Bioluminescence

I. Woodland Hastings

Bioluminescence

!. Introduction Although the emission by living organisms of light that is visible to other organisms is a rather rare occurrence, and in that sense a curiosity of nature, it has several different and fascinating functions. B ioluminescence is also a unique tool for investigating and understanding numerous different basic physiological processes, both cellular and organismic. It allows one to explore in one system a gamut of questions that confront biologists, ranging from gene expression and its regulation to enzymology, bioenergetics, physiology, function, ecology, and evolution (Hastings, 1983). Lastly, luciferases and associated proteins have recently been developed for use as reporters of gene expression. Such proteins may be visualized noninvasively from the same cell in vivo, and over an extended time course, for example, during development (Hastings et al., 1997; Chalfie and Kain, 1997). The phenomenon is not only rare; in the different groups that do emit light, the biochemical and physiological mechanisms responsible for it are very different, as are their specific functional roles. Indeed, bioluminescence is not an evolutionarily conserved function; in the different groups of organisms the genes and proteins involved are mostly unrelated and evidently originated and evolved independently. How many times this may have occurred is difficult to say, but it has been estimated that present-day luminous organisms come from as many as 30 different evolutionarily distinct origins (Harvey, 1952; Herring, 1978; Hastings, 1983; Hastings and Morin, 1991).

11. Physical and Chemical Mechanisms Bioluminescence does not come from or depend on light absorbed by the organism. It derives from an enzymatically catalyzed chemiluminescence, a highly exergonic (energyyielding) reaction in which chemical energy is transformed into light energy (McCapra, in Herring, 1978; Campbell, 1988; Wilson, 1985, 1995; Wilson and Hastings, 1998). Cell Physiology Sourcebook: A Molecular Approach, Third Edition

| | | 5

Thus in the reaction of substance A with substance B, one of the reaction products is formed in an electronically excited state (D*), which then emits a photon (hv). A+B~C+D* D * ~ D + hv Chemiluminescence is a special case of the more general phenomenon of luminescence, in which energy is specifically channeled to a molecule; excited state production is not dependent on the temperature of the molecule. Other kinds of luminescence include fluorescence and phosphorescence, in which the excited state is created by the prior absorption of light, or triboluminescence and piezoluminescence, involving crystal fracture and electric discharge, respectively. The color is a characteristic of the excited molecule, independent of how it was excited. Luminescence is contrasted with incandescence, in which excited states are produced by virtue of the thermal energy. An example is the light bulb, in which a filament is heated, and the color of the light depends on the temperature ("red hot" reflecting a lower temperature than "white hot"). The energy (E) of the photon is related to the color or frequency of the light, and is given by the equation E = hv, where h is Planck's constant and ~, is the frequency. In the visible light range, E is very large in relation to most biochemical reactions. Thus, the energy released by a mole of photons (6.02 x 1023) in visible wavelengths is about 50 kcal, which is much more than the energy from the hydrolysis of a mole of ATPmabout 7 kcal. A visible photon is thus able to do a lot of work (e.g., photosynthesis) or a lot of damage (mutation; photodynamic action, which can kill.) Conversely, it takes a highly exergonic reaction to create a photon. A question of fundamental importance, then, is what kind of chemical process possesses enough energy, and evidently in a single step (an important point), to "populate" an excited state. A clue is the fact that chemiluminescences in solution generally require oxygen, which in its reaction with a substrate, forms an organic peroxide. The energy from the breakdown of such peroxidesmwhich can generate up to Copyright 9 2001 by Academic Press. All rights of reproduction in any form reserved.

1 I 16

SECTION VIII Plant Cells, Photosynthesis, and Bioluminescence

100 kcal per mole--is ample to account for a product in an electronically excited state. A model of the reaction mechanism in such chemiluminescent reactions is referred to as chemically initiated electron exchange luminescence (CIEEL) (Schuster, 1979; Catalani and Wilson, 1989). In this mechanism, peroxide breakdown involves electron transfer with chemiexcitation. It is initiated by an electron transfer from a donor species (D) to an acceptor (A), which is the peroxide bond in this case. After electron transfer, the weak O-O bond cleaves to form products B and C-. The latter is a stronger reductant than A-, so the electron is transferred back to D with the concomitant formation of a single excited state D* and emission. Thus, A+D~A-+D

+ D*---,D+ht,

B+C-

C

The mechanisms of different bioluminescence reactions can possibly be accommodated within this general scheme. A useful way to think of chemiluminescence is to regard it as the reverse of a photochemical reaction, in which the excited state created by the absorption of a photon gives rise to chemical species capable of further reaction. But such species can also react reversibly to repopulate the excited state. Photosynthesis is a good example: the primary chemical states formed in photosynthesis are comparable to C- and D +, which may be the penultimate states in bioluminescence. In the primary step of photosynthesis, the energy of the excited state of chlorophyll (Chl*) gives rise to an electron transfer, with the consequent formation of a primary oxidant and a primary reductant (see Chapter 64). With A as the electron acceptor, these steps can be represented as Chl + ht, ~ Chl* Chl* + A = Chl +. + A-. stable oxidant stable reductant Most of these redox species give rise to stable products and ultimately to CO 2 fixation. However, some species recombine and reemit a "delayed light." This is essentially the reverse of the reaction, with the formation of the singlet excited state of chlorophyll and its subsequent emission of a photon. In bioluminescence, the substrates and enzymes, though chemically different in different organisms, are all referred to as luciferin and luciferase (DeLuca and McElroy, 1981). These are thus genetic terms, and to be correct and specific, each should be identified with the organism (Tables 1 and 2), thus firefly luciferin or bacterial luciferase. The luciferases, as well as the structures and reaction intermediates of the different known luciferins, will be discussed in connection with the individual groups of organisms.

I!I. Luminous Organisms: Abundance, Diversity, and Distribution B ioluminescence is indeed rare as measured by the total number of luminous species, but it is phylogenetically diverse, being found in more than 13 phyla (Herring, in Herring, 1978;

Herring, 1987); only the major ones are considered here (Table 1). These include bacteria, unicellular algae, and fungi, as well as animals ranging from jellyfish, annelids, and mollusks to shrimp, fireflies, echinoderms, and fish. Luminescence is unknown in higher plants and in vertebrates above the fish (Cormier, in Herring, 1978). It is also absent in several invertebrate phyla. In some phyla or taxa, a substantial population of the genera are luminous (e.g., ctenophores,--50%; cephalopods, >50%; echinoderms and annelids, .-4%). Commonly, all members of a luminous genus emit light, but in some cases there are both luminous and nonluminous species. The fact that the enzymes and substrates, as well as the physiological and functional aspects of bioluminescence, differ in the several major taxa is indicative of their independent evolutionary origins. In fact, there are chemically different systems found in different taxa within some phyla, so the total number of evolutionarily independent groups may be 30 or more (Hastings, 1983). Fewer than half of these have been studies in detail, and knowledge of the luciferins and luciferases is available for only about 10. Although luminescence is prevalent in the deep sea (Herring, 1985a), it is not associated especially with organisms that live in total darkness. There are no known luminous species either in deep freshwater bodies, such as Lake Baikal, Russia, or in the darkness of terrestrial caves. There are luminous dipteran larvae (Arachnocampa) that live near the mouths of caves in New Zealand and Australia (Fig. 1), but they also occur in culverts and the undercut banks of streams, where there is considerable daytime illumination. Although insect displays of bioluminescence are among the most spectacular, bioluminescence is relatively rare in the terrestrial environment (<0.2% of all genera). Some other terrestrial luminous forms are millipedes, centipedes, earthworms, and snails, but in none of these is the display very bright. For reasons that are still obscure, bioluminescence is most prevalent in the marine environment (Kelly and Tett, in Herring, 1978); it is greatest at midocean depths (200-1200m), where daytime illumination fluxes range between--10 -1 and 10-12 pW-cm -2. In these locations, bioluminescence in fish may occur in over 95% of the individuals and in 75% of the species; similar percentages are tabulated for shrimp and squid. The midwater luminous fish Cyclothone is considered to be the most abundant vertebrate on the planet. Where high densities of luminous organisms occur, their emissions can exert a significant influence on the communities and may represent an important component in the ecology, behavior, and physiology of these organisms. Above the below midocean depths, luminescence decreases to <10% of all individuals and species. It may be somewhat higher (--20%) at abyssal depths, whereas among coastal species, fewer than 2% are bioluminescent.

