Title: Gaussia princeps luciferase as reporter for transcriptional activity, protein secretion and protein-protein interactions in Salmonella enterica serovar Typhimurium

Gaussia princeps luciferase (Gluc) is widely used as a reporter in eukaryotes, but data about its applicability in bacteria is very limited. Here we show that a codon-optimized Gluc gene can be efficiently expressed in Salmonella enterica sv. Typhimurium (S. Typhimurium). To test different Gluc variants as transcriptional reporters, we used the siiA promoter of Salmonella pathogenicity island 4 (SPI-4) driving expression of either an episomal or a chromosomally integrated Gluc gene. Most reliable results were obtained from lysates of single-copy Gluc reporter strains. Given the small size, high activity and co-factor independence of Gluc, it might be especially suited to monitor secretion of bacterial proteins. We demonstrate its usefulness by luminescence detection of fusion proteins of Gluc and Cterminal portions of the SPI-4-encoded, type I-secreted adhesin SiiE in supernatants. The SiiE C-terminal moiety including immunoglobulin (Ig) domain 53 is essential and sufficient for mediating type I-dependent secretion of Gluc. In eukaryotes, protein-protein interaction studies based on split Gluc protein complementation assays (PCA) could be established. We adapted these methods for use in Salmonella, demonstrating the interaction between the SPI-1-encoded effector SipA and its cognate secretion chaperone InvB. In conclusion, the versatile Gluc can be used to address a variety of biological questions, thus representing a valuable addition to the toolbox of modern molecular biology and microbiology.