Activity and subunit functions of immobilized bacterial luciferase

Immobilized luciferase was studied with regard to its reactivity and subunit functions. When immobilized on a matrix (Sepharose 6B), neither the alpha nor the beta subunit alone exhibited luciferase activity. However, for both subunits (so attached), denaturation followed by renaturation in the presence of the second subunit resulted in the recovery of activity on the matrix. It was thus confirmed that both of the two different subunits (alpha and beta) are required for luciferase activity, even after immobilization. Recovery of activity was approximately the same or slightly less with alpha-immobilized luciferase compared with the beta-immobilized enzyme under our experimental conditions. Generally, immobilized luciferase exhibited both a lower FMNH₂ binding affinity and maximum light emission activity in comparison with free native luciferase, but surprisingly, it exhibited no change in the rate constant for the luminescence, this being a measure of the catalytic turnover time. The alpha-subunit-immobilized (renatured with beta) luciferase possessed a lower FMNH₂ binding affinity compared with beta-subunit-immobilized (renatured with alpha) luciferase. Since the protein attachment to the CNBr-activated Sepharose 6B occurs by way of an amino group of luciferase, it was suggested that the binding of FMNH₂ on luciferase, but not the subsequent catalytic steps, is dependent upon some exposed amino groups on both alpha and beta subunits.     

Bovine serum albumin interacts with bacterial luciferase

Bovine serum albumin (BSA) affects the amount of light obtained from bacterial luciferase by competing with luciferase for one of the luciferase substrates, the aldehyde. At low aldehyde concentrations BSA behaves as an inhibitor, but at high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal with a K_si = 3.36 μmol/l, approximately half the affinity of luciferase for decanal (K_M = 1.5 μmol/l). BSA also increased the rate of intermediate II dark decay. The data suggest that this involves a direct protein-protein (BSA-luciferase) interaction.     

Thermostability of bacterial luciferase expressed in different microbes

Bacterial luciferase was used to investigate the relationship between the thermostability of a cytoplasmic reporter molecule and cellular heat resistance. The luciferase activity of Vibrio fischeri was expressed in strains of Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Brochothrix thermosphacta following transformation with plasmid pSP13 carrying the luxAB genes. The thermostability of intracellular luciferase varied depending on the organism in which it was expressed, but was not related to the cellular heat resistance of the different organisms. Addition of xylitol to the heating medium protected against loss of viability and inactivation of intracellular luciferase. Glycerol also protected against loss of viability but was less effective at preventing thermal denaturation of luciferase.     

Intermediates in the bacterial luciferase reaction

Rapid formation of an unstable, intermediary state of an enzyme complex, which is obligatory in the bacterial luciferase reaction, was observed on aerobic oxidation of the luciferase-FMNH₂ complex. The rate of decay of this intermediate in the absence of aldehyde was measured. The value obtained coincided with that estimated from the decay of the peak intensity of luminescence observed on addition of aldehyde at intervals after mixing the luciferase-FMNH₂ complex with O₂. A sequential mechanism of the reaction of bacterial luciferase is discussed.     

Factors affecting the cellular expression of bacterial luciferase

The in vivo expression of cellular bacterial luciferase has been defined as the luciferase expression quotient, measured as the ratio of the bioluminescence intensity in vivo to the in vitro activity of luciferase in crude cell extracts. The expression is greater in the presence of inhibitors of the electron transport system such as cyanide and N-heptyl-4-hydroxyquinoline and also at lower oxygen tensions. The higher expression of the cellular luciferase under these conditions is postulated to be due to an increase in the intracellular levels of reduced coenzymes which enhance both the reduction of flavin and the reduction of fatty acid to aldehyde. Both FMNH₂ and aldehyde are substrates in the light emitting reaction.     

Alternative luciferase for monitoring bacterial cells under adverse conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Structure of bacterial luciferase

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.     

The isolation of a bacterial glycoprotein with luciferase activity

Luciferase from the luminescent bacteria Photobacterium leiognathi, strain S-1, has been isolated and purified from lysed cells and shown to be a glycoprotein. A soluble, non-carbohydrate containing luciferase was also isolated from the lysate. This latter luciferase amounts to less than 20% of the total luciferase activity. The purified glycoprotein is (1) susceptible to inactivation by lysozyme, (2) gives positive color reactions to the phenol, resorcinol, and anthrone tests for sugars, and (3) gives overlapping bands when stained for carbohydrate and protein on gel electrophoresis. Neither the soluble luciferase from S-1 nor a soluble luciferase isolated from Beneckea harveyi, mutant MB-20, give positive results to any of these carbohydrate tests.     