IV. Functions of Bioluminescence The functional importance of bioluminescence and its selection in evolution are believed to be based largely on its being detected by another organism; the response of that organism then favors in some way the luminous individual

65. Bioluminescence

TABLE 1

1 1 17

Luminous Organisms: Biochemical Mechanisms and Biological Functions

Type of organism

Representative genera

Luciferins and other factors (Emission max, nm)

Displays and functions

B acteria

Photobacterium Vibrio Xenorhabdus

Reduced ravin and long-chain aldehyde (475-535) Some with accessory emitters

Steady bright glow Autoinduction of luciferase Function as symbionts

Mushrooms

Panus, Armillaria Pleurotus

Unknown (535)

Steady dim glow; function unknown

Dinoflagellates

Gonyaulax Pyrocystis Noctiluca

Linear tetrapyrrole pH change (470)

Short (0.1 s) bright flashes function to frighten or deter

Aequorea Obelia Renilla

Ca2§ coelenterazine Imidazo pyrazine nucleus (460-510), some accessory emitters

Bright flash or train of flashes; function to frighten or deter

Mnemiopsis Beroe

Ca2§ coelenterazine (460)

Bright flashes; function to frighten or deter

Annelids Earthworms Marine polychaetes Syllid fireworm

Diplocardia Chaetoptorus Odontosyllis

N-Isovaleryl-3 amino propanal Unknown Unknown (480)

Cellular exudates, sometimes very bright; function to divert and to deter; others unknown

Mollusks Limpet Clam Squid

Latia Pholas Heteroteuthis

Aldehyde Clam luciferin, but structure is unknown, Cu 2+

Exuded luminescence in all three; photophores and symbiotic bacteria in some squid; diversion, decoy

Vargula Meganyctiphanes

!midazolopyrazine nucleus (465) Linear tetrapyrrole (470)

Squirts enzyme and substrates; diversion, decoy Photophores; camouflage

Benzothiazole, ATE Mg 2+ Similar chemistry in all coleoptera

Flashes, specific kinetic patterns Communication: courtship, mating

Biochemistry unknown

Lure to attract prey

Cnidaria Jellyfish Hydroid Sea pansy Ctenophores

Crustacea Ostracod Shrimp (euphausids) Copepods

Insects Coleopterids (beetles) Firefly Photinus, Photuris Pyrophorus Click beetles Railroad worm Phengodes, Phrixothrix Diptera (flies) Arachnocampa Echinoderms Brittle stars Sea cucumbers

Ophiopsila Laetmogone

Biochemistry unknown Biochemistry unknown

Trains of rapid flashes; frighten, divert predators Unknown

Chordates Tunicates

Pyrosoma

Organelles evolved from bacteria (480-500)

Brilliant trains of flashes stimulated by light and other factors

Squalus

Biochemistry unknown

Unknown

Leiognathus Photoblepharon Cryptopsaras Porichthys

Symbiotic luminous bacteria (--490) Symbiotic luminous bacteria (~490) Symbiotic luminous bacteria (~490) Self luminous, Vargula type luciferin, nutritionally obtained

Camouflage, ventral luminescence To attract and capture prey

Cyclothone Neoscopelus Tarletonbeania

Self-luminous, biochemistry unknown Self-luminous, biochemistry unknown Self-luminous, biochemistry unknown

Many photophores, ventral and lateral Photophores: lateral, on tongue Sexual dimorphism; males have dorsal (police car) photophores

Fish Cartilaginous Bony Ponyfish Flashlight fish Angler fish Midshipman

Camouflage? courtship display?

Midwater fish

1 1 18

SECTION Vlll Plant Cells, Photosynthesis, and Bioluminescence

TABLE 2

Luciferases kDa

E.C. No.

Bacterial

--80 (c~, 41;/3, 39)

1.14.14.3

Dinoflagellate

---135

Coelenterate

--35

Mollusk

1.13.12.5 1.14.99.21

Firefly

--60

1.13.12.7

Crustacean

--68

1.13.12.6

(Buck, in Herring, 1978; Hastings, 1983; Buck, 1938; Herring, 1990). In higher animals luminescence is generally controlled neurally (Anctil, 1987). B ioluminescence is interesting biologically because it is a clear and well-documented example of a function that, while not metabolically essential, confers an advantage on the individual. Although bioluminescence has evidently arisen independently many times, it may also have been lost many times in different evolutionary lines, particularly where it was not a truly important function. Bioluminescence may be thought of as a bag of tricks: the light can be used in different ways and for different functions. Most of the perceived functions of bioluminescence may be classified under three main rubrics: defense, offense, and communication (Table 3). Important defensive strategies associated with bioluminescence are to frighten, to serve as a decoy, to provide camouflage, and to aid in vision. Organisms may be frightened or diverted by flashes, which are typically bright and brief (0.1 s); light is emitted in this way by many organisms, and experimental studies confirm that flashes can indeed frighten (Morin, 1983). On the other hand, a glowing object in the ocean often appears to attract feeders or predators. Although a luminous organism would evidently be at risk by virtue of this attraction, the fact can be used defensively if an organism creates a decoy light to attract the predator, and then slips off under the cover of darkness. This is exactly what quite a number of organisms do. A luminous squid in darkness squirts luminescence instead TABLE 3

FIGURE I. Luminous dipteran larvae (Arachnocampa) on the ceiling of a cave in New Zealand. of ink; ink would be useless in such a case. Some organisms sacrifice more than light; in scaleworms and brittle stars, a part of the body may be automized (broken off) and left behind as a luminescent decoy to attract the predator. In these cases the body part .flashes while still attached but glows after detachment, exemplifying that a flash deters whereas a glow attracts. A unique method for evading predation from below is to camouflage the silhouette by emitting light that matches the

Functions of B i o l u m i n e s c e n c e

Function

Strategy

Method

Defense

Frighten, startle Decoy, diversion Camouflage

Bright, brief flashes Glow, luminous cloud, sacrificial lure Ventral luminescence during the day, disrupting or concealing the silhouette seen from below

Offense

Frighten, startle Lure Vision

Bright flash may temporarily immobilize prey Glow to attract, then capture prey To see and capture prey

Communication

Courtship, mating

Specific flashing signals; patterns of light emission recognized by opposite sex

Dispersal, propagation

Glow to attract feeders

Bacteria ingested by feeder pass through gut tract alive and are therefore dispersed

