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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]
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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.
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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.
- Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from Photobacterium phosphoreum
- Chan Yong Lee , Edward A. Meighen
- J. Microbiol. 2000;38(2):80-87.
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Abstract
- The nucleotide sequence of the luxC gene coding for lux-specific fatty acyl-CoA reductase and the upstream DNA (325 bp) of the structural gene from bioluminescent bacterium, Photobacterium phosphoreum, has been determined. An open reading frame extending for more than 20 codons in 325 bp DNA upstream of luxC was not present in both directions. The lux gene can be translated into a polypeptide of 54 kDa and the amino acid sequences of lux specific reductases of P. phosphoreum shares 80, 65, 58, and 62% identity with those of the Photobacterium leiognathi, Vibrio fischeri, Vibrio harveyi, and Xehnorhabdus luminescens reductases, respectively. Analyses of codon usage, showing that a high frequency (2.3%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA and B genes, suggested that the AUA codon may play a modulator role in the expression of lux gene in E. coli. The structural genes (luxC, D, A, B, E) of the P. phosphoreum coding for luciferase ([alpha], [beta]) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in E. coli under the T7 phage RNA polymerase/promoter system and identification of the [^35 S]methionine labelled polypeptide products. The degree of expression of lux genes in this system, high level of luxA, B genes whereas low level of luxC, D genes, were consistent with the analyses of codon usage. High expression of the luxC gene could only be accomplished in a mutant E. coli 43R. Even in crude extracts, the acylated acyl-CoA reductase intermediate as well as acyl-CoA reductase activities could be readily detected.
- Bioluminescent Assay of Phospholipase C Using A Luminescent Marine Mutant Bacterium Vibrio harveyi M-17
- Ki Woong Cho , SangJun Mo , Hyi-Seung Lee , Jung-Rae Rho , Jongheon Shin
- J. Microbiol. 2000;38(3):150-155.
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Abstract
- A bioluminescent assay method for detecting the activity of phospholipase C (PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2-dimyristoyl phosphatidyl choline (DMPC) as a substrate were used in the demonstration, and the produced sn-1,2-dimyristoyl glycerol was further hydrolyzed with lipase from Candida cylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hydrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the amount of enzyme added. Activity measurement conditions (at 25 C, pH 6.5, 10 min fixed time assay) were established to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.
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