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.     

Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth

When a culture of Escherichia coli ML30 growing exponentially at 37 C in a glucose minimal medium was shifted abruptly to 10 C, growth decreased for about 4.5 hr. There was no net synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. The cells, however, respired at a rate characteristic of cells growing in the steady state at 10 C and were able to accumulate alpha-methyl-d-glucoside. When growth recommenced at 10 C, protein synthesis started at 4 hr, RNA synthesis, with a burst at 6 hr, and DNA synthesis, with a burst at 7 hr. One synchronous division occurred at about 11 hr after shifting to 10 C. There was no alteration in the steady-state RNA to protein ratio. The results are discussed in relation to other reported effects of shifts in environmental conditions. The lag at 10 C was dependent on prior conditions of growth at 37 C. Growth at 37 C under conditions giving catabolite repression were necessary for the lag to be established on shifting to 10 C.     

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.     

Autoinduction in a luminous bacterium: A confirmation of the hypothesis

In some luminous bacterial species, it is postulated that luciferase is “autoinduced” by a substance produced by the bacteria themselves. This hypothesis was confirmed. In experiments with growing cultures that were subjected to repeated subculturing into or dialysis against fresh medium, which should prevent the autoinducer from accumulating, the normal synthesis of luciferase and the development of luminescence did not occur.     

Involvement of cAMP and cAMP-dependent protein kinase in the initiation of DNA synthesis by rat liver cells

Addition of calcium to calcium-deprived cultures of T51B rat liver cells caused brief bursts of cAMP production and cAMP-dependent protein kinase activity which were followed almost immediately by a stimulation of DNA synthesis. PKInh, a specific polypeptide inhibitor of the catalytic subunits of cAMP-dependent protein kinases, inhibited the DNA-synthetic response to calcium addition without stopping the preceding cAMP surge. Addition of cAMP to the calciumdeprived cultures increased protein kinase activity and stimulated DNA synthesis, both of which were inhibited by PKInh. DNA synthesis in these cultures was not stimulated by adding type I cAMP-dependent protein kinase holoenzyme to the calcium-deficient medium, but it was stimulated by type II cAMP-dependent protein kinase holoenzyme or the catalytic subunit from either type I or type II holoenzyme. The stimulatory actions of the type II holoenzyme or the catalytic subunits were inhibited by PKInh. Thus, a burst of cAMP-dependent protein kinase activity was ultimately responsible for the stimulation of DNA synthesis in calcium-deprived T51B cells by calcium or cAMP and it might also be involved in the events leading to initiation of DNA synthesis in many, if not all, normally cycling cells.     

Dual effect of osmotic cell swelling on prolactin secretion by acutely dispersed adenohypophyseal cells

Cell swelling induced by acute exposure to the permeant molecule urea or by medium hyposmolarity evoked a prompt PRL secretory burst from dispersed rat anterior pituitary cells. However, during continuous exposure greater than or equal to 10 min to these conditions inhibition of basal and TRH-induced PRL secretion occurred and there was an "off" burst of PRL secretion following return to basal conditions. Compared with continuous TRH stimulation which causes biphasic PRL secretion with a rapid high amplitude first phase secretory burst followed by a sustained low level second phase of secretion, cell swelling induced only "first phase" secretion. Removing Ca2+ from the medium or adding 50 microM verapamil markedly depressed the "off" secretory burst following return to basal conditions but had no effect on the initial high amplitude burst. Our data suggest that the effect of cell swelling on PRL secretion is complex and that there are at least two mechanisms for PRL secretion in normal anterior pituitary cells; these are differently affected by cell swelling and Ca2+ influx.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens.

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Mutant Analysis and Enzyme Subunit Complementation in Bacterial Bioluminescence in Photobacterium fischeri

Chemical mutagens were used to obtain mutants deficient in bioluminescence in the marine bacterium Photobacterium fischeri strain MAV. Acridine dyes were effective in the production of dark mutants but not in the production of auxotrophs. These dark mutants were all of one type and appeared to contain lesions blocking the synthesis of luciferase. ICR-191 was especially effective in the production of aldehyde mutants, i.e., dark strains that luminesce when a long-chain aldehyde such as n-decanal is added to them. However, other mutant types were isolated after treatment with ICR-191. N-methyl-N'-nitro-N-nitrosoguanidine induced many bioluminescence-deficient types with respect to both the site of the lesion and the quantitative effect on the luminescent system. We characterized the dark and dim mutants with respect to their response to exogenous decanal, levels of in vivo and in vitro luminescence, and their rates of reversion to wild type. In addition, the luciferases of the mutant strains were examined by subunit complementation. On the basis of these analyses, we identified mutants which synthesize altered luciferase, strains which are deficient in synthesis of luciferase, and aldehyde mutants. The results of analysis of luciferase from the aldehyde mutants and the complementation studies indicate that the lesions in these strains are in the luciferase itself. Results obtained with wild-type cells grown in minimal medium, and aldehyde mutant cells grown either in complete or minimal medium, indicate that a "natural aldehyde factor" is involved in in vivo light emission. These same studies showed that the long-chain aldehyde(s) could only partially substitute for the natural "aldehyde factor." The possibility that the in vivo aldehyde factor is not a long-chain aldehyde is discussed.     