65. Bioluminescence color and intensity of the downwelling background light. By analogy with countershading in reflected light, this has been called counterillumination. Imagine a plane in the sky during the day. If it could emit light from its bottom surface matching the sky behind, it would be invisible from below. Actually, it is not necessary for the entire surface to emit light; emission by only a part would mean that the object would no longer look like a plane. This can be called disruptive illumination, and many luminous marine organisms, including fish, apparently use this to aid in escaping detection (McFall-Ngai and Morin, 1991). Another novel defensive strategy has been dubbed the burglar alarm: Dinoflagellates flash when grazed upon, which may enhance predation on the grazers, and thus reduce grazing on dinoflagellates (Abrahams and Townsend, 1993). There are also several ways in which luminescence can aid in predation. Several of these, such as helping in vision, may be of value for both offense and defense. For example, flashes, which are more typically used defensively, can be used offensively to temporarily startle or blind prey. A glow can also be used offensively; it can serve as a lure. The organism is attracted to the light but is then captured by the organism that produced the light. Camouflage may also be used offensively, allowing the luminous predator to approach its prey undetected. Vision is certainly useful offensively; prey may be seen and captured under conditions that are otherwise dark, as practiced by the flashlight fish Photoblepharon (Fig. 2). Communication involves information exchange between individual members of a species, and luminescence is used for this in several organisms, including annelids, crustaceans, insects, squid, and fishes. The most common such use of light is for courtship and mating, as in fireflies (Buck and Buck, 1976; Lloyd, 1977, 1980). But there are numerous examples in the ocean (Herring 1990). In the syllid fireworm Odontosyllis a truly extraordinary display occurs as the animals engage in mating, which occurs just post-twilight a few days after the full moon. Readily observed in many parts of the world (e.g., Bermuda), the females come to the surface

1 1 19

FIGURE 3.

Luminousmushrooms.

and swim in a tight circle. A male streaks from below and joins the female, with eggs and sperm shed in the ocean in a luminous circle. Another example occurs over shallow reefs in the Caribbean: male ostracod crustaceans produce complex species-specific trains of secreted luminous materialmladders of lightmwhich attract females (Morin and Cohen, 1991). There remain some luminous organisms for which it is difficult to determine what the function of light emission may be. In the luminous fungi (Wassink, in Herring, 1978) (Fig. 3), both the mycelium and mushrooms emit a continuous light, day and night, but is typically very dim. Moreover, the mycelium itself is almost never exposed: it is underground or inside a decaying tree. The role of luminescence in this and other such cases remains to be understood.

V. B a c t e r i a l L u m i n e s c e n c e A. Occurrence and Functions

FIGURE 2. The flashlight fish (Photoblepharon), showing the light organ (harboring luminous bacteria) just below the eye.

Luminous bacteria (Fig. 4) occur ubiquitously in the oceans and can be isolated from most seawater samples from the surface to depths of 1000 m or more. A primary habitat where most species abound is in association with another (higher) organism, dead or alive, where growth and propagation occur. Planktonic forms are readily isolated, but they do not grow in seawater, as it is a poor growth medium, so they may be viewed as having overflowed from primary habitats (Nealson and Hastings, 1991). The most exotic specific associations involve specialized light organs (e.g., in fish and squid) in which a pure

1120

SECTION VllI Plant Cells, Photosynthesis, and Bioluminescence

F I G U R E 4. Luminous bacteria, photographed by their own light (top) and by room light (bottom). Note the dark mutant near the center.

culture of luminous bacteria is maintained at a high density and at high light intensity. In teleost fishes, some 11 different groups carrying such bacteria are known (Fig. 5). Exactly how symbioses are achievedmthe initial infection, exclusion of contaminants, nutrient supply, restriction of

growth but bright light emissionmis not understood (Hastings et al., 1987). In such associations, the host receives the benefit of the light and may use it for one or more specific purposes; the bacteria in return receive a niche and nutrients. Direct (nonspecific) associations include parasitizations and commensals. Intestinal bacteria in marine animals, notably fish, are often luminous, and heavy pigmentation of the gut tract is often present, presumably to prevent the light from betraying the location of the fish to predators. Indeed, the light emission in these cases is thought to benefit the bacteria more directly, for example, by attracting predators and thereby promoting bacterial dispersion and propagation. Luminous bacteria growing on a substrate, be it a parasitized crustacean, the surface of a dead fish, or a fecal pellet, can produce a light bright enough to attract other organisms, presumably to feed on the material. Attraction to promote dispersal and thus propagation of bacteria may thus be viewed as yet another function of bioluminescence, although, because the luminous organism must survive ingestion, it is probably limited to bacteria. Terrestrial luminous bacteria are rare. The best known are those harbored by nematodes that are parasitic on insects such as caterpillars. The nematode carries the bacteria as symbionts and injects them into the host along with its own fertilized eggs. The bacteria grow and the developing nematode larvae feed on them. The dead but now luminous caterpillar (Fig. 6) attracts predators, which serves to disperse the nematode offspring, along with the bacteria. Luminous bacteria are also examples of organisms that can exploit--or at least survive in---different habitats, for example, in a light organ or free in seawater, in the planktonic environment. This versatility also includes the capacity to turn the luminescent system on and off, at both physiological and genetic levels (Hastings, 1987). Where advantageous, it is expressed at high levels; where not, the genes are repressed and energy is conserved.

B. Biochemistry Luminous bacteria typically emit a continuous light, usually blue-green. When strongly expressed, a single bacterium

LEIOGNATHID

~ stomach ~nt~stirte CERATIOID

OPISTHOPROCTID MERLUCCIID

APOGON ID

MONOCENTRID ;" TRACHIcHTHYID MACROURID ACROPoMATID

FIGURE 5. The "ichthylicht." A diagrammatic fish is used to indicate the approximate locations, sizes, and configurations of the light organs in the several groups of luminous fishes that culture luminous bacteria as a source of light for the organ.

65. Bioluminescence

FIGURE 6. bacteria.

1 121 may emit 104 o r 105 photons s-1. The system is biochemically unique and is diagnostic for a bacterial involvement in the luminescence of a higher organism, as endosymbionts, for example. The pathway itself (Fig. 7) constitutes a shunt of cellular electron transport at the level of ravin, and reduced ravin mononucleotide is the substrate (luciferin) that reacts with oxygen in the presence of bacterial luciferase to produce an intermediate peroxy ravin (Wilson and Hastings, 1998; Meighen and Dunlap, 1993; Baldwin and Ziegler, 1992; Hastings et al., 1985). This intermediate then reacts with a long-chain aldehyde (tetradecanal) to form the acid and the luciferase-bound hydroxy ravin in its excited state. Although there are two substrates in this case, the ravin can claim the name luciferin on etymological grounds, since it forms (bears) the emitter. The bioluminescence quantum yield has been estimated to be about 30%. The enzyme is an external flavin monooxygenase (EC 1.14.14.3). Curiously, no other enzymes of this general type have been found to emit light, even at very low quantum yields. The light-emitting steps have been modeled in terms of an electron exchange mechanism (see Section II), and the experimental evidence is consistent with this. There are enzyme systems that serve to maintain the supply of myristic aldehyde, and genes coding for these enzymes are part of the lux operon (Meighen, 1991; Fig. 8). The luciferases themselves are homologous heterodimeric (a-r) proteins (--80 kDa) in all species. They possess a single active center per dimer, mostly associated with the asubunit. Structurally, they appear to be relatively simple; that is, no metals, disulfide bonds, prosthetic groups, or nonamino acid residues are involved. The luciferase and the mechanism of the bacterial reaction have been studied in great detail. An interesting feature of this luciferase reaction is its inherent slowness: at 20 ~ the time required for a single catalytic cycle is about 20 s.

