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- 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
- 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.
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