A . D I S T R I B U T I O N , I S O L A T I O N , A N D C L A S S I F I C A T I O N O F L U M I N O U S B A C T E R I A
Luminous bacteria m a y be classified as parasitic, causing infection of various living animals, such as insects, fresh and salt water shrimp,
amphi-pods, etc; saprophytic, living on such dead m a t t e r as fish or m e a t ; or sym-biotic, those found in the luminous organs of fish or squid. T h e salt water luminous bacteria have been those most frequently studied and are rela-tively easy to isolate. Most will grow on ordinary nutrient agar with 3 % sodium chloride and a carbon source such as glucose or glycerol. There have been, however, a few fresh water forms isolated, and these have been reported to grow on nutrient agar with 0.9 % sodium chloride or none at all. Among the best sources of salt water luminous bacteria are dead fish or squid which have not been washed with fresh water. If such animals are placed in a 15 to 20° C. incubator overnight, one usually observes small luminous colonies developing on the surface of the organism. If one removes a small amount of this material to an agar plate, little difficulty is en-countered in obtaining a pure culture of these forms. F r o m such isolations one can obtain luminous bacteria of long or short rods, cocci or vibrios;
they m a y be quite motile or nonmotile. Probably the two forms which have received the greatest attention are those which are the most confused in classification.
Achromobacter fischeri has been used extensively in t h e laboratory during the past 25 years. I t is a motile rod approximately 0.9 b y 1.8 microns. I t is Gram-negative and requires approximately 2 . 8 % sodium chloride for optimum growth. I t is a nitrate reducer and its polar flagella and biochemi-cal characteristics classify it as a Pseudomonas. T h e temperature for op-timal luminescence is 25° C. T h e organism can be grown only aerobically unlike other closely related species. I t has been isolated from a n u m b e r of places b u t the original was obtained from a dead herring from the sea water at Kiel. T h e official name for Achromobacter fischeri now listed in
"Bergey's M a n u a l of Determinative Bacteriology" is Bacterium phos-phor escens indigenus (Eisenberg). However, most workers in the field have used the earlier and more familiar name.
T h e second species of luminous bacteria which has been studied in great detail in the laboratory is Photobacterium phosphoreum. I t will grow either aerobically or anaerobically. However, luminescence occurs only in the presence of oxygen. T h e temperature optimum for light emission is a p -proximately 15° C. I t is readily isolated from dead fish and meat and from time to time has been given the following names: Micrococcus phosphoreus (Cohn), Bacterium phosphorescens (Fisher), Photobacterium phosphorescens (Beijerinck), Streptococcus phosphoreus (Trevisan), and Bacillus phos-phoreus (Mace).
I t is evident t h a t much confusion exists in the literature on the naming of these various forms. Some of the confusion in classification has been due in part to the fact t h a t a luminous bacterium isolated from different sources has invariably been given new names. For example, luminous bacteria have been isolated from diseased insect larvae and have been given t h e
506 W . D . M C E L R O Y
name Bacterium hemophosphoreum (Pfeiffer and Stammer), from midges—
Bacterium chironomi (Issatschenko), from marine crustaceans—Bacterium giardi (Kruse), from fresh water fish—Bacterium hippanici (Issatschenko), from luminous clams such as Pholas dactylis—Bacterium pholas (DuBois), and from deep sea fish—Coccobacillus collorhynchus. I n addition, there are those interesting luminous bacteria which inhabit special glands in the deep sea fish Physiculus japonicus; these have been named Micrococcus physiculus.
Two fresh water species have been studied extensively, Vibrio albensis and Vibrio phosphorescens. T h e optimum sodium chloride concentration for growth and luminescence is approximately 0.9 %. Both species are Gram-negative and motile. Morphologically they look very much alike, how-ever, W a r r e n ' s4 9 studies on the antigenic properties of these two forms indicate a definite difference. There are other reasonably well-defined species of luminous bacteria and the reader is referred to "Bergey's M a n u a l of Determinative Bacteriology" for this information. However, it is apparent t h a t despite extensive investigations of the cultural characteristics of these various forms the separation of luminous bacterial species is in a rather unsatisfactory state. I t is certain t h a t too much attention has been paid to light emission as a unique and distinguishing characteristic. T h e earlier belief t h a t all luminous bacteria must be closely related taxonomically can no longer be accepted.
B . E V O L U T I O N A R Y S I G N I F I C A N C E O F T H E L I G H T - E M I T T I N G R E A C T I O N
As H a r v e y1 has often emphasized, a glance a t the evolutionary tree will reveal luminous species scattered in about half of the phyla with no a p -parent rhyme or reason. I n the course of evolution ap-parently light pro-duction has appeared again and again, and the origin of this light-emitting process has fascinated a number of workers. T h e ability to produce light does not confer a great survival value on the organisms endowed with it since there are m a n y more nonluminous than luminous forms. Secondarily, however, this ability m a y be adapted to uses which do confer a selective advantage on the luminous organism. I n the case of the firefly the light emission has been restricted to particular organs and the yellow flashing is used for the identification of t h e species to ensure sexual reproduction.
One would hardly question the long-range survival advantage of this unique ability. Luminous bacteria, on the other hand, probably do not obtain any selective advantage under most conditions from their ability to luminesce. R a t h e r their light emission has been regarded as an acci-dental mutation in which the energy liberated b y a terminal flavin oxidase is channeled into an excited state of a molecule which subsequently emits light. Certainly the ability to emit light in a number of organic oxidations
is not unique. I t is very likely t h a t most organisms emit a very weak luminescence. Certainly those luminous bacteria which grow in fish light organs derive an advantage through a symbiotic life.
A mechanism of energy liberation and conservation as phosphate bond energy is reasonably well understood a t the present time. However, as Szent-Gyorgyi5 0 has recently emphasized, t h e actual mechanism of utiliza
tion of this energy is poorly understood. I t m a y be t h a t , in t h e transition of oxidation-reduction reactions in which energy is liberated, a n excited state does appear with a very short half-life; b u t it is this excited state which is concerned with the important processes of muscle contraction and other cellular functions t h a t depend upon energy utilization, rather t h a n energy conservation. If in t h e process of mutation and evolution an organism acquires t h e ability to t r a p these excited states it is possible t h a t luminescence will occur. Only because of the extremely low concentration of these excited states in normal oxidation-reduction reactions, and be
cause of their channeling into other processes in t h e cell, is there a failure to see light emission. Workers have often compared light emission b y or
ganisms to t h e process of light absorption—photosynthesis. I n t h e latter process light q u a n t a are absorbed, exciting t h e chlorophyll molecule, lead
ing eventually to the formation of a reducing as well as a n oxidizing sub
stance. I n this excited state t h e electrons can be passed to a number of acceptors. Light emission is, in essence, a reversal of this process and it is extremely likely t h a t the excited state in t h e case of bioluminescent or
ganisms is drained off in a wasteful side reaction. I n other cases t h e excita
tion state m a y be used in other biosynthetic pathways.
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