Luminous caterpillars; caused by parasitic luminous

SUBSTRATE ~

NADH ~ FMN

CYTOCHROMES ~

flavin reductase

ADP +p

02 ATP

FMNH 2

f AMP+PP NADP+ " ~ ~ / ,

,--~- RCHO

O2 luciferase

myds!ic acid reductas~ ATP . ~ NADPH

N

~__ RCOOH

NH

'-/~L-, OH~ ~ J LIGHT + H20 + FMN

FIGURE 7. The luciferase reaction in bacteria. In the electron transport pathway, adenosine triphosphate (ATP) is generated; luciferase shunts electrons at the level of reduced flavin (FMNH2) directly to molecular oxygen. In the mixed function, oxidation with long-chain aldehyde, hydroxy-FMN is produced in its excited state (*) along with long-chain acid. The FMN product is reduced again and recycles, and the aldehyde is regenerated from the acid.

I 122

SECTION VIII Plant Cells, Photosynthesis, and Bioluminescence

Accessory emitter proteins

lump P lux Y

luciferase o~ B

auto induction

nonfluorescent flavoprotein

riboflavin synthesis (?)

C

R

I

D

C

I

I

A I

I

B

(N)

I

E I

G I

H I

J

r

transcription

al eh, e synthe,is and recycling

in

-"

reductase (?)

F I G U R E 8. Organization of the lux genes in Vibrio fischeri. The operon on the right, transcribed from the 5' to the 3' end, carries genes for synthesis of autoinducer (lux I), for luciferase ce and/3 peptides (lux A and B), and for aldehyde production (lux C, D, and E). The operon on the left encodes for a gene (lux R), which encodes for a receptor molecule that binds autoinducer; the complex controls the transcription of the right operon. Other genes, lux F (N), G and H (right), are associated with the operon but with still uncertain functions, genes for accessory emitter proteins also occur (left).

The luciferase peroxy flavin itself has a long lifetime; at low temperatures (0 to - 2 0 ~ it has been isolated, purified, and characterized. It can be further stabilized by aldehyde analogs such as long-chain alcohols and amines, which bind at the aldehyde site.

C. Regulation of Bacterial Luminescence Typically, bacteria are unable to regulate emission on a fast time scale (ms, s), as in organisms that emit fashes. However, bacteria are generally able to control the development and expression of luminescence at both physiological and genetic levels. The most unique of these mechanisms is "autoinduction," in which the transcription of the luciferase and aldehyde synthesis genes of the lux operon is regulated by a gene product of the operon itself. A substance produced by the cells called autoinducer (Fig. 9) is a product of the lux I gene (Nealson and Hastings, 1991). The ecological implications are evident: in planktonic bacteria, a habitat where luminescence has no value, autoinducer cannot accumulate, and no luciferase synthesis occurs. However, in the confines of a light organ, high autoinducer levels are reached and the luciferase genes are transcribed. Interestingly, it has recently been discovered that similar mechanisms control the expression of other specific genes in bacteria, and this has been dubbed quorum sensing (Hastings and Greenberg, 1999; Fuqua et al., 1994). There are a number of other control mechanisms that serve to regulate the transcription of the lux operon, including glucose (catabolic repression), nutrient levels, iron, and oxygen. Each of these factors represents a different mechanism for the physiological control of gene expression, and each has implications concerning the ecology of luminous

0

o

II

II

CH2- CH /

CH3- CH2- CH2-C -CH 2 - C - N H - C H C

J

O

II

o

F I G U R E 9.

Structure of autoinducer from Vibriofischeri.

bacteria and the function of their luminescence. But there is also control at the genetic level: in some species of bacteria there are "dark" (very, very dim) mutants that may arise spontaneously (see Fig. 4). In these the synthesis of the luminescent system fails altogether to occur, irrespective of conditions, and this is an inheritable property. However, the lux genes are not lost and revertants occur. Thus, the organism can compete under conditions where luminescence is not advantageous, yet be able to produce luminous forms and populate the appropriate habitat when and where it is found. Indeed, the bacterial lux genes may occur in many bacterial strains but not be expressed. This means that there may be many more potentially luminous bacteria than would be deduced from the luminescence of colonies on plates.

VI. D i n o f l a g e l l a t e L u m i n e s c e n c e A. Occurrence and Function Dinoflagellates occur ubiquitously in the oceans as planktonic forms and contribute substantially to the so-called "phosphorescence" commonly seen at night (especially in summer) when the water is disturbed. They occur primarily in surface waters and many species are photosynthetic. In the phosphorescent bays (e.g., in Puerto Rico and Jamaica), high densities of a single species (Pyrodinium bahamense) usually occur. The so-called "red tides" are blooms of dinoflagellates. About 6% of all dinoflagellate genera contain luminous species, but since there are no luminous dinoflagellates among the freshwater species, the proportion of luminous forms in the ocean is higher. As a group, dinoflagellates are important as symbionts, notably for contributing photosynthesis and carbon fixation in animals, but unlike bacteria, no luminous dinoflagellates are known from symbiotic niches. Since dinofagellates are stimulated to emit light when predators (e.g., crustacea) are active, predators on the crustacea might be alerted, resulting in a reduced predation on dinoflagellates (Abrahams and Townsend, 1993). Predation on

65. Bioluminescence

1 123

dinoflagellates may also be impeded more directly, since the flash could startle or divert the predator. The response time to stimulation (ms) is certainly fast enough to have this effect.

B. Biochemistry, Cell, and Molecular Biology Luminescence in dinoflagellates is emitted from many small (~0.5 pm) cortical structures, identified as a new type of organelle, termed the seintiilon (flashing unit) (Hastings and Dunlap, in DeLuca and McElroy, 1986). They occur as outpocketings of the cytoplasm into the cell vacuole, like a balloon, with the neck remaining connected (Fig. 10). Scintillons contain only dinoflagellate luciferase and luciferin (with its binding protein), other cytoplasmic components being somehow excluded. The genes for luciferase and luciferin binding protein have been cloned and sequenced; the first ~100 N-terminal amino acids of the two exhibit about 50% identity; neither has sequence similarity to other known proteins. The luciferase is unusual in that after the first ~100 residues it comprises three contiguous intramolecularly conserved domains, each with a catalytic site, thus three active centers in a single molecule (Li et al., 1997; Wilson and Hastings, 1998). Scintillons can be identified by immunolabeling with antibodies raised against the luminescence proteins (Nicolas et al., 1987), and visualized by their bioluminescent flashing after stimulation, as well as by the fluorescence of luciferin (Fig. 11). Dinoflagellate luciferin is a novel tetrapyrrole related to chlorophyll (Fig. 12). Activity can be obtained in extracts made at pH 8 simply by shifting the pH from 8 to 6; it occurs in both soluble and particulate (scintillon) fractions, suggesting that during extraction some scintillons are lysed, whereas others seal off at

cytoplasmic membrane

th//teca/~ '::":. . . . . .

potential

I~:.!=..~:i=.[" triggHrs proton flux

F I G U R E 1 1. A Gonyaulax cell visualized by fluorescence microscopy, showing scintillons by the fluorescence of dinoflagellate luciferin.

the neck and form closed vesicles. With the scintillon fraction, the in vitro activity occurs as a flash (~100 ms), very close to that of the living cell, and the kinetics are independent of the dilution of the suspension. For the soluble fraction, the kinetics are dependent on dilution, as in an enzyme reaction. A distinctive feature is that at pH 8 the luciferin is bound to the luciferin binding protein, which thereby prevents it from reaction with luciferase, but it is free at pH 6 (Fig. 12B). The fashing of dinoflagellates in vivo is postulated to result from a transient pH change in the scintillons, triggered by an action potential in the vacuolar membrane which, while sweeping over the scintillon, opens ion channels that allow protons from the acidic vacuole to enter (see Fig. 10 and Section III).

