Bacterial luciferase: FMNH2-aldehyde oxidase.
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Flavin Mononucleotide↗
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Vibrio ("Beneckea") vulnificus, the bacterium associated with sepsis, septicaemia, and the sea.
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Humans↗
Two epidemics of diarrhoeal disease possibly caused by Plesiomonas shigelloides.
Two epidemics of water-borne diarrhoeal disease involving a total of 1000 persons are reported. In both epidemics, none of the usual bacterial entero-pathogens were recognized and Plesiomonas shigelloides was the only suspect aetiological agent isolated. The ecology of P. shigelloides was investigated in these outbreaks. It was recognized as an inhabitant of fresh surface water and its presence was closely related to warm weather.
Bacterial Infections↗
Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.
Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.
Flavin Mononucleotide↗
Activity and stability of the luciferase--flavin intermediate.
A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.
Anaerobiosis↗
Immobilization of bacterial luciferase and FMN reductase on glass rods.
Bacterial luciferase and NAD(P)H: FMN oxidoreductase isolated from Beneckea harveyi were covalently linked via diazotization to arylamine porous glass beads which had been cemented onto plain glass rods. These immobilized enzymes are individually active and also function to produce light via a coupled reaction utilizing NADH or NADPH. These enzymes have properties similar to the soluble forms with regard to pH and substrate optima and also exhibit linearity in peak intensity of the initial flash of light emitted as a function of NADH or NADPH concentration. Linearity with NADH is obtained in the range of 1 pmol to 50 nmol, and between 10 pmol to 200 nmol for NADPH. The bound enzymes are stable and reusable. This immobilized system offers a rapid and inexpensive m
Enzymes, Immobilized↗
Inhibition and stimulation of the development of the bioluminescent system in Beneckea harveyi by cyclic GMP.
The development of the luminescence system in Beneckea harveyi is controlled by cyclic nucleotides at the level of transcription. In the wild type, it is repressed by exogenously added guanosine 3':5'-cyclic monophosphate and this repression is overcome by the addition of adenosine 3':5'-cyclic monophosphate. These observations alone support a model in which these nucleotides act antagonistically. On the other hand, in a mutant requiring adenosine 3':5'-cyclic monophosphate for maximum luminescence, guanosine 3':5'-cyclic monophosphate stimulates the synthesis of the luminescence system at low concentrations and inhibits it at higher concentrations. These results are apparently not consistent with a model involving a simple antagonistic effect of guanosine 3':5'-cyclic monophosphate on the action of adenosine 3':5'-cyclic monophosphate.
Cyclic AMP↗
Evidence for tetradecanal as the natural aldehyde in bacterial bioluminescence.
Dim aldehyde mutants of the luminous bacterium Beneckea harveyi emit light with exogenously added long-chain aliphatic aldehyde. In one class of these mutants, luminescence is also stimulated by myristic (tetradecanoic) acid. In such mutants the amount of light obtained by the addition of a small (limiting) amount of either tetradecanal or myristic acid may be increased 60-fold by cyanide and other agents that block respiration. This indicates that the fatty acid product of the luminescent reaction is recycled. The effect, like the stimulation by exogenous fatty acid, exhibits specificity for the 14-carbon compound, suggesting that tetradecanal is the natural aldehyde. In those aldehyde mutants that are not stimulated to emit light by fatty acids, and thus presumably lack the recycling system, the chain-length-specific stimulation by cyanide does not occur.
Aldehydes↗
Covalent structure of subunits of bacterial luciferase: NH2-terminal sequence demonstrates subunit homology.
The heterodimeric subunit structure of bacterial luciferase was demonstrated more than 10 years ago. The enzymes from both Beneckea harveyi and Photobacterium fischeri have since been studied in detail; they each consist of two nonidentical subunits, designated alpha and beta. Both are required for bioluminescence activity, with the active center apparently confined to the alpha subunit. Amino acid sequence analysis of the NH2 termini of the alpha and beta subunits of the B. harveyi and P. fischeri luciferases not only confirms the earlier observation that the alpha subunits are homologous but also demonstrates that the NH2-terminal sequences of the beta subunits of the luciferases from the two genera are homologous. Furthermore, within each luciferase, the NH2-terminal sequences of the alpha and beta subunits are similar, suggesting the possibility that the genes coding, for alpha and beta may have arisen by gene duplication, presumably prior to divergence of the lines leading to present-day luminous bacteria.
Amino Acid Sequence↗
Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole.
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Alcaligenes↗
Adenylate energy charge during batch culture of Beneckea natriegens.
