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′-monphosphate (cyclic AMP) to snnthesize 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 ffter autoinduction had occurred. The induction time by cyclic AMP ranged between 6 and 10 min at 27°C.     

Regulation of luminescence by cyclic AMP in cya-like and crp-like mutants of Vibrio fischeri.

Mutants of Vibrio fischeri MJ-1 (wild type) apparently deficient in adenylate cyclase (cya-like) or cyclic AMP receptor protein (crp-like) were isolated and characterized. Compared with MJ-1, the mutants produced low levels of luminescence and luciferase. Addition of cyclic AMP restored wild-type levels of luminescence and luciferase in the cya-like mutant but not in the crp-like mutant. The results are consistent with the hypothesis that in V. fischeri cyclic AMP and cyclic AMP receptor protein are required for induction of the luminescence system.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: glucose-negative mutant and regulation of intracellular cyclic AMP.

Glucose-negative mutants of Mycoplasma capricolum were selected for growth on fructose in the presence of the toxic glucose analog alpha-methyl-D-glucopyranoside. The mutants are defective in the phosphoenolpyruvate:sugar phosphotransferase system for glucose. One mutant, pts-4, was studied in detail. It lacks the glucose-specific, membrane-bound enzyme II, IIGlc, as well as the general, low-molecular-weight, phosphocarrier protein, HPr. In place of the latter, however, it has a fructose-specific protein, HPrFru. Consistent with these changes, the mutant lost the ability to grow on glucosamine and maltose but retained its ability to grow on sucrose. In the glucose-negative mutant, glucose did not regulate the intracellular concentration of cyclic AMP. The intracellular concentration of cyclic AMP in M. capricolum is regulated by the presence of metabolizable sugars. In the wild-type, both glucose and fructose reduced the intracellular concentration of cyclic AMP; however, in the glucose-negative mutant, glucose no longer regulated the intracellular level of cyclic AMP.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3'',5''-monophosphate.

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.     

Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Galphas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Galpha subunits. The Galphas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT(1) or MT(2) melatonin receptors with Galphas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Galpha C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Galpha chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Galphas chimaeras coupled. Recognition of the C-terminal five amino acids of the Galpha subunit is a requisite for coupling to a receptor, but it is not the sole determinant.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

Extracellular cyclic AMP during development of the cellular slime mold Polysphondylium violaceum: comparison of accumulation in the wild type and an aggregation-defective mutant.

Cyclic AMP was synthesized by Polysphondylium violaceum after starvation and during the preaggregation stage of development. Most of the newly synthesized cyclic AMP accumulated in the extracellular medium, with very little change in the intracellular cyclic AMP concentration. The addition of 10(-3) to 10(-6) M exogenous cyclic AMP to starved amoebae caused a 20 to 50% decrease in the number of aggregation centers formed compared with untreated controls. An aggregation-defective mutant of P. violaceum (strain aggA586) excreted or accumulated very little cyclic AMP. Strain aggA586 aggregated normally in the presence of a dialyzable, excreted product (D factor) produced by wild-type amoebae. When the mutant was incubated with D factor, cyclic AMP accumulated in the medium, and the amount accumulated depended on the amount of D factor added to the mutant amoebae.     

Utilization of gluconate by Escherichia coli. A role of adenosine 3'':5''-cyclic monophosphate in the induction of gluconate catabolism.

1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.     

Regulation of uracil uptake in Escherichia coli by adenosine 3',5'-monophosphate.

Culture of a wild-type strain of Escherichia coli in the presence of cyclic AMP leads to an impairment of uracil uptake. Half maximum inhibition of uracil uptake was observed at 1.5 mM cyclic AMP. The effect seems to be specific since no inhibition was found in cultures supplemented with ATP, ADP or 5'-AMP. Similarly the inhibition was not observed in cultures of a mutant deficient in the cyclic AMP receptor protein. The inhibition in uracil uptake, found in bacteria cultured in the presence of cyclic AMP, is not a consequence of a reduction in the growth rate. On the other hand, this inhibition was observed only in cultures containing glucose or pyruvate as carbon source.     

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.     

Cyclic AMP and the positive inotropic effect of norepinephrine and phenylephrine.

Time-response studies of the effects of norepinephrine and phenylephrine revealed that both agonists caused an increase in cyclic AMP levels before increases in contractile force in either the electrically stimulated left atria or spontaneously beating right atria of the rat. Norepinephrine caused a nearly sixfold increase in cyclic AMP, whereas phenylephrine produced only a 50% increase in the nucleotide. Pretreatment with reserpine did not affect the norepinephrine cyclic AMP response; however, the phenylephrine cyclic AMP response was abolished. Reserpine pretreatment did not significantly affect the contractile responses of either amine. In the presence of propranolol, norepinephrine was found to have the ability to produce an increace in contractile force in which cyclic AMP was apparently not involved. The time course of the contractile response induced by adrenergic amines was found to be remarkably influenced by the chronotropic response in spontaneously beating preparations while the cyclic AMP response was not greatly affected. This difference in the contractile response may be due to the ability of the chronotropic response to influence the flux of calcium through the cell membrane.     

Temperature-dependent alteration of cellular morphology by cholera toxin in rat liver epithelial cells which are ts for maintenance of transformed properties

Cholera toxin via its ability to increase intracellular cyclic AMP levels can induce drastic changes in cell morphology. This report describes a temperature sensitive mutant of chemically transformed rat liver epithelial cells which only display cell shape alterations in response to cholera toxin at the permissive temperature. Shift up-shift down experiments indicate that the change in the response occurs fairly rapidly, i.e., within 2 hours at the new temperature. The behavior of the temperature sensitive cells at the nonpermissive temperature mimics that of the untransformed rat liver epithelial cells (i.e., no morphological change in response to cholera toxin) while at the permissive temperature the positive cell shape change is identical to that exhibited by chemically transformed rat liver epithelial cells. The temperature sensitive response to cholera toxin is not a function of cyclic AMP production, since the amount of cyclic AMP found as a function of either time or concentration of cholera toxin is quite similar in cells treated at either temperature.     

Gonadotropin stimulation of cyclic AMP levels in Chinese hamster ovary cells in culture.

When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 micrograms/ml hCG. The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.     

Effect of adenosine 3',5'-cyclic monophosphate derivatives on alpha-amylase release, protein kinase and cyclic nucleotide phosphodiesterase activity from rat parotid tissue.

Several 8-substituted derivatives of cyclic AMP were tested for their effects on alpha-amylase release. None of the 8-substituted compounds were more active than N6,O2-dibutyryl- or N6-monobutyryl adenosine 3',5'-monophosphate in causing alpha-amylase release. The rat parotid was found to contain a high (105 muM) and a low (1.15 muM) Km cyclic AMP phosphodiesterase activity. All of the 8-substituted cyclic AMP compounds inhibited the hydrolysis of 1 muM cyclic AMP. However, there was only a partial correlation between the ability to cause alpha-amylase release and inhibit cyclic AMP hydrolysis. Extracts of parotid tissue contained a cyclic AMP-dependent protein kinase activity. None of the compounds were as effective as cyclic AMP in activating the protein kinase. As in the case of inhibition of cyclic AMP hydrolysis, the ability of the 8-substituted cyclic AMP compounds to increase protein kinase activity did not correlate with their effects on alpha-amylase release. It is concluded that factors in addition to the in vitro inhibition of cyclic AMP hydrolysis and activation of protein kinase are important in determining the net result of the 8-substituted cyclic AMP compounds on parotid gland function. These additional factors might include differences in the rate of uptake and differences in rats of conversion to compounds with modified activity.     

Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling.

Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.