C. Control of Dinoflagellate Luminescence: The Circadian Clock

FIGURE 10. Scintillons of dinoflagellates represented as organelles formed as cytoplasmic outpocketings hanging in the acidic vacuole.

The composition of the medium and nutrient conditions apparently have little effect on the development and expression of bioluminescence in dinoflagellates. However, in Gonyaulax polyedra and some other dinoflagellates, luminescence is regulated by day-night light-dark cycles and an internal circadian biological clock mechanism (Morse et al.,

1124

SECTION VIII Plant Cells, Photosynthesis, and Bioluminescence lOO

71

.

~

~

CH2H 8o

21

,,i--, om

Me

-~

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~ N H

NaO2C 19 ~~P------NH Me,,,,,,

18~

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NaO2C

B L B P - LH2 ~

H+

LBP + LH 2 ~

02

0, 0

2

4

6

8

10 Days

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18

F I G U R E 13. The circadian rhythm of the steady glow of bioluminescence in Gonyaulax. A culture grown in a 24-hour light-dark cycle was transferred at zero time to constant conditions (19 ~ dim white light); measurements were made about once every hour for 18 days. The average period length was 22.8 hours.

hv + L = O + H20

luciferase (pH 7.5)

~ 4o 20

HN

172

c: 60 .~_

(pH 6)

FIGURE 12. (A) Structure of dinoflagellate luciferin, a tetrapyrrole (fluorescence maximum, -475 nm). (B) Reaction steps for Gonyaulax bioluminescence. Luciferin (LH2) is attached to luciferin binding protein (LBP) at pH 8, but is free at pH 6 and thus able to be oxidized by luciferase; the product has a carbonyl at position 132.

1990). The flashing response to mechanical stimulation is far greater during the night than during the day, and a steady low-level emission (glow) exhibits a peak toward the end of the night phase. The regulation is attributed to an endogenous mechanism; cultures maintained under constant conditions (light, temperature) continue to exhibit rhythmicity for weeks (Fig. 13), but with a period that is not exactly 24 hours; it is only about (circa) one day (diem), thus the origin of the term. The nature of this circadian clock remains one of the real enigmas in physiology. In humans and other higher animals, where it regulates the sleep-wake cycle and many other physiological processes, the mechanism involves the nervous system (Hastings et al., 1991). But it also occurs in plants and unicellular organisms, including Euglena, Chlamydomonas, and Paramecium. In the case of G. polyedra it is known that daily changes occur in the cellular concentrations of luciferase, luciferin, and its binding protein; the proteins are synthesized and destroyed each day. Hence, the biological clock exerts control at a very basic level by controlling gene expression.

ers, are well known. The organisms are mostly sessile or sedentary, and upon stimulation emit light as flashes (Fig. 14A). Bioluminescence is absent in sea anemones and corals. Hydroids occur as plantlike growths, typically adhering to rocks below low tide level in the ocean. When they are touched, a sparkling emission is conducted along the

A

L_J 250 ms

...............

-

|

250 ms

250 ms

D

VII. Coelenterates and Ctenophores A. Occurrence and Function Luminescence is common and widely distributed in these groups (Cormier, in Herring, 1978; Herring, in Herring, 1978). In the ctenophores (comb jellies), luminescent organisms constitute over half of all genera, whereas in the coelenterates (cnidaria) the figure is about 6%. Luminous hydroids, siphonophores, sea pens, and jellyfish, among oth-

!

.....

I

250 ms F I G U R E 14. Bioluminescent flashes. (A) A train of flashed in the hydrozoan Obelia geniculata following a single electrical stimulus. Spontaneous flashes from three species of fireflies: (B) Photinus evanescens (C) P. marginellis, and (D) P versicolor.

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colony; repetitive waves from the origin may occur. Luminous jellyfish (such as Pelagia noctiluca) are well known; the bright flashing comes from photocytes along the edge of the umbrella at the base of the tentacles. Aequorea, a hydromedusan that is very abundant during the summer in the ocean off the northwest United States (San Juan Islands region), has been the material used for much of the research on the biochemistry of the system (Shimomura, 1985). The sea pansy, Renilla, which occurs near shore on sandy bottoms, has also figured importantly in the elucidation of the biochemistry of coelenterate luminescence (Cormier, 1981).

B. Biochemistry, Cell Biology, and the Control of Flashing Photocytes occur as specialized cells located singly or in clusters in the endoderm. They are commonly controlled by epithelial conduction in hydropolyps and siphonophores and by a colonial nerve net in anthozoans. The light may be emitted as one to many flashes per stimulus. The putative neurotransmitter involved in luminescence control in Renilla is adrenaline or a related catecholamine. The luciferin from coelenterates, coelenterazine, possesses an imidazolopyrazine skeleton (Fig. 15A). It is notable for its widespread phylogenetic distribution, but whether the reason is nutritional or genetic (hence, possible evolutionary relatedness) has not yet been elucidated. In some cases (e.g., Renilla), the sulfated form of luciferin may occur as a precursor or storage form and is convertible to active luciferin by sulfate removal with the cofactor 3',5'diphosphadenosine. The active form may also be sequestered by a Ca2+-sensitive binding protein, analogous to

the dinoflagellate binding protein. In this case Ca 2§ triggers the release of luciferin and flashing. Another, more novel type of control of the reaction occurs in other cnidaria (e.g., Aequorea); this involves a luciferase-peroxyluciferin intermediate poised for the completion of the reaction. The photoprotein aequorin (Shimomura, 1985; Charbonneau et al., 1985), isolated from the jellyfish Aequorea (in the presence of EDTA to chelate calcium), emits light simply upon the addition of Ca 2§ which is presumably the trigger in vivo (Blinks et al., 1982; Cormier et al., 1989). This luciferin and the luciferase (EC 1.13.12.5) react with oxygen to form the peroxide in a calcium-free compartment (the photocyte), where it is stored. An action potential allows Ca 2+ to enter and bind to the protein, changing its conformational state and allowing the reaction to continue, but without the need for free oxygen at this stage. An enzyme-bound cyclic peroxide, a dioxetanone, is a postulated intermediate; it breaks down with the formation of an excited emitter, along with a molecule of CO 2. Coelenterate luciferase possesses homology with calmodulin (Lorenz et al., 1991). In several coelenterates, the light emission occurs at a longer wavelength in vivo than in vitro. This is attributed to energy transfer from the excited luciferase-bound emitter to the fluorophore of the accessory green fluorescent protein (GFP), now widely used as a reporter of gene expression (Chalfie and Kain, 1997; Hastings et al., 1997). It had been reported in the early literature that coelenterates could emit bioluminescence without oxygen. The explanation is now evident: the animal contains the luciferase-bound peroxyluciferin (analogous to the bacterial flavin peroxide) in a stored and stable state, and only calcium is needed for the light-emitting step.

