Gaussia princeps luciferase as a 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 are very limited. Here we show that a codon-optimized Gluc gene can be efficiently expressed in Salmonella enterica serovar 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 cofactor 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 C-terminal 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.
Junior Research Group 3, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
Thorsten
T
Wille
Kathrin
K
Blank
Christiane
C
Schmidt
Vivien
V
Vogt
Roman G
RG
Gerlach
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D033342:Copepoda / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D008156:Luciferases