J. Woodland Hastings

Biochemistry and Physiology of Bioluminescent Bacteria

Publisher Summary This chapter describes the biochemistry and physiology of bioluminescent bacteria. The function of bioluminescent bacteria is to emit light in biological systems. The chapter explains the variety of habitats in which these bacteria are found, the light-emitting system may play an important role in their ecology or physiology. The luciferase in bacteria, unlike that of any other luminous group (except, perhaps, the fungi), is related to the respiratory pathway, functioning as a shunt for electrons directly to oxygen at the level of reduced flavin. This luciferase is an external flavin mono-oxygenase or mixed-function oxidase, electrons for reduction of flavin mononucleotide (FMN) are provided by the reducing power derived from the electron-transport pathway. The light-emitting reaction then proceeds via the reaction of molecular oxygen with reduced flavin to form an intermediate luciferase-flavin peroxy species, whose breakdown provides energy sufficient to leave one of the products in an electronically excited singlet state, with subsequent light emission. The bacterial (luciferase-bound) peroxide chromophore, which has been isolated and characterized, provides a model in this respect for the different bioluminescent reactions.

NOVEL DINOFLAGELLATE CLOCK-RELATED GENES IDENTIFIED THROUGH MICROARRAY ANALYSIS1

In a genome‐wide study of circadian gene expression, we constructed microarrays containing 3500 cDNAs from the dinoflagellate Pyrocystis lunula Schütt and compared the abundance of transcripts at circadian times separated by 12 h. About 3% of the unique genes screened were identified as circadian controlled, more than 50% of which could be identified with diverse known genes. Most were preferentially expressed in either early subjective day or late subjective night. Light exposures at times expected to induce phase shifts in the rhythm revealed 30 differentially expressed genes, including some potentially participating in photic entrainment and others in pathways connecting a central oscillator to output rhythms. Those that appeared in both screens were considered as possible core clock genes, but there were no similarities with such genes from other organisms. This is the first report of circadian regulation of transcript levels in a dinoflagellate.

A substrate-binding protein in the Gonyaulax bioluminescence reaction.

Abstract Gonyaulax luciferase, the enzyme involved in the luminescence reaction of the dinoflagellate Gonyaulax polyedra , can be obtained as either A-luciferase (molecular weight about 150,000), or B-luciferase (molecular weight 35,000), by extracting the cells at pH 8, or at pH 6, respectively. Luciferin, the substrate in the luminescence reaction, occurs in extracts at pH 8 bound to a protein (molecular weight about 100,000) which has no enzymatic activity in the luminescent reaction. The luciferin is released at pH 6.5. An inhibitor of the B-luciferase reaction (molecular weight about 100,000) which is effective at pH 8 but not at pH 6.5, can also be isolated from Gonyaulax extracts. Evidence is presented that only free luciferin can react with luciferase, and that the inhibitor is identical with the binding protein, which acts by binding luciferin reversibly at pH 8. It is suggested that the binding protein may have a role in the control of the in vivo bioluminescent flash.

Voltage-gated proton channel in a dinoflagellate

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

What is the clock? Translational regulation of circadian bioluminescence

An oscillation in the cellular level of specific proteins in the unicellular marine alga Gonyaulax is correlated with the prominent circadian rhythm of bioluminescence of living cells and persists under constant conditions. This regulation involves a daily bout of synthesis of a specific protein, which is controlled by the circadian clock at the translational level.

Light to Hide by: Ventral Luminescence to Camouflage the Silhouette

The so-called pony fish of the tropical and subtropical Indo-Pacific region can emit light from a broad area of its ventral surface. An experimental analysis of this luminescent system supports the hypothesis that it functions by emitting light during the daytime, which matches the background light and thereby obscures the silhouette of the animal.

The inhibition of a biological clock by actinomycin D.

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The specificity of symbiosis: Pony fish and luminescent bacteria

Luminescent bacteria isolated from light organs of seven different species (3 genera) of fishes of the family Leiognathidae were subjected to taxonomic analysis. Of the 733 isolated all but seven were identified as Photobacterium leiognathi; the others are considered to be either chance contaminants of the sampling procedure or transients within the organ. In most fish, the luminous organ appeared to contain a single predominating strain of P. leiognathi with small numbers of one to three other strains of the same species, differing by only one or two characters.

Low oxygen is optimal for luciferase synthesis in some bacteria

The synthesis of the bioluminescent systems in many strains of two species of the genus Photobacterium which were isolated as symbionts is greater at low oxygen concentrations, where aerobic growth is blocked. In strains of two other species, one Photobacterium of symbiotic orgin, and one (genus Beneckea) whose luminous members are not known to be involved in symbiotic associations, a different response is observed. At low oxygen concentrations, where there is an inhibition of growth, there is also a similar decrease in the synthesis, of the luminescent system. These species-specific differences may indicate important ecological differences along with distinctive differences in the molecular control mechanisms involved in the synthesis of luciferase.

The structure and organization of the luciferase gene in the photosynthetic dinoflagellate Gonyaulax polyedra

The structural features of dinoflagellate nuclei are distinct from those of other eukaryotes in several respects, and the mechanisms of DNA replication and transcription are almost completely unknown. In this study we investigated the structure and organization of the gene coding for luciferase (LCF), the enzyme catalyzing the bioluminescent reaction in the dinoflagellate Gonyaulax polyedra. The genomic lcf sequence, including its flanking regions, were completely determined. The transcription initiation site was identified using primer extension and RNase protection assays. Sequence analysis shows that, like the luciferin-binding protein gene (lbp) from G. polyedra, lcf does not contain introns. Analysis of results from genomic Southern blots, inverse PCR, and sequencing revealed that the lcf gene is organized as tandem repeats in the genome. The spacer region between the lcf genes, which very likely contains the promoter elements necessary for transcription initiation, has no TATA box or other known promoter elements or consensus sequences. However, a conserved sequence motif was identified by comparing the two intergene spacer regions of lcf and the peridinin chlorophyll protein gene, pcp; a novel 13 nt sequence, CGTGAACGCAGTG, which might be a dinoflagellate promoter, was found to be present in both.