O--O

J I OY I

0

O R3

R3

N,~ j N H

I~N~~R2

02

"

.....

=-

+ CO2

Coelenterate luciferin

(Renilla, Aequorea, etc.)

o-

g

oI

o

o-o

o OAMP

.o

S

SI -

Firefly luciferyl adenylate

R - - - ~ ~ 02

s

, + CO2

,-o AMP

Emitter

J

FIGURE 15. (A) The structure of coelenterate luciferin (coelenterazine) and the light-emitting reaction, showing the postulated cyclic peroxide intermediate and excited state in the light-emittingreaction. (B) The structures of firefly luciferin, showing the postulated cyclic peroxide intermediate and excited state.

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VIII. Fireflies A. Occurrence and Function Only about 100 genera of insects are classed as luminous out of a total of approximately 70000 insect genera (Lloyd, in Herring, 1978). But when it occurs, the luminescence is impressive, most notably in the many species of beetles, fireflies, and their relatives. Fireflies themselves possess ventral light organs on posterior segments (Fig. 16). The South American railroad worm, Phrixothrix, has paired green lights on the abdominal segments and red head lights, while the click and fire beetles, Pyrophorini, have both running lights (dorsal) and landing lights (ventral). The dipteran cave glow worm (see Fig. 1) exudes beaded strings of slime from its ceiling perch, serving to entrap minute flying prey, which are attracted by the light emitted by the animal. The variety of different fireflies, with their different habitats and behaviors, is impressive. The major function of light emission in fireflies is for communication during courtship, in which one sex emits a flash as a query, to which the other responds, usually in a species-specific pattern (Case, 1984; Lloyd, 1977, 1980). Some flashing patterns are shown in Figs. 14B-14D. Two signal system types have been distin-

FIGURE 16.

Ventrallight organs of fireflies.

guished. In the first, one sex (usually female) is stationary and emits light or a flashing signal to which the other sex is attracted. In the second, one sex (usually the flying male), emits a species-specific flash, while the other emits a species-specific flash response. The time delay between the two may be a signaling feature; for example, it is 2 s in some North American species. But the flashing pattern (e.g., trains distinctive in duration and/or intensity) is also important in some cases, as is the kinetic character of the individual flash (duration; onset and decay kinetics). In some species, flickering occurs in the flashes, sometimes at frequencies higher than those detectable by the human eye (~40 Hz). In other cases, the communication patterns and their interpretations are not as clear or as readily classified. Fireflies in Southeast Asia are particularly noteworthy in this respect, especially the synchronous flashing of Pteroptyx spp. These fireflies form congregations of many thousands in single trees, where the males produce an all-night-long display, with flashes every 1-4 s, dependent on species (Buck and Buck, 1976). This appears to serve to attract females to the tree.

B. Biochemistry The firefly system was the first in which the biochemistry was well characterized. It had been known since before 1900 that cell-free extracts could continue to emit light for several minutes or hours, and that after the complete decay of the light, emission could be restored by adding a second extract, prepared using boiling water to extract the cells (then cooled). The enzyme luciferase was assumed to be in the first (cold water) extract (with all the luciferin substrate being used up during the emission), whereas since the enzyme was denatured by the hot water extraction, some substrate was left intact. This was referred to as the luciferinluciferase reaction, and it was already known in the first part of this century that luciferins and luciferases from the different major groups would not cross-react, indicative of their independent evolutionary origins (Harvey, 1952). In 1947 it was discovered that the addition of adenosine triphosphate (ATP), a "high-energy" intermediate, to an "exhausted" cold water extract resulted in an enormous bioluminescence response. This response showed that luciferin had not actually been consumed in the cold water extract; ATP could not be the emitter, since it did not have the appropriate fluorescence. For some time, ATP was believed to be providing the energy for light emission. But, as noted already, the energy available from ATP hydrolysis is only about 7 kcal per mole, whereas the energy of a visible photon is 50 kcal or more per mole. It was soon discovered that firefly luciferin, later shown to be a unique benzothiazole (Fig. 15B), was still present in large amounts in the exhausted cold water extract and that it was ATP that was consumed, but available, in the hot water extract. ATP was shown to be required to form the luciferyl adenylate intermediate, which then reacted with oxygen to form a cyclic luciferyl peroxy species, which broke down to yield CO 2 and an excited state of the carbonyl product (McElroy and DeLuca, in Herring, 1978). Luciferase catalyzes both the luciferin activation and the subsequent steps leading to the excited product.

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In reactions in which luminescence has decreased to a low level (this may continue for days), it was long ago found that emission is greatly increased by coenzyme A, but the reason for this was obscure. The discovery that long-chain acylCoA synthetase (EC 6.2.1.3) has homologies with firefly luciferase (EC 1.13.12.7) both explains this observation and indicates the evolutionary origin of the gene. Firefly luciferase has been cloned and expressed in other organisms, including Escherichia coli and tobacco. In both cases, luciferin must be added exogenously; tobacco plants "light up" when the roots are immersed in luciferin (Ow et al., 1986). There are some beetles in which the light from different organs is a different color. The same ATP-dependent luciferase reaction with the same luciferin occurs in the different organs, but the luciferases are slightly different, coded by different (but homologous) genes (Wood et al., 1989). They are presumed to differ with regard to the site that binds the luciferin, which could thereby alter the emission wavelength.

C. Cell Biology and Regulation of Flashing The firefly light organ comprises a series of photocytes arranged in a rosette, positioned radially around a central tracheole, which supplies oxygen to the organ (Ghiradella, 1998). The organ itself comprises a series of such rosettes, stacked side by side in many dorsoventral columns. Photocyte granules or organelles containing luciferase have been identified with peroxisomes on the basis of immunochemical labeling. It is still not known how flashing is controlled in fireflies (Case and Strause, in Herring, 1978). Although flashing is initiated by a nerve impulse that travels to the light organ, most of the nerve terminals in the light organ are not on photocytes but on special tracheolar cells, which control the supply of oxygen. This accounts for the considerable time delay between the arrival of the nerve impulse at the organ and the onset of the flash. The possibility that the flash is somehow directly triggered by an action potential thus seems unlikely. Also, none of the ions typically gated by membrane potential changes (Na +, K +, and Ca 2+) appear to be likely candidates for controlling luminescence chemistry. An alternate theory is that the flash is controlled by the availability of oxygen, which is required in the luminescence reaction. A comparison of different species shows a strong positive relationship between the extent of the tracheal supply system in the adults and the flashing ability. On the other hand, the mechanism must account for the rapid kinetics, complex waveforms, multiple flashes, and high-frequency flickering, all of which seem unlikely to be regulated by a gas in solution. However, although oxygen might diffuse slowly, it reacts very rapidly chemically in this system. The half rise time of luminescence with the anaerobic enzyme system (luciferase-luciferyl adenylate) is about 10 ms. The mechanism is still being actively investigated.

