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[8] Techniques for cloning and analyzing bioluminescence genes from marine bacteria
Abstract
This chapter discusses the techniques for cloning and analyzing bioluminescence genes from marine bacteria. The isolation by recombinant DNA techniques of genes for bioluminescence (lux) from marine bacteria has resulted in a rapid expansion of knowledge of the biochemical activities necessary for light production and of the regulatory mechanisms, which govern the expression of these functions. The chapter describes a variety of genetic methodologies for cloning DNA fragments encoding luminescence functions, eliciting expression of luminescence genes, defining individual lux genes and transcriptional units containing lux genes, identifying the products of cloned lux genes, exploring the regulatory control of lux genes, and using lux gene fusions to measure transcriptional control of other gene systems. Most genetic analysis has been performed with luminescent Vibrio, and the attention is confined to bacteria of this genus.
... There have also been some short reviews describing applications of the lax genes (77,87,124,138). Techniques for cloning the lux genes and screening for the luminescent phenotype have been described in detail (45,95,130); additional methodology is detailed in specific papers. A list of references on the molecular biology of the luminescent systems from different organisms including bacteria has recently been published (137). ...
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
The bioluminescent bacteria comprise one of several groups of luminous organisms. Significant differences exist between the bioluminescence reactions of different organisms, including the structure and properties of the luciferases and substrates. Molecular oxygen is the only common feature of bioluminescence reactions, indicating that the luminescent systems in most organisms may have evolved independently. Luminescent bacteria are present in marine environment, freshwater, and terrestrial habitats. They can occur as free-living forms, saprophytes, commensal symbionts, parasites of animals, and specific light-organ symbionts. The luminescence produced by these bacteria, because of its inherent beauty and ease of detection, has attracted scientific attention. With the use of molecular approaches to study the luminescence systems of these bacteria, population biology, ecology, and molecular mechanisms of luminescence (lux) gene regulation can be studied. This chapter describes the current status of bioluminescent systems of luminous bacteria, emphasizing the biochemistry, lux gene organization, and the physiological and genetic regulation of lux gene expression. The effects of oxygen on luminescence illustrate the application of bacterial luminescence system as a sensor of specific molecules that affect metabolic function and gene expression. Knowledge of the basic biochemistry, molecular biology, and physiology of luminescent bacteria is thus not only of interest but of importance for future scientific endeavors.
A mixed-sequence synthetic oligonucleotide probe was used to isolate a clone containing the gene encoding the alpha subunit of bacterial luciferase from Vibrio harveyi and part of the gene coding for the beta subunit. DNA sequence analysis has allowed us to determine that the genes are closely linked on the bacterial chromosome and transcribed in the same direction. Comparison of the sequences in the regions preceding the two structural genes has revealed considerable homology and has identified sites that may be involved in the expression of the genes. Identification of a clone from a clone bank of total genomic DNA from this organism shows that mixed probes can be successfully used to isolate a gene of interest from any bacterium provided some protein sequence for the gene product is available.
Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.
Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.
Coliphage P1 was used to transduce derivatives of transposons Tn5 and mini-Mu into marine Vibrio spp. Transposon Tn5 encoding tetracycline resistance (Tn5-132) was used to isolate mutants of Vibrio harveyi defective in genes for bioluminescence (lux). Insertion of transposon Tn5-132 into the lux gene region was demonstrated by intraspecific transduction with phage hv-1 and by Southern blot hybridization. Transposon mini-Mu, modified to specify tetracycline resistance, was employed to mutagenize genes for lateral flagella synthesis of Vibrio parahaemolyticus. Mini-Mu contains the lacZ structural gene, and transposition results in transcriptional fusion of Vibrio genes with the transposon lacZ gene. Thus, in these fusions, lacZ expression was proportional to the level of transcription of the target gene. Regulation of lateral flagella gene expression was studied in vivo by measuring beta-galactosidase activity, and conditions which activate transcription of these genes were identified. A method for gene cloning with transposon-induced mutations is discussed.
The luminous bacteria have been isolated from marine environments all over the world, both from seawater and from the light organs of certain fish and squid, and from nonmarine habitats as well. The recent taxonomic studies have placed the luminous bacteria in several major groups: marine forms in the genera Photobacterium, Beneckea and Alteromonas, and nonmarine forms in the genera Vibrio and Xenorhabdus. Among the marine luminous bacteria, Photobacterium species occur as symbionts within specialized light organs of higher organisms; Beneckea species have not been found associated with light organs, but members of both genera occur as gut symbionts. The ecology of the luminous bacteria and its relationship to the control of synthesis and expression of the luminescent system has recently been reviewed in some detail. This chapter discusses the present understanding of the reaction catalyzed by bacterial luciferase and the enzyme
A 50-fold (or greater) increase in the production of phage 21 repressor was obtained by construction of a plasmid in which the 21cl (repressor) gene could be transcribed from λP
L. The enhancement due to increased 21cI gene copy number and transcription from λP
L were at least five-fold and ten-fold, respectively. The plasmid was constructed in vitro by recombination of EcoRI-generated DNA fragments. The use of the DNA fragment containing λP
L in obtaining expression of cloned genes is discussed.
Two copies of the structural gene for the elongation factor EF-Tu have been identified in Escherichia coli: one near rif and the other near str. The latter seems to belong to a single transcriptional unit together with the genes for ribosomal protein S7, S12 (str) and the elongation factor EF-G (fus).