The value of the adenylate energy charge, i.e. ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]), during batch culture of Beneckea natriegens remained relatively constant during the exponential and early stationary phases of the growth cycle. During exponential growth the intracellular ATP content remained constant, the amount of ATP in the culture increasing proportionally with growth; these conditions were unaltered during growth in the presence of added cyclic AMP. On cessation of growth, significant variation in bacterial ATP content was observed depending on whether growth of the cultures terminated due to exhaustion of carbon or nitrogen from the medium, and on the presence or absence of added cyclic AMP.
Adenine Nucleotides↗
Steps in the population of the emitter in the bacterial luciferase reaction.
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Luciferases↗
Chloramphenicol bioassay.
An accurate plate diffusion bioassay for chloramphenicol is described, in which the fast-replicating Beneckea natriegens and 1.5% salt agar are used. Zones of inhibition were well defined after 3 h, and the limit of sensitivity of the method was around 2 mug/ml. The concurrent presence of gentamicin did not influence the assay. The assay is simple to carry out and duplicate assays can be performed with as little as 100 mug of capillary blood.
Biological Assay↗
Physiology and ecology of bacteriophages of the marine bacterium Beneckea natriegens: salinity.
The effects of variation in ionic levels on the stability and replication of two bacteriophages (nt-1 and nt-6) host specific for the marine bacterium Beneckea natriegens were examined. Monovalent cations influenced the adsorption of the nt-1 but not the nt-6 phage; however, one-step growth studies showed that NaCl was required for replication of both phage. The NaCl optimum for nt-1 production was 0.25 M NaCl, the same as the growth optimum for B. natriegens. However, the optimum for nt-6 production was 0.16 M NaCl. These NaCl optima for host and phage are at estuarine rather than oceanic levels. The nt-1 phage was better suited to replicate at NaCl levels typical of higher salinity areas (18-35%) and the nt-6 phage was better suited to replicate at lower salinities (5-18%). The nt phage were more resistant to low NaCl levels than their host bacterium and appeared limited to marine waters by the lower survival salinity of B. natriegens coupled with phage inactivation processes occurring in natural estuarine waters.
Adsorption↗
Viscometric assay of bacterial alginase.
Equations defining the reaction of microbial alginase on commercial sodium alginate are presented with respect to the viscometric assay of enzyme activity. The negative log of K was found to be linearly related to the concentration of algin, where K is defined as the change in the reciprocal of relative viscosity with time. The negative log of K was also found to be linearly related to the amount of enzyme in reaction mixtures when the substrate concentration was held constant.
Alginates↗
Pyruvate production and excretion by the luminous marine bacteria.
During aerobic growth on glucose, several species of luminous marine bacteria exhibited an imcomplete oxidative catabolism of substrate. Pyruvate, one of the products of glucose metabolism, was excreted into the medium during exponential growth and accounted for up to 50% of the substrate carbon metabolized. When glucose was depleted from the medium, the excreted pyruvate was promptly utilized, demonstrating that the cells are capable of pyruvate catabolism. Pyruvate excretion is not a general phenomenon of carbohydrate metabolism since it does not occur during the utilization of glycerol or maltose. When cells pregrown on glycerol were exposed to glucose, they began to excrete pyruvate, even if protein synthesis was blocked with chloramphenicol. Glucose thus appears to have an effect on the activity of preexisting catabolic enzymes.
Aerobiosis↗
Mutants of luminous bacteria with an altered control of luciferase synthesis.
Arginine is known to increase the luminescence in vivo and in vitro of the marine bacterium Beneckea harveyi growing in minimal medium. Mutants in which this arginine effect is either diminished, or absent were isolated as bright clones on a minimal medium after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. On a minimal medium both with and without added arginine and also on complex medium, these "minimal bright" mutants produce higher levels of luminescence than the wild type both in vivo and in vitro. This is attributed to the production of an increased amount of luciferase, which itself is wild type in terms of its specific activity.
Arginine↗
Cloning of Beneckea genes in Escherichia coli.
Genes from Beneckea harveyi, a luminescent marine bacterium, were cloned in Escherichia coli. This was done by producing randomly sheared fragments of Beneckea DNA and inserting them into the EcoRI site of plasmid pMB9 by the adenine-thymine joining procedure. The hybrid plasmids were used to transform E. coli C600 SF8. Among the transformants selected for tetracycline resistance, one clone that appeared to complement a leucine tb mutation was identified. The transformants were screened for the presence of Beneckea 5S genes. Four of these clones were analyzed in detail by hybridization with 16S, 23S, and 4S Beneckea RNA. The observations suggest that the ribosomal genes in Beneckea are linked, but are present in a different order than those in E. coli.
DNA, Bacterial↗