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A. Mollusks Snails (gastropods), clams (bivalves), and cephalopods (squid) have bioluminescent members (Young and Bennett, 1988). The squid luminous systems are by far the most numerous and diverse, in both form and function, rivaling the fishes in these respects. As is also true for fishes, some squid use symbiotic luminous bacteria (Ruby, 1996), but most are self-luminous, indicating that bioluminescence had more than one evolutionary origin within the class. Many squid possess photophores, which may be used in spawning and other interspecific displays (communication). Photophores are compound structures with associated optical elements such as pigment screens, chromatophores, reflectors, lenses, and light guides. They may emit different colors of light and are variously located near the eyeball, on tentacles, on the body integument, or associated with the ink sac or other viscera. In some species, luminescence intensity has been shown to be regulated in response to changes in ambient light, indicative of a camouflage function. Along the coasts of Europe a clam, Pholas dactylus, inhabits compartments that it makes by boring holes into the soft rock. When irritated, these animals produce a bright cellular luminous secretion, squirted out through the siphon as a blue cloud. This animal and its luminescence have been known since Roman times, and the system was used by DuBois in his discovery and description of the luciferinluciferase reaction in the 1880s (see Section VIII). Well ensconced in its rocky enclosure, the animal presumably uses the luminescence to somehow deter or thwart would-be predators. The Pholas reaction has been studied extensively, but the structure of the luciferin, which involves a protein-bound chromophore, remains unknown. The luciferase is a coppercontaining large (>300-kDa) glycoprotein (Henry et al., 1975). It can serve as a peroxidase with several alternative substrates, indicating the involvement of a peroxide in the light-emitting pathway; the superoxide ion is apparently involved in the reaction. There are luminous species in several families of gastropods; a New Zealand pulmonate limpet, Latia neritoides, is notable as the only known luminous eukaryote that can be classed as a truly freshwater species. It also secretes a bright luminous slime (green emission, ~'max = 535 nm), whose function may be similar to that of the Pholas emission. Its luciferin is an enol formate of an aldehyde, but the emitter and products in the reaction are unknown. In addition to its luciferase (M r ~ 170 kDa; EC 1.14.99.21), a "purple protein" (M r ~ 40 kDa) is required, but only in catalytic quantities, suggesting that it may be somehow involved as a recycling emitter.

B. Annelids IX. Other Organisms: Other Chemistries The four systems already described are known best, but several others have been studied in some detail. Several of these are briefly described below.

The annelids also include many luminous species, both marine and terrestrial (Herring, in Herring 1978). Chaetopterus are marine polychaetes that construct and live in U-shaped tubes in sandy bottoms; they also exude luminescence upon stimulation, but the chemistry of the reaction

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has eluded researchers. Other marine polychaetes include the Syllidae, such as the Bermuda fireworm mentioned earlier, and the polynoid worms, which shed their luminous scales as decoys. Extracts of the latter have been shown to emit light upon the addition of superoxide ion. More but still limited knowledge is available concerning the biochemistry of the reaction in terrestrial earthworms, some of which are quite large, over 60 cm in length (Wampler in DeLuca and McElroy, 1981). Upon stimulation they exude coelomic fluid from the mouth, anus, and body pores. This exudate contains cells that lyse to produce a luminous mucus, emitting in the bluegreen region. However, the exudate from animals deprived of oxygen does not emit, but will do so after the admission of molecular oxygen to the free exudate. In Diplocardia longa the cells responsible for emission have been isolated; luminescence in extracts involves a coppercontaining luciferase M r -~ 300 kDa), and the luciferin (Nisovaleryl-3-amino-l-propanal). The in vitro reaction requires H202, not free O 2. C. Crustaceans

Many crustaceans are luminescent (Herring, 1985b). The cypridinid ostracods such as Vargula (formerly Cypridina) hilgendorfii are small organisms that possess two glands with nozzles from which the luciferin and luciferase (EC 1.13.12.6) are squirted into the seawater, where they react and produce a spot of light, useful either as a decoy or for communication (Morin and Cohen, 1991). Cypridinid luciferin and its reaction have differences and similarities to those of the coelenterazine system (Cormier, in Herring, 1978). The luciferin in both is a substituted imidazopyrazine nucleus that reacts with oxygen to form an intermediate cyclic peroxide, which then breaks down to yield CO 2 and an excited carbonyl. However, the cypridinid luciferase gene has been cloned and appears to have no homologies with the gene for the corresponding coelenterate proteins, and calcium is not involved in the cypridinid reaction. The two different luciferases reacting with similar luciferins have apparently had independent evolutionary origins, indicative of convergent evolution at the molecular level. Euphausiid shrimp possess compound photophores with accessory optical structures and emit a blue, ventrally directed luminescence. The system is unusual because both luciferase and luciferin cross-react with the dinoflagellate system. This cross-taxon similarity indicates another possible exception to the rule that luminescence in distantly related groups had independent evolutionary origins. The shrimp might obtain luciferin nutritionally, but the explanation for the occurrence of functionally similar proteins is not evident. One possibility is lateral gene transfer; convergent evolution is another. Analyses of gene structures for homologies should provide insight into this question. D. Fish

Bioluminescence in fish is highly diverse and occurs in both teleost (bony) and elasmobranch (cartilaginous) fish. Partly because animals have not been so readily available,

SECTION VIII Plant Cells, Photosynthesis, and Bioluminescence

relatively little is known about their physiology and biochemistry, but many have been described (Herring, 1982). As noted in the section on bacteria, many fish obtain their light-emitting ability by culturing luminous bacteria in special organs (see Figs. 2 and 5), but most are self-luminous. Self-luminous species include Porichthys, the midshipman fish, so-called because of the array of its photophores distributed linearly along the four pairs of lateral lines, much as are the buttons on a military uniform. Because it occurs close to shore, it has been the object of considerable study, and more is known about the physiological control of luminescence in Porichthys than in any other fish. Biochemically it is less well characterized, but its luciferin and luciferase cross-react with the cypridinid ostracod crustacean system already described. This was an enigma until it was discovered that Puget Sound fish have photophores but are nonluminous; however, they can emit if injected with cypridinid luciferin or fed the animals, showing that luciferin may be obtained nutritionally. Did the luciferase in this fish originate independently to make use of the available substrate, or was the ability to synthesize luciferin lost secondarily from a complete system? If the latter, this would be analogous to the loss of the ability to synthesize vitamins in mammals. Open sea and midwater species include the sharks (elasmobranchs), some of which may have several thousand photophores. The teleosts include the gonostomatids such as Cyclothone, with simple photophores, and the hatchet fish, having compound photophores with elaborate optical accessories; emission is directed exclusively downward, indicative of a camouflage function of the light. Fish often possess different kinds of photophores located on different parts of the body, particularly ventrally and around the eyes, evidently with different functions. One interesting arrangement, known in both midwater squid and myctophids, makes use of a special photophore positioned to shine on the eye or a special photoreceptor. Its intensity parallels that of the other photophores, so it provides information to the animal concerning its own brightness, thus allowing it to match the intensity of its own counterillumination to that of the downwelling ambient light. Another clear case of functional use is in Neoscopelus; in addition to the many photophores on the skin, they also occur on the tongue, allowing it to attract prey to just the right location. Sexual dimorphism is also frequent in fish luminescence. Males and females of the myctophid Tarletonbeania were originally thought to be different species. Only one (now known to be the male) has caudal luminous organs, and the occurrence of these fish was known only from stomach contents of predator fish; only the female could be captured in nets, but never together with a male. The apparent explanation is that when a predator attacks, the males dart off in all directions with their dorsal lights flashing, like a police car, leading the predators on a wild chase (and sometimes getting caught), but leaving the females, who remain in place, safe from the predator in the cover of darkness, yet easy to catch in a net. A number of self-luminous fish eject luminous material; in the searsid fishes this is cellular in nature, but it is not bacterial, and its biochemical nature is not known. Such animals may also possess photophores.

