Control of Neurospora crassa morphology by cyclic adenosine 3'', 5''-monophosphate and dibutyryl cyclic adenosine 3'', 5''-monophosphate.

The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.     

Control of cyclic adenosine 3'',5''-monophosphate levels by depolarizing agents in fungi.

It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.     

Antagonism by dibutyryl adenosine cyclic 3'',5''-monophosphate and testololactone of concanavalin A capping

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.     

Suppression of defects in cyclic adenosine 3'',5''-monophosphate metabolism in Escherichia coli.

Strain MM6-13 (ptsI suc lacI sup) of Escherichia coli contains a suppressor of the succinate-negative phenotype. In MM6-13, sup caused enhanced growth in glycerol, maltose, melibiose, and succinate media and increased activity of beta-galactosidase and tryptophanase relative to an isogenic strain without sup. In strain A61 (cya sup), sup partially suppressed cya. Cyclic guanosine monophosphate increased beta-galactosidase activity sevenfold in A61 and enabled this strain to grow on maltose, galactose, succinate, and arabinose. Strain A61 responded to much lower concentrations of cyclic adenosine monophosphate than cyclic guanosine monophosphate. It appears that sup is located in the crp locus. These results suggest that sup mutants have an altered cyclic adenosine monophosphate receptor protein which is activated by cyclic guanosine monophosphate and has an increased affinity for cyclic adenosine monophosphate.     

Metabolism of cyclic adenosine 3'',5''-monophosphate and induction of tryptophanase in Escherichia coli.

The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell.     

Respiratory capacity, cyclic adenosine 3'',5''-monophosphate, and morphogenesis of Mucor racemosus.

A variety of cultural conditions were examined to determine the relationship between respiratory capacity and the growth of Mucor racemosus in the yeast and mycelial form. The results show that both yeasts and hyphae can develop when the respiratory capacity is low (e.g., in N2). In addition, the yeast form of the fungus could be grown in air in the presence of cyclic adenosine 5'-monophosphate with high respiratory rates characteristic of air-grown mycelia. These results indicate that their is not an obligatory relationship between respiratory capacity and morphogenesis in M. racemosus. Low intracellular levels of cyclic adenosine 5'-monophosphate, however, were correlated with aerobic mycelial development, whereas yeast development under CO2 was characterized by higher cyclic adenosine 5'-monophosphate levels.     

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.     

Role of cyclic adenosine 3'',5''-monophosphate on cessation of respiration in ultraviolet-irradiated Escherichia coli.

The addition of cyclic adenosine 3',5'-monophosphate (cAMP) to ultraviolet-irradiated Escherichia coli B/r cultures causes additional cells to cease respiring and to die. These effects of cAMP are greater on glucose-grown cells, where the effects of ultraviolet radiations alone are smaller and where the intracellular concentrations of cAMP are known to be lower.     

Deficient cyclic adenosine 3'',5''-monophosphate control in mutants of two genes of Neurospora crassa.

Strains of Neurospora crassa mutant in either of two genes, Crisp-1 (cr1) and Frost (fr), showed no increase of cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels when subjected to several treatments which produce large increases of cyclic AMP in wild-type Neurospora. Evidently, the previously reported deficiencies of adenylate cyclase in these mutants were sufficient to block the normal increases. This fact suggests that both mutants could be used to help determine which control phenomena involve cyclic AMP and to interrupt the control of established cyclic AMP-regulated functions. Earlier studies had suggested an interdependence of the cyclic AMP level and the electric potential difference across the plasma membrane of Neurospora. Present experiments, therefore, employed several strains with the cr1 mutation to test for possible roles of cyclic AMP in recovery and oscillatory behavior of the Neurospora membrane potential. The results showed all such phenomena to be normal in the adenylate cyclase-defective strains, which demonstrates that variations of cyclic AMP are not obligatorily involved in the apparent control processes. Evidence is also presented that the induction of both glucose transport system II and the alternative oxidase do not require elevated cyclic AMP levels.     

Purine-containing compounds, including cyclic adenosine 3'',5''-monophosphate, induce fruiting of Myxococcus xanthus by nutritional imbalance.

Induction of Myxococcus xanthus fruiting by a number of different purine-containing compounds, including cyclic adenosine 3',5'-monophosphate, is defective in a mutant resistant to 2,6-diaminopurine. Furthermore, the purine-induced fruiting of wild-type cultures is uniquely blocked by a low concentration of added glycine. These results imply that different purine-containing compounds induce fruiting through a single mechanism involving nutritional imbalance.     

Identification of adenosine 3'',5''-monophosphate in Mycobacterium smegmatis.

Cyclic adenosine 3',5'-monophosphate isolated from Mycobacterium smegmatis cells was identified by thin-layer chromatography, stepwise conversion to adenosine 5'-monophosphate and adenosine, ultraviolet absorption spectrum, phosphate analysis, and detection by two relatively specific radioisotopic methods.     

Properties of adenyl cyclase and cyclic adenosine 3'',5''-monophosphate receptor protein-deficient mutants of Escherichia coli.

Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants.     

Role of the rel gene product in the control of cyclic adenosine 3'',5''-monophosphate accumulation.

The presence of a relA mutant allele affects the kinetics of cyclic adenosine 3',5'-monophosphate accumulation during downshift from glucose to succinate. The nucleotide accumulates at the normal rate early in the downshift transition but continues to accumulate for a longer time in the relA mutant, leading to a two- to threefold excess by the end of the diauxic lag. Evidence is presented that this effect occurs independently of the accumulation of ppGpp.     

Inosine 5''-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5''-monophosphate.

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.     

Inhibition of pancreatic ribonuclease by 2''-5'' and 3''-5'' oligonucleotides.

Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.     

Siderophore production by Proteus mirabilis

Studies on the isolation and characterization of Proteus mirabilis siderophores provided no evidence that these bacteria synthesize catechol- or hydroxamate-type siderophores. However, gas chromatograph analysis in conjunction with mass spectroscopy revealed the presence of alpha-hydroxyisovaleric acid, a previously unknown metabolite. Additional substantiating evidence for the presence of alpha-hydroxyisovaleric acid in these bacteria was obtained from experiments involving the use of thin-layer chromatography and an ultraviolet absorption spectrum. This compound was found to be capable of removing iron from the synthetic chelator, ethylene-diamine-di-orthohydroxyphenyl acetic acid, and supplying that iron to the bacteria both in a solid agar medium and in a liquid medium. Proteus mirabilis was found to possess an enzyme capable of catalyzing the reaction by which alpha-hydroxyisovaleric acid is converted to alpha-ketoisovaleric acid, an intermediate in the valine biosynthetic pathway.