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
A rapid, direct method for screening single plaques of Agt recombinant phage is described. The method allows at least 10(6) clones to be screened per day and simplifies physical containment of recombinants.
Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.
A simple and sensitive radioimmunoassay is described which can detect insoluble membrane proteins in single colonies of Escherichia coli K-12. The method involves transfer of colonies onto filter paper, extraction with organic solvents, exposure to radioiodinated specific immunoglobulin, and autoradiography. Some 30,000 colonies can easily be screened within a week. The method should be applicable for shot-gun-type cloning experiments aiming at genes for insoluble membrane proteins and when selection for a corresponding wild-type allelc is not possible.
DNA coding for the alpha and beta subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the alpha subunit as a hybridization probe, we identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the alpha and beta subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to and released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase alpha and beta subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.
Light is produced by recombinant Escherichia coli that contain lux genes cloned from the marine bacterium Vibrio fischeri.
The bioluminescence phenotype requires genes for regulatory and biochemical functions, the latter encoded by five lux genes
contained in a single operon. These lux genes were disconnected from their native promoter and inserted into the transposon
mini-Mu. The resulting transposon, mini-Mulux, could induce mutations by insertional inactivation of a target gene, and the
lux DNA was oriented to align target gene transcription with that of the lux genes. Genes in Escherichia coli and Vibrio parahaemolyticus
were mutagenized, and mutants containing transposon-generated lux gene fusions produced light as a function of target gene
transcription. Light production offers a simple, sensitive, in vivo indicator of gene expression.
A clone of DNA, obtained from the luminescent bacterium Vibrio fischeri ATCC 7744 and inserted into pBR322, was found to express luminescence in Escherichia coli. Polypeptides involved in biosynthesis of the fatty aldehyde substrate for the light reaction were identified by fatty acid acylation of proteins synthesized in E. coli from the recombinant plasmid. The cloned region was similar to that reported for the V. fischeri MJ1 luminescence system (Engebrecht et al., Cell 32:773-781), except for some differences in endonuclease restriction sites and the requirement of a lower temperature for the expression of light in our cloned system. Fatty acid reductase activity could be detected in extracts of E. coli harboring the recombinant plasmid but not in extracts of the parental V. fischeri strain. Using in vivo labeling with [3H]tetradecanoic acid, we showed that the acylated polypeptides synthesized in the cloned system corresponded to the labeled polypeptides in V. fischeri (34, 42, and 54 kilodaltons) and that they could only be detected after induction of luminescence. These results provide direct evidence that the genes coding for the fatty acid reductase polypeptides are an integral part of the luminescence operon in the V. fischeri luminescence system.
The DNA encoding the luciferase alpha and beta subunits in the luminous marine bacterium Vibrio harveyi (strain 392) is contained within a 4.0-kilobase HindIII fragment. DNA from V. harveyi was digested with HindIII, and the resulting fragments were inserted into the HindIII site of plasmid pBR322. The recombinant plasmids were introduced by transformation into Escherichia coli RR1. The colonies were supplied with n-decanal, the substrate for the bioluminescence reaction, and 12 colonies (of ca. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, has been studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V. harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene. Using antiluciferase antibody to probe proteins transferred from sodium dodecyl sulfate-polyacrylamide gels to nitro-cellulose paper, we have shown that the E. coli transformants produce luciferase that cross-reacts with antiluciferase antibody and is the same molecular weight as V. harveyi luciferase. No alpha subunit could be detected by using antiluciferase antibody in lysates of a subclone, pTB104, which is identical with pTB7 except for deletion of the beta-subunit gene. Thus, the alpha subunit may be unstable and be degraded unless it is associated with beta. The bioluminescence emission spectra of V. harveyi and of E. coli transformants carrying pTB7 are indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)
RNA extracted from the luminescent bacterium Vibrio harveyi was translated in an Escherichia coli system. RNA from highly luminescent cells produced both alpha and beta subunits of luciferase in vitro, as confirmed by immunoprecipitation and partial proteolysis. RNA from cells before induction of luminescence did not direct luciferase synthesis.
A multipurpose cloning site has been introduced into the gene for β-galactosidase (β-D-galactosidegalactohydrolase, EC 3.21.23)
on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375–377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the
lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II
ware removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety
of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined
rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding
the new array of restriction sites.
Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions.
Recombinant E. coli that produce light were found in a clone library of hybrid plasmids containing DNA from the marine bacterium Vibrio fischeri. All luminescent clones had a 16 kb insert that encoded enzymatic activities for the light reaction as well as regulatory functions necessary for expression of the luminescence phenotype (Lux). Mutants generated by transposons Tn5 and mini-Mu were used to define Lux functions and to determine the genetic organization of the lux region. Regulatory and enzymatic functions were assigned to regions of two lux operons. With transcriptional fusions between the lacZ gene or transposon mini-Mu and the target gene, expression of lux operons could be measured in the absence of light production. The direction of transcription of lux operons was deduced from the orientation of mini-Mu insertions in the fusion plasmids. Induction of transcription of one lux operon required a function encoded by that operon (autoregulation). From these and other regulatory relationships, we propose a model for genetic control of light production.
Genes for the luciferase enzyme of Vibrio harveyi were isolated in Escherichia coli by a general method in which nonluminous, transposon insertion mutants were used. Conditions necessary for light production in E. coli were examined. Stimulation of transcription of the genes for luciferase (lux A and lux B) was required for efficient synethesis of luciferase. To enhance transcription bacteriophage promoter elements were coupled to the cloned lux gene fragments.