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X. Applications of Bioluminescence Instrumentation for measuring light emission is very sensitive and free of background and interference characteristic of many other analytical techniques (Wamplar, in Herring, 1978; Kricka and Whitehead, 1984). A typical photon counting instrument can readily detect an emission of about 104 photons s-1, which corresponds to the transformation of 105 molecules s-1 (or 6 x 106 min-1) if the quantum yield is 10%. (Those bioluminescent reactions that have been measured have quantum yields of this or higher; the firefly yield is about 90%.) A substance at a concentration of 10-9 M could readily be detected in a single cell 10 lam in diameter by this technique. For these and other reasons, luminescence has come into widespread use (Roda et al., 1999; Hastings et al., 1997). Luminescent tags have been developed that replace radioactivity with as high a sensitivity. Since the different luciferase systems have different specific chemistries, quantitative determinations of many different specific substances can be accomplished. One of the first and still widely used assays uses firefly luciferase to detect ATP. Many different enzymes use or produce ATP, so their activities may be followed using this assay. With bacterial luciferase, any reaction that produces or utilizes NAD(H), NADP(H), or long-chain aldehyde, either directly or indirectly, can be coupled to this light-emitting reaction. A photoprotein from the scaleworm has been used to detect superoxide ion; the purified photoprotein aequorin is widely used to detect intracellular Ca 2+ and its changes under various experimental conditions (e.g., during muscle contraction). The protein is relatively small, nontoxic, and it may be readily injected into cells, reporting Ca 2+ over the range of 3 x 10-7 to 10-4 M. The luminescence of bacteria has also been used as a very sensitive test for oxygen, making use of the fact that the K m for 0 2 in that reaction is extremely low. An oxygen electrode-incorporating luminous bacteria has been developed recently. Luciferase genes have also been exploited as reporters for many different specific genes. Such systems are noninvasive and nondestructive, and the relevant activity can be measured in the living cell and in the same cell over the course of time. Recent studies of circadian rhythms have made use of the expression of the bacterial lux gene for the study of rhythms in cyanobacteria (Kondo et al., 1993), the firefly luc gene for rhythms in higher plants (Millar et al., 1995), and the aequorin gene for tracking intracellular calcium rhythms in Arabidopsis (Johnson et al., 1995). As indicated earlier, the gfp gene is in wide use in many different types of applications (Chalfie and Kain, 1997). These many diverse applications illustrate the fact that studies of bioluminescence, sometimes thought to be of little importance, have contributed to knowledge in ways not foreseen at the time the investigations were being made. Basic knowledge is a powerful tool in more than one respect.

XI. Summary The emission of visible light by living organisms is an unusual phenomenon, both in terms of its relative rarity and

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with respect to the biochemical and regulatory mechanisms involved. But where it does occur, bioluminescence is sometimes spectacular and can usually be inferred to have functional importance---a consequence of the fact that another organism detects and responds to the light. The uses of the light may be classified under three headings: defense, offense, and communication. Light may be used defensively to startle or frighten (flashes), to divert predators, as a decoy, or to provide camouflage. Offensively, light may be used as a lure, to attract and convert would-be predators into prey. Communication occurs in courtship and mating displays. Biologically, the single most striking aspect of bioluminescence is its wide and diverse phylogenetic distribution and the independent evolutionary origin of different systems. The ability to emit light occurs in some 13 different phyla, and apparently originated many times, perhaps 30 or more. This is reflected not only in the gene and protein structures, but also in its biological, biochemical, and functional diversity, as well as its sporadic phylogenetic distribution. Another unusual and unexplained fact is that bioluminescence is primarily a marine phenomenon. Although there are terrestrial forms, it is virtually absent in fresh water; only one such species is known. It is also not confined to or especially prevalent in animals that live in complete darkness (caves, the deep ocean). B ioluminescence is an enzymatically catalyzed chemiluminescence, a chemical reaction that emits light. The enzymes involved are all referred to generically as luciferases, somewhat unfortunately, because they are not conserved evolutionarily, and are thus structurally different in different groups of organisms. The genes coding for several of the luciferases have been cloned and sequenced, confirming that they possess no homologous regions in common. The substrates, generally called luciferins, react with molecular oxygen to form intermediate luciferase-bound peroxides, which break down to give a product in an excited state, which subsequently emits light. In the marine environment, luminous bacteria are ubiquitous as planktonic forms in seawater, and they are also responsible for the light emission of many species of higher organisms, usually as symbionts. Terrestrial forms are not common, but do occur as symbionts in nematodes, as an agent in the nematode's parasitization of insects. All species use the same biochemical system, a shunt of the electron transport pathway, in which reduced flavin and aldehyde are oxidized by molecular oxygen to give a luciferase-bound flavin intermediate in an excited state. Luminous species are versatile with respect to the alternate uses of light emission in different situations, the exploitation of alternate habitats, and the capacity to turn the synthesis of the system on and off at both the physiological and genetic levels. The genes involved may be widely distributed and mobile. Dinoflagellates are unicellular algae; in the ocean these organisms are largely responsible for the sometimes brilliant sparkling "phosphorescence" seen at night when the water is disturbed, and also for "red tides." Their luminescent flashing originates from novel cellular organelles called scintilIons, formed as spherical outpocketings of the cytoplasm into the vacuole. They contain dinoflagellate luciferase and

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SECTION VIII Plant Cells, Photosynthesis, and Bioluminescence

luciferin (a novel reduced linear tetrapyrrole), the latter bound to a second protein. Flashing is triggered by an action potential in the vacuolar membrane that causes a transient pH change in the scintillon, releasing the luciferin from its binding protein. Luminescence in some dinoflagellates is controlled by a cellular circadian biological clock, which causes the synthesis and destruction of the components to occur on a daily cycle. There are many luminous comb jellies, hydroids, siphonophores, sea pens, and jellyfish. Upon stimulation, light is generally emitted as brief bright flashes or trains of flashes. The luminescence originates in specialized cells called photocytes, triggered by a conducted epithelial or nerve action potential. The luciferin is a substituted imidazole called coelenterazine, which, the name notwithstanding, occurs in several other phyla; its oxidation results in light emission. The reaction, catalyzed by coelenterate luciferase, is regulated by Ca 2+. This is a case where the evolutionary origin of the luciferase is known; it bears homology with calmodulin. Fireflies typically emit light as flashes, which are used as species-specific signals for communication in courtship. The light organ is a complex structure with photocytes arranged in a rosette pattern, invested with tracheoles to transport the required molecular oxygen directly to the cells. It may be that oxygen ultimately regulates flashing, though a nerve impulse initiates the process. The firefly reaction is unique in having a requirement for ATP, which serves to "activate" the luciferin (a benzothiazole). The luciferyl adenylate is thus the "true" substrate that reacts with oxygen, forming an intermediate cyclic peroxide whose breakdown results in light emission. Firefly luciferase also shows homologies with another protein, namely, long-chain acylCoA synthetase. Other major luminous groups include the mollusks (snails, clams, squid), annelid worms (both marine and terrestrial), crustacea (shrimp and ostracods), echinoderms (brittle stars, starfish, sea cucumbers), and fish, both cartilaginous (sharks) and teleost (bony fishes). Of all the groups, fish and squids have the greatest variety of luminous systems; some make use of symbiotic luminous bacteria as a source of light, whereas others are self-luminous. Luminous organisms are most abundant at midwater depths (500-1000 m) in the open ocean.

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