A cyanide-aldehyde complex inhibits bacterial luciferase.

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.     

Separation of bacterial luciferase from oxidoreductases by affinity chromatography

The NADH-dependent and NADPH-dependent oxidoreductase activities associated with bacterial luciferase in Vibrio (Beneckea) harveyi can simultaneously be removed from purified luciferase through a Blue Sepharose CL-6B column. The result achieved with this one-step affinity chromatography is similar to that obtained with two sequential "reverse-affinity" chromatographies.     

Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase.

Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.     

Single bacterial cell detection using a mutant luciferase

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10⁻¹⁸ mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.     

Action of phenobarbital on bacterial luciferase of Photobacterium fischeri

The effect of phenobarbital, a typical substrate of monooxygenases from higher organisms, on bioluminescence of the marine bacterium Photobacterium fischeri and bacterial luciferase was studied. Phenobarbital was shown to be an effective quenching agent owing to the interaction with cytochrome P-450, a terminal luciferase component. A competitive interrelation was found between phenobarbital and an aliphatic aldehyde, the substrate of the luminescent reaction.     

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.     

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.     

Controlled expression of click beetle luciferase using a bacterial operator-repressor system

Abstract The bioluminescent phenotype conferred by luciferase genes in a particular bacterium has demonstrated to be one of the most versatile and useful methods to detect microorganisms. Genetic constructions derived from miniTn5 vectors have been constructed for the introduction and stable maintenance of the click beetle luciferase gene, lucOR , in various Gram-negative bacteria. To attenuate the expression in the environment where the marked strain has to survive (and to allow sensitive detection when desired) a DNA fragment containing the repressor gene lacI ^q and a P _trc:: lucOR fusion was cloned onto a suicide plasmid. This construction is able to express high luciferase levels only when induced by IPTG. Matings between Escherichia coli containing the suicide transposoon vector and different recipient bacteria gave transposition frequencies from 10⁻⁷ to 10⁻⁵. Strains with miniTn5- lucOR insertions showed luciferase activity induced by IPTG addition. The stringency of the regulation and the intensity of light emission depended on the tagged strain. This system allows stable maintenance of the marker and tight control of luciferase expression in the environment.     

Activity coupling and complex formation between bacterial luciferase and flavin reductases.

Luminous bacteria contain several species of flavin reductases, which catalyze the reduction of FMN using NADH and/or NADPH as a reductant. The reduced FMN (i.e. FMNH(2)) so generated is utilized along with a long-chain aliphatic aldehyde and molecular oxygen by luciferase as substrates for the bioluminescence reaction. In this report, the general properties of luciferases and reductases from luminous bacteria are briefly summarized. Earlier and more recent studies demonstrating the direct transfer of FMNH(2) from reductases to luciferase are surveyed. Using reductases and luciferases from Vibrio harveyi and Vibrio fischeri, two mechanisms were uncovered for the direct transfer of reduced flavin cofactor and reduced flavin product of reductase to luciferase. A complex of an NADPH-specific reductase (FRP(Vh)) and luciferase from V. harveyi has been detected in vitro and in vivo. Both constituent enzymes in such a complex are catalytically active. The reduction of FRP(Vh)-bound FMN cofactor by NADPH is reversible, allowing the cellular contents of NADP(+) and NADPH as a factor for the regulation of the production of FMNH(2) by FRP(Vh) for luciferase bioluminescence. Other regulations of the activity coupling between reductase and luciferase are also discussed.     

Mechanism of inhibition of bacterial luciferase by anaesthetics.

The effects of volatile anaesthetics on bacterial luciferase were studied $\text{in vitro}$. It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% $\text{in vivo}$ and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.     

Photokinetic microanalysis of NADP+, using bacterial luciferase

Summary Bioluminescence photokinetic assay of NADP⁺ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. The analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP⁺ as measurable product and for nucleotide assay in cell samples of 0.5–10 g dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP⁺.Previously, FMN, NADH, NAD⁺ and NADH have been analysed with the bacterial luciferase system. Its applicability has now been extended by the assay of NADP⁺.