Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi

Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.     

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.     

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.     

Bovine and feline gastrin cDNA sequences and the amino acid and nucleotide sequence homologies among mammalian species.

The complete nucleotide sequences of cDNAs encoding bovine and feline preprogastrins have been cloned from the antral mucosa mRNA. The gastrin mRNA of each animal encodes a preprogastrin of 104 amino acids consisting of a signal peptide, a prosegment of 37 amino acids, and a gastrin 34 sequence, followed by a glycine (the amide donor). The cleavage following a pair of lysine residues yields gastrin 17. We found that pairs of arginine residues flanking gastrin 34, the typical processing site sequence of all other preprogastrins and many peptide hormones, were arginines in the bovine preprogastrin, but the first basic amino acid pair had changed to Arg-Trp (57-58 residues) instead of Arg-Arg in the feline preprogastrin. Comparison of these amino acid and nucleotide sequences with published mammalian sequences showed extensive homology in the coding (63 to 73% amino acid identity) and in the untranslated regions (67 to 89% identity). Prosequence, the most variable region, shows greater amino acid difference between bovine and human preprogastrin (54% identity), and between bovine and rat preprogastrin (54% identity) than between other species (62 to 82% identity).     

The amino acid sequences of cytochrome c from four plant sources

Proposed amino acid sequences of cytochrome c from nasturtium (Tropaeolum majus L.), box-elder (Acer negundo L.), elder (Sambucus nigra L.) and parsnip (Pastinaca sativa L.) are presented. Because of the very limited amounts of cytochrome available from some plant sources, peptides derived from the cytochromes c have been sequenced by the semi-quantitative dansyl-Edman technique (Gray & Hartley, 1963) without supporting quantitative amino acid analyses. Because of the qualitative nature of the work, the sequences proposed must be regarded as tentative. Considerations of homology, although useful as a guide, have been kept to a minimum in the construction of sequences. Only the nasturtium sequence relies on considerations of homology for a complete ordering of the peptides. Where material permitted, each residue of a proposed sequence was determined at least once from both a tryptic and a chymotryptic peptide.     

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.     

Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.     

The nucleotide sequence of the luxE gene of Vibrio harveyi and a comparison of the amino acid sequences of the acyl-protein synthetases from V. harveyi and V. fischeri

The luxE gene of bioluminescent bacteria encodes the acyl-protein synthetase component of the fatty acid reductase complex. The complex is responsible for converting tetradecanoic acid to the aldehyde which serves as a substrate in the luciferase-catalyzed reaction. The nucleotide sequence of the luxE gene of Vibrio harveyi was determined and the amino acid sequence of the acyl-protein synthetase deduced. The protein consists of 378 amino acid residues and has a molecular weight of 42,965 daltons. Alignment of the V. harveyi enzyme with the V. fischeri acyl-protein synthetase showed 62% identity.     

Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens

The entomopathogenic bacterium Xenorhabdus luminescens produces a red pigment and an antibiotic in insect carcasses in which it grows and in axenic cultures. The pigment was purified and identified as the anthraquinone derivative 1,6-dihydroxy-4-methoxy-9,10-anthraquinone, which exhibits a pH-sensitive color change, i.e., it is yellow below pH 9 and red above pH 9. The antibiotic was also purified and identified as the hydroxystilbene derivative 3,5-dihydroxy-4-isopropylstilbene.     

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.     

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.     

The nucleotide sequence of a cytoplasmic tRNAPhe from Scenedesmus obliquus and comparison with a tRNATyr species.

The nucleotide sequence of a cytoplasmic tRNAPhe from the eukaryotic green alga Scenedesmus obliquus was determined as: pG-G-C-U-U-G-A-U-A-m2G-C-U-C-A-G-C-D-Gm-G-G-A-G-A-G-C-m22G-p si-psi-A-G-A-Cm-U-G - A-A-m1G-A-psi-C-U-A-C-A-G-m7G-N-m5C-C-C-C-A-G-T-psi-C-G-m1A-U-m5C-Cm-U-G -G-G-U- C A-G-G-C-C-A-C-C-A-OH. The structure has some notable features. Unlike other tRNAPhe species from plant sources, it has an unmodified G as the first residue of the anticodon and m1G rather than a Y derivative as the residue following the anticodon. The sequence m5C(60)-Cm(61) is unique to this tRNA. The sequence of S. obliquus tRNAPhe shows close homology with S. obliquus tRNATyr.     

Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luminescens.

The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.     

Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens.

The entomopathogenic bacterium Xenorhabdus luminescens produces a red pigment and an antibiotic in insect carcasses in which it grows and in axenic cultures. The pigment was purified and identified as the anthraquinone derivative 1,6-dihydroxy-4-methoxy-9,10-anthraquinone, which exhibits a pH-sensitive color change, i.e., it is yellow below pH 9 and red above pH 9. The antibiotic was also purified and identified as the hydroxystilbene derivative 3,5-dihydroxy-4-isopropylstilbene.     

Motifer, a search tool for finding amino acid sequence patterns from nucleotide sequence databases.

Motifer is a software tool able to find directly in nucleotide databases very distant homologues to an amino acid query sequence. It focuses searches on a specific amino acid pattern, scoring the matching and intervening residues as specified by the user. The program has been developed for searching databases of expressed sequence tags (ESTs), but it is also well suited to search genomic sequences. The query sequence can be a variable pattern with alternative amino acids or gaps and the sequences searched can contain introns or sequencing errors with accompanying frame shifts. Other features include options to generate a searchable output, set the maximal sequencing error frequency, limit searches to given species, or exclude already known matches. Motifer can find sequence homologues that other search algorithms would deem unrelated or would not find because of sequencing errors or a too large number of other homologues. The ability of Motifer to find relatives to a given sequence is exemplified by searches for members of the transforming growth factor-beta family and for proteins containing a WW-domain. The functions aimed at enhancing EST searches are illustrated by the 'in silico' cloning of a novel cytochrome P450 enzyme.     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

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.