Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (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 beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.     

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.     

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.     

Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.     

Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo

Bacterial luciferase is a heterodimeric enzyme comprising two nonidentical but homologous subunits, alpha and beta, encoded by adjacent genes, luxA and luxB. The two genes from Vibrio harveyi were separated and expressed from separate plasmids in Escherichia coli. If both plasmids were present within the same E. coli cell, the level of accumulation of active dimeric luciferase was not dramatically less than within cells containing the intact luxAB sequences. Cells carrying the individual plasmids accumulated large amounts of individual subunits, as evidenced by two-dimensional polyacrylamide gel electrophoresis. Mixing of a lysate of cells carrying the luxA gene with a lysate of cells carrying the luxB gene resulted in formation of very low levels of active heterodimeric luciferase. However, denaturation of the mixed lysates with urea followed by renaturation resulted in formation of large amounts of active luciferase. These observations demonstrate that the two subunits, alpha and beta, if allowed to fold independently in vivo, fold into structures that do not interact to form active heterodimeric luciferase. The encounter complex formed between the two subunits must be an intermediate structure on the pathway to formation of active heterodimeric luciferase.     

Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase alpha and beta subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate that neither the specific activity nor the size of the alpha (362 amino acid residues, Mr 41,389) and beta (324 amino acid residues, Mr 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the alpha and beta subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.     

Structure and properties of luciferase from Photobacterium phosphoreum

The nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits, respectively, of luciferase from Photobacterium phosphoreum have been determined. The predicted amino acid sequences of the alpha and beta subunits were shown to be significantly different from other bacterial luciferases with 62 to 88% identity with the alpha subunits and 47 to 71% identity with the beta subunits of other species. Expression of the different luciferases appear to correlate with the number of modulator codons. Kinetic properties of P. phosphoreum luciferase were shown to reflect the bacterium's natural cold temperature habitat.     

Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers

A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.     

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence in different marine bacteria.     

Isolation of the lux genes from Photobacterium leiognathi and expression in Escherichia coli

Genes necessary for luminescence (lux genes) in the marine bacterium Photobacterium leiognathi, strain PL721, were isolated and expressed in Escherichia coli. A 15-kb fragment obtained from a partial digestion of PL721 DNA with HindIII was cloned into the plasmid pACYC184, resulting in the hybrid plasmid pSD721. When pSD721 was transformed into E. coli ED8654, the resulting transformants were luminous with no additions to the cells, indicating that it contained the structural genes coding for the alpha and beta subunits of luciferase (luxA and luxB), and for components involved in aldehyde biosynthesis. Hybridization analysis with luxA and luxB 32P probes confirmed the location of these two genes on the 15-kb insert. When pSD721 was transformed into four different strains of E. coli, luminescence expression varied widely in amount and in pattern. In some strains, luminescence developed like an autoinducible system, and at maximum induction was very bright, even with no addition of aldehyde, while in others, luminescence was 100-fold less, and no induction was seen. In no case was luminescence affected by shifts in temperature, osmolarity, or iron concentration. These results indicate that, while the complete lux regulon is apparently contained on the 15-kb cloned fragment, the regulation of the lux regulon in pSD721 is subject to host controls by E. coli, controls which vary widely among different E. coli strains.     

Cloning and expression of the lux-operon of Photorhabdus luminescens, strain Zm1: nucleotide sequence of luxAB genes and basic properties of luciferase

A chromosomal fragment of bacteria Photorhabdus luminescence Zm1, which contains the lux operon, was cloned into the vector pUC18. The hybrid clone containing plasmid pXen7 with the EcoRI fragment approximately 7-kb was shown to manifest a high level of bioluminescence. By subcloning and restriction analysis of the EcoRI fragment, the location of luxCDABE genes relative to restriction sites was determined. The nucleotide sequence of the DNA fragment containing the luxA and luxB genes encoding alpha- and beta-subunits of luciferase was determined. A comparison with the nucleotide sequences of luxAB genes in Hm and Hw strains of Ph. luminescence revealed 94.5 and 89.7% homology, respectively. The enterobacterial repetitive intergenic sequence (ERIC) of 126 bp typical for Hw strains was identified in the spacer between the luxD and luxA genes. The lux operon of Zm1 is assumed to emerge through recombination between Hm and Hw strains. Luciferase of Ph. luminescence was shown to possess a high thermal stability: its activity decreased by a factor of 10 at 44 degrees C for 30 min, whereas luciferases of marine bacteria Vibrio fischeri and Vibrio harveyi were inactivated by one order of magnitude at 44 degrees C for 1 and 6 min, respectively. The lux genes of Ph. luminescence are suggested for use in gene engineering and biotechnology.     

Random and site-directed mutagenesis of bacterial luciferase: investigation of the aldehyde binding site

Numerous luciferase structural gene mutants of Vibrio harveyi have been generated by random mutagenesis and phenotypically characterized [Cline, T.W., & Hastings, J.W. (1972) Biochemistry 11, 3359-3370]. All mutants selected by Cline and Hastings for altered kinetics in the bioluminescence reaction had lesions in the alpha subunit. One of these mutants, AK-20, has normal or slightly enhanced thermal stability and enhanced FMNH2 binding affinity but a much-reduced quantum yield of bioluminescence and dramatically altered stability of the aldehyde-C4a-peroxydihydroflavin-luciferase intermediate (IIA), with a different aldehyde chain length dependence from that of the wild-type luciferase. To better understand the structural aspects of the aldehyde binding site in bacterial luciferase, we have cloned the luxAB genes from the V. harveyi mutant AK-20, determined the nucleotide sequence of the entire luxA gene, and determined the mutation to be TCT----TTT, resulting in a change of serine----phenylalanine at position 227 of the alpha subunit. To confirm that this alteration caused the altered kinetic properties of AK-20, we reverted the AK-20 luxA gene by oligonucleotide-directed site-specific mutagenesis to the wild-type sequence and found that the resulting enzyme is indistinguishable from the wild-type luciferase with respect to quantum yield, FMNH2 binding affinity, and intermediate IIA decay rates with 1-octanal, 1-decanal, and 1-dodecanal. To investigate the cause of the AK-20 phenotype, i.e., whether the phenotype is due to loss of the seryl residue or to the properties of the phenylalanyl residue, we have constructed mutants with alanine, tyrosine, and tryptophan at alpha 227.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.     

Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.     

Expression of the cloned subunits of bacterial luciferase from separate replicons

The lux A and lux B genes of Vibrio harveyi, encoding the alpha and beta subunits of bacterial luciferase, were cloned individually into Escherichia coli in two different compatible plasmids. Active luciferase was formed in an amount equal to that produced in cells carrying a plasmid with the cloned genes on a single fragment.