The beta subunit polypeptide of Vibrio harveyi luciferase determines light emission at 42 degrees C.
The nucleotide sequence of the luxA and luxB genes encoding the alpha beta heterodimeric luciferase from thermotolerant Vibrio harveyi CTP5 was determined. The DNA sequence of the CTP5 luxA and luxB genes is identical to the DNA sequence of the luxA and luxB genes from mesophilic V. harveyi MAV (B 392), with minor exceptions. The sequence differences result in 5 amino acid substitutions in the alpha subunit polypeptide and 7 amino acid substitutions in the beta subunit polypeptide. Escherichia coli cells grown on solid medium and expressing CTP5 or MAV luxAB genes emit similar amounts of light at 37 degrees C, while at 42 degrees C cells containing CTP5 luxAB genes show more than tenfold increased bioluminescence compared to cells with MAV luxAB genes. When grown in liquid medium E. coli cells with CTP5 or MAV luxAB genes emit equivalent amounts of light at 37 degrees C; however, in liquid medium at 42 degrees C cells containing CTP5 luxAB genes show only three times higher bioluminescence than cells with MAV luxAB genes. Expression of T7 promoter-linked hybrid luxAB transcriptional units luxACTP5-luxBMAV and luxAMAV-luxBCTP5 in E. coli reveals that (i) the MAV luxB gene product is responsible for the decreased activity of MAV luciferase at 42 degrees C; (ii) the CTP5 luxB gene encodes the information required for most of the increased activity of CTP5 luciferase relative to MAV luciferase at 42 degrees C; and (iii) E. coli cells containing MAV luxB gene show an increase in bioluminescence when grown in liquid medium at 42 degrees C, which coincides with elevated GroEL chaperonin levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Plant Molecular Genetics, University of Alberta, Edmonton, Canada.
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Escher
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O'Kane
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Szalay
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D018834:Chaperonin 60; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000032:analysis; D015151:Immunoblotting; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D014158:Transcription, Genetic; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology
D000596:Amino Acids; D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D008156:Luciferases