Construction, characterization and exemplificative application of bioluminescent Bifidobacterium longum biovar longum.
The aim of this work was to construct a bifidobacterial biosensor that could be used to analyze the metabolic state of cells. We transformed by electroporation the human intestinal bacterium Bifidobacterium longum biovar longum with a vector (pGBL8b) containing the insect luciferase gene from a click beetle (Pyrophorus plagiophthalamus) and studied the basic parameters affecting light production in the bioluminescent phenotype. We detected a minimum of 4000 cells, which indicates that the insect luciferase expression in Bifidobacterium longum is extremely good, and a measurement requires only a few minutes of incubation in ambient oxygen conditions. A pH of 7.0 was optimal for incorporating the substrate d-luciferin, and the substrate saturation effect occurred at 125 microM. We employed bioluminescent B. longum for a quick test of the efficacy of different carbohydrates to preserve cell physiology under acidic conditions. The prebiotic compounds Actilight and lactulose were the most active in preventing loss of intracellular ATP during incubation at pH 3. Glucose and inulin were less active, though still effective. In sum, our results show that bioluminescent B. longum, transformed with the pGBL8b plasmid, is a valuable tool for rapidly studying the physiological state of anaerobic bacterial cells under different environmental conditions.
Guglielmetti
Simone
S
Department of Food Science and Microbiology, University of Milan, Italy. simone.guglielmetti@unimi.it
Ciranna
Alessandro
A
Mora
Diego
D
Parini
Carlo
C
Karp
Matti
M
D001644:Bifidobacterium / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D049409:Luciferases, Firefly / Q000378:metabolism*; D049408:Luminescent Agents; D008163:Luminescent Measurements; D019936:Probiotics; D014169:Transformation, Bacterial
D049408:Luminescent Agents; D049409:Luciferases, Firefly