Organization of the lux structural genes of Vibrio harveyi. Expression under the T7 bacteriophage promoter, mRNA analysis, and nucleotide sequence of the luxD gene.
The structural genes (luxA-E) of the Vibrio harveyi luminescent system coding for the luciferase (alpha, beta) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in Escherichia coli under the T7 phage promoter providing a convenient method for identifying and locating these genes. luxD which codes for the acyltransferase enzyme producing fatty acids for the luminescent reaction was located immediately above the luciferase genes (luxA, B) with the two other fatty acid reductase genes (luxC, E) flanking these genes, in the same order as found for the Vibrio fischeri luminescent system. By hybridization with luxC DNA probes, a set of mRNAs coding for this gene was detected; part of this set of mRNAs extended downstream and complemented the set of mRNAs previously detected for the other lux structural genes. The luxD gene from a mutant (M17) which requires tetradecanoic acid for light emission was cloned into the T7 system, and upon expression it could be demonstrated that the lack of activity was due to synthesis of a full-length nonfunctional protein and not to introduction of a stop codon. The nucleotide sequence of the luxD gene of the native and mutant strains was determined and shown to consist of an open reading frame of 915 bases preceded by a Shine-Dalgarno sequence with very high homology to the ribosome binding site found for luxA and B. The 3'-sequence was identical to a 669-base open reading frame upstream of the luxA gene reported earlier by Cohn et al. (Cohn, D.H., Mileham, A.J., Simon, M.I., Nealson, K.H., Rausch, S.K., Bonam, D., and Baldwin, T.O. (1985) J. Biol. Chem. 260, 6139-6146). The luxD mutant arose by a single point mutation of G to A resulting in a change of glycine to glutamic acid.
McGill University, Department of Biochemistry, Montreal, Quebec, Canada.
C M
CM
Miyamoto
M
M
Boylan
A F
AF
Graham
E A
EA
Meighen
D000215:Acylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D005786:Gene Expression Regulation; D005796:Genes; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis*; D014733:Vibrio / Q000235:genetics*
D012333:RNA, Messenger