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Summary Superoxide dismutases have been identified in a number of marine bioluminescent bacteria. Except for one case, all of these bacteria contain a ferri protein enzyme which belongs to one of two classes characteristic of symbiont or of free living bacteria. The ferri-superoxide dismutases from P. leiognathi (symbiont) and from P. sepia (free living) have been purified and characterised and a number of properties defined. Although the two enzymes are closely similar, various differences exist. A second coppercontaining superoxide dismutase is present in P. leiognathi but is less sensitive to increase in partial pressure of oxygen than is the ferri-enzyme, the level of which is strongly stimulated by increase in pO 2 . Inhibition of the autoxidation of ascorbic acid and of pyrogallol by superoxide dismutase demonstrates the role of superoxide radicals in these autoxidations.

A newly developed procedure is described for the isolation and purification on a large scale of luciferases from two different bioluminescent bacteria, Pf and MAV. In only three major steps luciferase can be obtained in greater than 95% purity. An improved method for the isolation of subunits in milligram quantities by column chromatography in 5 m urea is also described; upon recombination of the subunits with the removal of urea, renatured luciferase with a high specific activity is obtained. These procedures should be generally applicable with luciferases from different bacterial strains, including mutant luciferases. The two luciferases have been used to produce specific antiluciferases, which, by precipitation patterns on Ouchterlony plates, appear distinctively different. Nevertheless, a degree of homology has been demonstrated by cross-reaction in an antibody activity inhibition assay. Interaction between subunits from the different strains has also been demonstrated by inhibition of renaturation (from 5 m urea) of one of the luciferases by a subunit of the other luciferase, indicating that inactive hybrids may be formed.

Summary An oxidoreductase which catalyses the reduction of flavin mononucleotide has been isolated from various strains of bioluminescent bacteria. This enzyme has a molecular weight of 23,500 and is involved in the bacterial luminescent system. However, non-luminescent bacteria also contain reductases with very similar properties which function equally well in the luciferase system with emission of light. Purification and characteristics of the reductases are described and a mechanism for bacterial luminescence proposed in which the role of reduced flavin mononucleotide is that of an oxidant (in the presence of oxygen).

Mutants of a marine luminous bacterium, in which the ability to emit light is conditional upon temperature, have been isolated. The mutants obtained fall into three classes, which are readily distinguishable by both in vivo and in vitro criteria. In one class an altered, more temperature-sensitive luciferase is produced; in a second, the luciferase is actually not produced at the higher temperature. The third is defective in the production of an aldehyde-like factor that is a known requirement in the in vitro reaction.

In bioluminescent bacteria growing in shake flasks, the enzyme luciferase has been shown to be synthesized in a relatively short burst during the period of exponential growth. The luciferase gene appears to be completely inactive in a freshly inoculated culture; the pulse of preferential luciferase synthesis which occurs later is the consequence of its activation at the level of deoxyribonucleic acid transcription which is attributed to an effect of a “conditioning” of the medium by the growing of cells. Although cells grown in a minimal medium also exhibit a similar burst of synthesis of the luminescent system, the amount of synthesis is quantitatively less, relative to cell mass. Under such conditions, added arginine results in a striking stimulation of bioluminescence. This is attributed to a stimulation of existing patterns of synthesis and not to induction or derepression per se.

The Leiognathid (pony) fish, which occur in the shallow coastal waters of the tropical and subtropical Indo-Pacific region, are capable of emitting a bright light from their ventral surface. In these small fish, as in several other (but not all) bioluminescent fish, the source of light is symbiotic luminous bacteria, maintained within a special organ (Harvey, 1952, 1958 ; Buchner, 1965). The evidence that bacteria are involved as symbionts has come from microscopic observations to gether with the fact that cultures of luminous bacteria have been obtained from the organs (Harms, 1928 ; Haneda, 1940, 1950) . In the present experiments addi tionai proof of the bacterial origin of the light is presented, together with evidence that the symbiotic bacteria are distinct from many of the free living luminescent bacteria which may be isolated directly from sea water in the same area. In different grOU@)5of fish there are very different and sometimes highly elabo rate types of organs and modes for display of the bacterial light. In pony fish the system involves several special and unusual elements. The organ itself, which surrounds the esopilagus like a donut, and communicates with it †? zA±a paired ducts (Haneda, 1940, 1950) is literally packed with bacteria. Upon dissection it is always fotlnd to be emitting light, brightly and continuously, irrespective of the tinle of day or other environmental factors. The light reaches the outside (ventral) surface via indirect and somewhat sophisticated optics. The gut tract makes a loop into the wall of the swim (air) bladder at the site of the light organ. Tile organ thus faces directly into the swim bladder, and is provided with an eyelid-like flap which can control the amount of light shining into the air-filled bladder. The swim bladder is internally reflecting, being lined with guanine crystals—the same material which is responsible for tue silvery skin of many fish (Denton, 1970, 1971). The ventral portion of the swim bladder is only partially reflective (“half silvered―), and to it attach specialized translucent ienticular muscle cells. The optical arrangement thus takes the light from a small source and causes it to be evenly diffused over a larger area, namely most of the ventral part of the body. As shown below, it was possible to estimate the total number of viable bacteria within an organ, and to compare this with the weight of the organ. Such counts indicate that a large percentage, if not all, of the bacteria which are crammed into the ducts of the organ are both viable and bioluminescent. The isolated symbiotic bacteria were compared with free living bacteria isolated directly from sea water in the same area where the fish were collected. Although tilebacteriafrom the two sourceshad similarcolonialmorphology, and were the same in certain other special respects, tile two appeared to possess distinctly differ ent types of luciferases, corresponding to types previously known from different strains (Hastings, Weber, Friedland, Eberhard, Mitchell and Gunsalus, 1969), hut not previouslycorrelatedwith symbioticand freelivinglifestyles.

Luciferase synthesis is repressed when bioluminescent bacteria are inoculated into fresh medium but is induced after the cells have grown in the medium for some time. In minimal medium, an activator which leads to induction of the enzyme is released into the medium by the bacteria. Complete medium contains a dialyzable and quite stable inhibitor which leads to repression of luciferase. The bacteria remove the inhibitor from the medium and also produce activator, thus allowing synthesis of the enzyme. Two unidentified nonluminescent strains of bacteria were unable to remove the inhibitor. Two different bioluminescent strains, Photobacterium fischeri and P. fischeri strain MAV, produce specific activators that are ineffective with cells of the other strain. The two activators are different with respect to heat stability, but both are small molecules. The activators can be assayed on the basis of their ability to counteract the inhibitor. Identification of the inhibitor and the activators may allow the bioluminescent system to be linked to other metabolic processes of the cells.

Quantitative analyses of the phospholipids of three strains of marine bioluminescent bacteria were carried out after separation by two-dimensional thin layer chromatography. The phospholipids of all three species consisted of about 75% phosphatidyl ethanolamine, 13% phosphatidyl glycerol and 7% cardiolipin. The composition was only slightly affected by drastic changes in the growth conditions. One of the species contained poly-β-hydroxybutyrate. The fatty acids of another species contained principally straight and branched-chain 16 and 18 carbon fatty acids. No clue as to the nature of the elusive “aldehyde factor” of bacterial bioluminescence was found by analysis of aldehyde deficient mutants, indicating that possibly the factor is not a major phospholipid of these bacteria.