Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae.

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.

Introduction to beetle luciferases and their applications.

All beetle luciferases have evolved from a common ancestor: they all use ATP, O2, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.

Bioluminescent click beetles revisited.

In studying beetle bioluminescence in the early 1960s, Dr. McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light. They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases. We have recently cloned cDNAs from this beetle species which code for at least four different luciferases. The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli. The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few. Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.

Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors.

Eleven complementary DNA (cDNA) clones were generated from messenger RNA isolated from abdominal light organs of the bioluminescent click beetle, Pyrophorus plagiophthalamus. When expressed in Escherichia coli, these clones can elicit bioluminescence that is readily visible. The clones code for luciferases of four types, distinguished by the colors of bioluminescence they catalyze: green (546 nanometers), yellow-green (560 nanometers), yellow (578 nanometers), and orange (593 nanometers). The amino acid sequences of the different luciferases are 95 to 99 percent identical with each other, but are only 48 percent identical with the sequence of firefly luciferase (Photinus pyralis). Because of the different colors, these clones may be useful in experiments in which multiple reporter genes are needed.

Two kinetically distinguishable ATP sites in firefly luciferase.

Results are presented which indicate that firefly luciferase has two catalytically active sites. One site, Km of 1.1 X 10(-4) M ATP, is responsible for the initial flash and is apparently product inhibited for further light production. The second site, Km of 2 X 10(-5) M ATP, catalyzes the continuous low production of light. ATP or AMP is a potent inhibitor of the initial flash when LH2-AMP is used to initiate the light reaction but appears to have no affect on the second site low level light emission. Both sites must be occupied by ATP for the formation of one L-AMP. Thus, ATP appears to function both as a catalytically active substrate and a regulator for light emission.

Firefly and bacterial luminescence: basic science and applications.

The basic chemistry of the reactions leading to light emission in the firefly and in bacteria are briefly reviewed. With excess firefly reagents, the light intensity is proportional to the ATP concentration. For this reason, the reagents have been used for ATP determination in a number of important biological systems. A number of such applications are reviewed. With excess bacterial reagents, the light intensity is directly proportional to the reduced pyridine nucleotide concentration (NADH or NADPH). The applications of this system for studying reactions involving dehydrogenases using NAD or NADP as electron acceptors are presented. Many assays have now been developed using enzymes immobilized on Sepharose. The advantages of using the immobilized enzymes are greater stability of the immobilized enzymes over the soluble forms; increased sensitivity of detection relative to the soluble forms, and reusability of the immobilized enzymes. A comparison of the immobilized bioluminescent assay for 7 alpha-hydroxysteroid with gas-liquid chromatography and radioimmunoassay is presented. Coimmobilized enzymes can be packed in a flow cell and used in an automated instrument with good reproducibility. It is likely that future developments of bioluminescent assays for ATP or NAD(P)H will be with immobilized enzymes using an automated instrument.

The Role of 1N-Ethenoadenosine Triphosphate and 1N-Ethenoadenosine Monophosphate in Firefly Luminescence.

1,N(6)-Ethenoadenosine triphosphate (epsilon-ATP) has been found to be inactive as a substrate for firefly luciferase. However, chemically synthesized luciferyl-epsilonAMP is oxidized by luciferase and light is emitted. The color of the light is red in contrast to the normal yellow-green observed with luciferyladenylate. Although epsilonATP will not serve as a substrate in the activation reaction, the reversal of the activation, pyrophosphorolysis of dehydroluciferyladenylate, will occur with the ethenoadenosine analog.