Proteolytic inactivation of the luciferase from the luminous marine bacterium Beneckea harveyi.

The enzymatic activity of bacterial luciferase from Beneckea harveyi (a heterodimer, Mr = approximately 79,000) is rapidly lost upon treatment with trypsin or chymotrypsin. Under nondenaturing conditions, the proteolytically inactivated molecule has the same apparent molecular weight as the native enzyme, and appears to be relatively stable to further proteolytic degradation. Gel electrophoresis in sodium dodecyl sulfate of the products of this digestion shows that only the alpha subunit is degraded during the time of these experiments, and its rate of loss is the same as the rate of loss of light-producing activity. The action of either protease produces a species with mobility indicative of a molecular weight of about 28,000 and smaller fragments, and an unaltered beta subunit.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

Differential synthesis of the polypeptides of aldehyde dehydrogenase and NAD(P)H:flavin oxidoreductase in the bioluminescent bacterium Beneckea harveyi.

The proteins of the bioluminescent bacterium Beneckea harveyi have been labelled with [3H]leucine prior to the induction of bioluminescence, and with [14C]leucine during the development of the bioluminescent system. An aliphatic aldehyde dehydrogenase and a NAD(P)H:flavin oxidoreductase, two enzymes that may be directly involved in the metabolism of the substrates (aldehyde, FMNH2) for the luminescent reaction catalyzed by luciferase, were purified and the isotope ratios of their respective polypeptide chains determined after sodium dodecyl sufate gel electrophoresis. A comparison of these isotope ratios to (a) the isotope ratios of the induced polypeptide chains of luciferase, purified in the same experiment, and (b) the average isotope ratio for the proteins synthesized in concert with growth has provided direct evidence that the synthesis of aldehyde dehydrogenase but not NAD(P)H:flavin oxidoreductase is induced during the development of bioluminescence.     

Tryptophan biosynthesis in the marine luminous bacterium Vibrio harveyi.

Tryptophan biosynthetic enzyme levels in wild-type Vibrio harveyi and a number of tryptophan auxotrophs of this species were coordinately regulated over a 100-fold range of specific activities. The tryptophan analog indoleacrylic acid evoked substantial derepression of the enzymes in wild-type cells. Even higher enzyme levels were attained in auxotrophs starved for tryptophan, regardless of the location of the block in the pathway. A derepressed mutant selected by resistance to 5-fluorotryptophan was found to have elevated basal levels of trp gene expression; these basal levels were increased only two- to threefold by tryptophan limitation. The taxonomic implications of these and other biochemical results support previous suggestions that the marine luminous bacteria are more closely related to enteric bacteria than to other gram-negative taxa.     

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

An adenosine 3',5'-monophosphate-requiring mutant of the luminous bacteria Beneckea harveyi.

We have isolated a mutant of the luminous bacterium Beneckea harveyi, which requires exogenous adenosine 3',5'-monophosphate (cyclic AMP) to synthesize luciferase and emit light. The mutant was pleiotropic, lacking not only the ability to luminesce, but also the capacities to form flagella and the ability to utilize a variety of carbohydrates for growth. All these deficiencies could be corrected by added cyclic AMP. The cyclic AMP-induced de novo synthesis of luciferase was possible only after autoinduction had occurred. The induction time by cyclic AMP ranged between 6 and 10 min at 27 degrees C.     

Induction of fatty aldehyde dehydrogenase activity during the development of bioluminescence in Beneckea harveyi.

Bioluminescence rises very rapidly in the later stages of growth of $\text{Beneckea harveyi}$ due to the induction of luciferase activity. This enzyme catalyzes the $\text{in vitro}$ oxidation of FMNH₂ and a long chain aliphatic aldehyde resulting in the emission of light. The present experiments report the discovery of an aldehyde dehydrogenase in $\text{Beneckea harveyi}$ which is remarkably similar to luciferase in its specificity for long chain aliphatic aldehydes. Furthermore, the activity of this enzyme is shown to be induced at the same time as luciferase thus providing strong evidence for a functional implication of aldehyde dehydrogenase in the bioluminescent system of $\text{Beneckea harveyi}$.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

Differential regulation of enzyme activities involved in aldehyde metabolism in the luminescent bacterium Vibrio harveyi.

The effects of catabolite repression and nutrient abundance on the activities of Vibrio harveyi enzymes known to be related to aldehyde metabolism were investigated. The growth of cells in complex medium containing glucose, which decreases in vivo luminescence and luciferase synthesis, also resulted in decreases in the specific activities of V. harveyi aldehyde dehydrogenase and acyl carrier protein acyltransferase as well as in the degree of fatty acylation of three bioluminescence-specific polypeptides (32, 42, and 57 kilodaltons), as monitored by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This repression was partially alleviated in glucose medium containing cyclic AMP. The acylation of the above-mentioned proteins, in addition to light emission and luciferase and acyltransferase activities, was also repressed when cells were grown in minimal medium, with partial recovery of these functions upon the addition of arginine. In contrast, aldehyde dehydrogenase activity was increased in minimal medium. These results suggest that the 42-, 57-, and 32-kilodalton proteins, which are responsible for the supply and reduction of fatty acids to form aldehydes for the luciferase reaction, are regulated in the same way as luciferase under the above-described conditions. However, aldehyde dehydrogenase, whose role in V. harveyi aldehyde metabolism is not yet known, is regulated in a different way with respect to nutrient composition.     

Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi.

Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction.     

Transhydrogenase activity in the marine bacterium Beneckea natriegens.