Managanese stimulates the oxidative brust of differentiated HL-60 cells

Incubation with manganese results in a twofold increase in the oxidative burst of differentiated HL-60 cells. This stimulation was characterized by examining the dose response, length of incubation time, and specifity of manganese. Managese only stimulated the burst in cells induced to differentiated with retinoic acid and not in undifferentiated HL-60 cells. Incubation with manganese did not result in a greater number of differentiated cells. The maximum stimulation occurred at 0.2 μmol/L manganese. Stimulation of the oxidative burst required 96 h of incubation with manganese, since cells incubated with the same levels of manganese for the last 24 h of culture did not result in any stimulation. Magnesium, present in the incubation medium at physiological serum levels (820 μmol/L) also stimulated the oxidative burst, whereas iron (0.3 μmol/L), zinc (18 μmol/L), and copper (12 μmol/L) had no effect. To determine whether manganese and magnesium stimulated the burst differently, the initial rates of superoxide anion production was determined. The initial rate of the reaction proceeded rapidly in cells incubated with managnese, whereas there appeared to be a lag before magnesiumtreated cells produced superoxide anion. Thus, manganese seems to stimulate the oxidative burst differently than magnesium.     

Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis.     

Uncoupling of the calcium-induced terminal differentiation and the activation of membrane-associated transglutaminase in murine keratinocytes by type-beta transforming growth factor

Calcium is an important regulator of terminal differentiation of cultured epidermal cells. In order to investigate the relationship between the termination of proliferative activity and the process of keratinization, we studied the time course of events induced by a sudden increase of extracellular calcium (calcium-switch) in cultures of established murine skin keratinocytes (BALB/c MK-1). These cells displayed density-dependent growth arrest without undergoing terminal differentiation in the presence of serum- and mitogen-free medium with a calcium concentration less than 0.10 mM. The calcium-switch alone was sufficient to induce a dose-dependent burst of DNA synthesis, which was followed by a state in which the cells became progressively refractory to mitogenic stimulation with epidermal growth factor. Treatment of cultures with type beta transforming growth factor during the first 6- to 10 h following the calcium-switch completely eliminated the initial burst of DNA synthesis as well as the terminal differentiation in response to calcium. On the other hand, the calcium-switch also caused the induction of a four- to fivefold increase of the activity of the membrane-associated form of transglutaminase that is required for keratinization, which was not affected by the presence of type beta transforming growth factor. These observations suggest that type beta transforming growth factor regulates the calcium-induced terminal cell division independently of the induction of phenotypic markers of keratinization, such as transglutaminase.     

Iron represses bioluminescence and affects catabolite repression of luminescence inVibrio harveyi

Bioluminescence and the synthesis of luciferase inVibrio harveyi growing in a minimal medium are repressible by iron; this is not significantly reversed by cyclic adenosine 3,5-monophosphate (cAMP). Cultures grown with added iron emit less light and possess less luciferase per cell than those grown under conditions of limiting iron; this may have significance in relation to the function of luciferase as an electron carrier. With iron, and with glycerol as the sole carbon and energy source, the addition of glucose causes further repression, both transient and permanent, and this is only partially reversible by cAMP. Without iron, glucose addition results in only a small and transient repression, but this is fully reversible by cAMP. The inability of cAMP to reverse iron-influenced repression may be explained by both a low rate of transport of cAMP into the bacteria and increased intracellular levels of cyclic nucleotide phosphodiesterase.     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Functional Organization of the Cardiac Ganglion of the Lobster, Homarus americanus

External recording and stimulation, techniques were used to determine which neurons and interactions are essential for production of the periodic burst discharge in the lobster cardiac ganglion. Burst activity can be modulated by brief single shocks applied to the four small cells, but not by similar stimulation of the five large cells, suggesting that normally one or more small cells primarily determine burst rate and duration. Repetitive electrical stimulation of large cells initiates spike activity in small cells, probably via excitatory synaptic and/or electrotonic connections which may normally act to prolong bursts and decrease burst rate. Transection of the ganglion can result in burst activity in small cells in the partial or complete absence of large cell spike activity, but large cells isolated from small cell excitatory synaptic input by transection or by application of dinitrophenol do not burst. Generally, transections which decrease excitatory feedback to small cells are accompanied by an increase in burst rate, but mean spike frequency over an entire burst cycle stabilizes at the original level within 10–30 min for various groups of cells whose spike-initiating sites are still intact. These and previous results suggest that the system is two layered: one or more small cells generate the burst pattern and impose it on the large cells which are the system's motorneurons.     

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.     

The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection.

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.     

Effects of aldehyde and internal ions on bioluminescence expression of Photobacterium phosphoreum

In a complex medium, cells of Photobacterium phosphoreum (strain 496) grow equally well with 1% and 3% NaCl, but luminescence occurs only with 3% NaCl in the medium. However, the suppression of luminescence is not attributable to the lack of luciferase; log phase cells growing in 1% NaCl will develop luminescence following a shift to 3% NaCl, which is accompanied by an increase of intracellular potassium. Tetradecanal stimulates bioluminescence in a 1% NaCl culture, and also in the presence of nalidixic acid, an inhibitor or gyrase. It is thus suggested that the suppression of luminescence in 1% NaCl or in 3% NaCl with nalidixic acid is due to a deficiency in the synthesis of intracellular aldehyde. The increase in intracellular potassium that occurs upon shifting from 1% to 3% NaCl may also relate to aldehyde synthesis gene expression via activation of gyrase, or via an increase in negative supercoiling of the chromosome. However, since an initial decrease of light intensity is still observed during culture even with the addition of tetradecanal, an additional factor related to cell density must also be involved in bioluminescence expression.Abbreviations nal nalidixic acid - nal-r nalidixic acid resistant strain