter vectors for transcriptional analysis in drug resistant pathogens.

In this work, we report the construction of four bacterial luciferase-based promoter probe vectors with an expanded set of selectable markers, designed to facilitate their use in antibiotic-resistant bacteria. These vectors contain the low-copy-number, broad-host-range pBBR origin of replication and an origin of transfer, allowing efficient conjugative transformation into various bacterial genera. The broad host range origin also enables their use in bacterial strains that harbor other plasmids, as the pBBR origin is compatible with a wide variety of other plasmid replication systems. The utility of these vectors was demonstrated by quantifying capsule gene expression in both classical and hypervirulent Klebsiella pneumoniae strains lacking tolC, which encodes the outer membrane pore protein for tripartite transport systems. Our results revealed that the tolC mutation reduced capsule gene expression, highlighting a critical role for tolC in K. pneumoniae pathobiology and the utility of bioluminescence for studying gene expression in real time. These new vectors provide a flexible platform for circumventing antibiotic resistance phenotypes and studying gene expression across diverse bacterial species, including strains containing additional plasmids.