Journal of Microbiology

Research Support, Non-U.S. Gov't

Coregulation of lux Genes and Riboflavin Genes in Bioluminescent Bacteria of Photobacterium phosphoreum: Nack-Do Sung , Chan Yong Lee; J. Microbiol. 2004;42(3):194-199.; DOI: https://doi.org/2090 [pii]

Abstract: Investigation of the expression of the riboflavin (rib) genes, which are found immediately downstream of luxG in the lux operon in Photobacterium phosphoreum, provides more information relevant to the evolution of bioluminescence, as well as to the regulation of supply of flavin substrate for bacterial bioluminescence reactions. In order to answer the question of whether or not the transcriptions of lux and rib genes are integrated, a transcriptional termination assay was performed with P. phosphoreum DNA, containing the possible stem-loop structures, located in the intergenic region of luxF and luxE ([omega]_A), of luxG and ribE ([omega]_B), and downstream of ribA ([omega]_C). The expression of the CAT (Chloramphenicol Acetyl Transferase) reporter gene was remarkably decreased upon the insertion of the stem-loop structure ([omega]_C) into the strong lux promoter and the reporter gene. However, the insertion of the structure ([omega]_B) into the intergenic region of the lux and the rib genes caused no significant change in expression from the CAT gene. In addition, the single stranded DNA in the same region was protected by the P. phosphoreum mRNA from the S1 nuclease protection assay. These results suggest that lux genes and rib genes are part of the same operon in P. phosphoreum

Bioluminescent Assay of Bovine Liver Riboflavin Kinase Using a Bacterial Luciferase Coupled Reaction: Ki Woong Cho; J. Microbiol. 2000;38(2):74-79.

Abstract: For the demonstration of a novel riboflavin kinase assay method based on the bacterial bioluminescence, partially purified riboflavin kinase was prepared from bovine liver through ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. Using bacterial luciferase from Photobacterium phosphoreum and the dithionite reduction method, an easy, safe, and fast assay method was established. The optimal temperature, pH, Km values for riboflavin and ATP of bovine liver riboflavin kinase determined with this luminescence method were 35 C, pH 7, 15.3 uM and 8.3 uM, respectively. The detection limit of FMN produced by riboflavin kinase was in the range of 200 pM to 4 uM which is comparable to the HPLC-fluorescence detection method, while the detection time for each assay was less than 15 sec compared to the HPLC method which requires at least 10 min for completion.

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