The marine bacterium, Beneckea natriegens, which has previously been reported not to form transhydrogenase, has been shown to synthesize a soluble energy-independent transhydrogenase (NADPH:NADP+ oxidoreductase, EC 1.6.1.1), though no energy-linked activity could be detected. The transhydrogenase is induced maximally in stationary phase cells and its formation is 70-90% repressed by raising the medium phosphate level from 0.33 to 3.3 mM. The enzyme is inhibited by arsenate, inorganic ortho- and pyrophosphate and by a range of organic phosphate-containing compounds, including 2'-AMP, which is an activator of several bacterial transhydrogenases.     

Bioluminescent method of determining picomolar amounts of nicotinamide-adenine dinucleotide using an immobilized extract of the luminescent bacterium Beneckea harveyi

The luminous bacteria Beneckea Harveyi were immobilized on BrCN-sepharose and cellulose films activated with cyanuric chloride. Preparations with high luciferase and FMN-reductase activities were obtained, which showed no background luminescence without NADH being added. The storage conditions for the preparations obtained were optimized, and their kinetic parameters and thermostability were studied. Standard curves for NADH determining within the concentration range 1 nM-1 microM were plotted with the detection level of 1 picomol NADH. The preparations are very promising for bioluminescent assay due to their high activity, simple production, high stability during storage and a possibility for the repeated use.     

Distribution of the Luminous Bacterium Beneckea harveyi in a Semitropical Estuarine Environment

Bioluminescent bacteria were found in the water column, sediment, shrimp, and gastrointestinal tract of marine fishes from the semitropical estuarine environment of the East Lagoon, Galveston Island, Tex. Populations in the water column decreased during cold weather while sedimentary populations persisted. The highest percentages of luminous organisms were isolated from the gastrointestinal tract of marine fishes, where they persisted during 5 days of starvation. The presence of chitin temporarily increased intestinal populations. All isolates were Beneckea harveyi, whose natural habitat appears to be the gut of fishes and whose free-living reservoir appears to be marine sediments.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

Regulation of aspartokinase activity in the genus Beneckea and marine,luminous bacteria

Summary The pattern of allosteric regulation of aspartokinase activity was determined in species of Beneckea and in the marine, luminous bacteria. The results indicated that these organisms have at least three isofunctional aspartokinases of which the first is inhibited by l-threonine, the second is inhibited by l-lysine, and the third is unaffected by either l-threonine or l-lysine. The homoserine dehydrogenase activity is clearly separable from the l-lysine-sensitive aspartokinase, that may be associated with one (or both) of the other isofunctional aspartokinases.The species of Beneckea and luminous bacteria studied varied in the relative levels of the three isofunctional aspartokinases. B. parahaemolytica, B. alginolytica, B. pelagia, B. neptuna, B. harveyi, B. nigrapulchrituda, B. natriegens, and Photobacterium fischeri had predominant levels of the l-lysine-sensitive activity; B. campellii, P. phosphoreum, and P. mandapamensis had predominant levels of the l-threonine-sensitive activity; while in B. nereida these two activities were approximately equivalent. Species of Beneckea could be distinguished from P. fischeri on the basis of the sensitivity of their aspartokinase activities to inhibition by l-lysine. In P. fischeri 10.3×10^-5 M l-lysine was required for a 50% inhibition of the l-lysine-sensitive enzyme, while in species of Beneckea 0.5–2.7×10^-5 M l-lysine was required to achieve the same level of inhibition.Non-standard abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - GC guanine plus cytosine     

A calorimetric investigation of the growth of the luminescent bacteria Beneckea harveyi and Photobacterium leiognathi.

Direct calorimetric determinations of the rate of heat production along with simultaneous determinations of the rate of photon emission and the number of viable cells have provided insight into the growth of Beneckea harveyi and Photobacterium leiognathi. These experiments were performed with a Tronac isothermal microcalorimeter modified with a fiber optic light guide to allow in situ detection of light. Escherichia coli and a dark variant of P. leiognathi were also examined to provide points of reference. It is demonstrated that B. harveyi seems to pause in the rate of metabolic heat production at the same point in time that the enzyme luciferase begins to be synthesized. This effect is not removed if B. harveyi is grown in conditioned medium. The thermograms for all species are correlated with cell generation time. The heat production per cell indicates that uncrowded cultures produce more heat than older, more crowded cultures, supporting the original observation of Bayne-Jones and Rhees (1929). These observations reopen for examination the suggestion that living systems tend toward a state of minimum metabolism per unit mass.     

Growth, luminescence, respiration, and the ATP pool during autoinduction in Beneckea harveyi.

The bacterial bioluminescence system is unusual because it is self-induced. In the late logarithmic phase of growth, upon the accumulation of an autoinducer, the synthesis of the components of the system is initiated. We were interested in determining what effect this burst of synthesis and activity has on cellular energy metabolism. The ATP pool of the luminous bacterium Beneckea harveyi was found to dip 10- to 20-fold during the luminescence period, while the respiration per unit cell mass (optical density) increased but by much less. The dip in the ATP pool did not occur in four different types of dark mutants, including one that was temperature conditional and another that was conditional upon added cyclic AMP for luminescence. However, it is neither the synthesis nor the activity of luciferase that is responsible for the ATP dip; the dip does not occur in certain dark "aldehyde" mutants which nevertheless synthesize normal levels of luciferase, whereas it does occur at 36 degrees C in a temperature-sensitive luciferase mutant which forms normal levels of inactive luciferase. Results with other aldehyde mutants implicate the pathway involved in the synthesis of the aldehyde factor with the ATP dip.