Generation of Fluorescent Bacteria with the Genes Coding for Lumazine Protein and Riboflavin Biosynthesis

Abstract

Lumazine protein is a member of the riboflavin synthase superfamily and the intense fluorescence is caused by non-covalently bound to 6,7-dimethyl 8-ribityllumazine. The pRFN4 plasmid, which contains the riboflavin synthesis genes from Bacillus subtilis, was originally designed for overproduction of the fluorescent ligand of 6,7-dimethyl 8-ribityllumazine. To provide the basis for a biosensor based on the lux gene from bioluminescent bacteria of Photobacterium leiognathi, the gene coding for N-terminal domain half of the lumazine protein extending to amino acid 112 (N-LumP) and the gene for whole lumazine protein (W-LumP) from P. leiognathi were introduced by polymerase chain reaction (PCR) and ligated into pRFN4 vector, to construct the recombinant plasmids of N-lumP-pRFN4 and W-lumP-pRFN4 as well as their modified plasmids by insertion of the lux promoter. The expression of the genes in the recombinant plasmids was checked in various Escherichia coli strains, and the fluorescence intensity in Escherichia coli 43R can even be observed in a single cell. These results concerning the co-expression of the genes coding for lumazine protein and for riboflavin synthesis raise the possibility to generate fluorescent bacteria which can be used in the field of bio-imaging.

Development of Fluorescent Bacteria with Lux and Riboflavin Genes

“…In a series of studies regarding the generation of fluorescent bacteria, we initially started to work with pRFN4 plasmids by inserting the lumazine protein gene from P. leiognathi to test fluorescence intensities and single-cell imaging [ 10 ]. By binding one molecule of lumazine to the protein, the fluorescence intensity of N-LumP is increased because of its lumazine chromophore [ 12 ].…”

“…Proteins are as follows: lumazine protein (LuxL), α and β subunits of luciferase (LuxAB), fatty acid reductase complex (LuxCDE), non-fluorescent flavoprotein (LuxF), flavin reductase (LuxG), GTP cyclohydrolaseII (RibA), dihydroxy-butanone 4-phosphate synthase (RibB), lumazine synthase (RibH), and riboflavin synthase (RibE). Modified from reference [ 10 ].…”

Single-cell level LasR-mediated quorum sensing response of Pseudomonas aeruginosa to pulses of signal molecules

“…To explore the kinetics of this transition, tracking the population’s state is necessary. Luminescence (either natural, as in Vibrio species 9 – 11 , or due to synthetic constructs 12 – 14 ) is ideal for this as it enables both single-cell and population-level monitoring.…”