Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Effects of colicins K and E1 on the glucose phosphotransferase system.

1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of alpha-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of alpha-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake. 2. Colicins K and E1 decreased the apparent Km for alpha-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins. 3. In the presence of colicins, alpha-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated alpha-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound. 4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.     

Insulin action on Escherichia coli. Regulation of the adenylate cyclase and phosphotransferase enzymes.

Insulin on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotransferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, the rate of alpha-methylglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. In vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin on E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of alpha-methylglucoside.     

Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast

A mutation (pde1) was detected by suppressor activity on the CYR3 mutation which caused cAMP requirement for growth at 35 degrees C by the alteration of cAMP-dependent protein kinase. The pde1 mutant produced a significantly reduced level of cyclic nucleotide phosphodiesterase activity when assayed with 500 microM cAMP. Two cyclic nucleotide phosphodiesterases, I and II, were identified. Approximate molecular weights of these enzymes were 60,000 and 110,000, and the apparent Km values were 100 and 0.4 microM, respectively. The pde1 mutant was deficient in phosphodiesterase I activity. The cells carrying the pde1 mutation accumulated several times over the intracellular cAMP found in wild type cells. Phosphodiesterase I was not essential for growth of yeast cells, but controlled the intracellular cAMP levels in wild type and various mutant strains.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

Cyclic AMP-induced cytolysis in S49 cells: selection of an unresponsive "deathless" mutant.

Wild-type S49 lymphoma cells respond to cyclic adenosine 3', 5'-monophosphate (cAMP) by inducing cAMP phosphodiesterase, halting growth in the G1 phase of the cell cycle and subsequently dying. By using a counter selection procedure, we have isolated a new class of mutants of S49 cells termed "deathless" that are resistant to cytolysis, but otherwise respond like the wild-type cells to cAMP. Upon removal of the cyclic nucleotide, D-cells resume their normal growth. Unlike all other cAMP-resistant mutants of S49 cells isolated until now, the D- mutant has a functionally normal cAMP-dependent protein kinase and retains normal ability to induce phosphodiesterase and arrest cell growth in G1. It is probable that the altered gene product of the D- mutant is distal to protein kinase and in a biochemical pathway separate from that of cAMP induction of phosphodiesterase or growth arrest. The D- mutant may facilitate studies of the mechanism of cAMP-induced cytolysis and growth regulation in S49 cells.     

Cyclic AMP phosphodiesterase in Salmonella typhimurium: characteristics and physiological function.

The physiological function of cyclic AMP (cAMP) phosphodiesterase in Salmonella typhimurium was investigated with strains which were isogenic except for the cpd locus. In crude broken-cell extracts the properties of the enzyme were found to be similar to those reported for Escherichia coli. The specific activity in the mutant was less than 1% that in the wild type. Rates of cAMP production in the mutant were as much as twice those observed in the wild type. The amount of cAMP accumulated when cells grew overnight with limiting glucose was 4.5-fold greater in the mutant than in the wild type. The intracellular concentration of cAMP in the two strains was measured directly, using four different techniques to wash the cells to remove extracellular cAMP. The cAMP level in the cpd strain was only 25% greater than in the wild type. The functional concentration of the cAMP receptor protein-cAMP complex was estimated indirectly from the specific activity of beta-galactosidase in the two strains after introducing F'lac. When cells were grown with carbon sources permitting synthesis of different levels of cAMP, the specific activity of the enzyme was at most 25% greater in the cpd strain. The cpd strain was more sensitive to the effects of exogenous cAMP. Exogenous cAMP relieved both permanent and transient catabolite repression of the lac operon at lower concentrations in the cpd strain than in the wild type. When cells grew with glucose, glycerol, or ribose, exogenous cAMP inhibited growth of the mutant strain more than the wild type.     

Regulation of cyclic AMP synthesis in Escherichia coli K-12: effects of the rpoD800 sigma mutation, glucose, and chloramphenicol

An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C. In the present study we characterize the nature of the inhibition. The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP). We inferred that the inhibition might be mediated by a decreased intracellular concentration of cAMP. Consistent with this inference, the rate of cAMP accumulation in mutant cells after a temperature upshift was depressed relative to that in wild-type cells. Glucose and chloramphenicol, two agents known to inhibit differentially beta-galactosidase mRNA synthesis, caused a similar inhibition in the rate of cAMP accumulation. Thus, three diverse stimuli, glucose, chloramphenicol, and a temperature-sensitive sigma mutation, appear to affect beta-galactosidase synthesis by regulating the synthesis of cAMP.     

Isolation of a catabolite repression mutant of yeast as a revertant of a strain that is maltose negative in the respiratory-deficient state.

A character originating from Saccharomyces cerevisiae 1403-7A is described which interferes with maltose growth in the respiratory-deficient state. This character is inherited in an apparently non-Mendelian way, but at present no statement can be made concerning the localization of this character on a plasmid or the involvement of multiple genes. As a revertant of this character, a flaky mutant was isolated, showing a heavy flocculation during growth on liquid medium and resistance to catabolite repression for maltase, alpha-methyl-glucosidase, invertase, and succinate dehydrogenase. In wild-type cells, repression (caused by growth on 2% glucose) and derepression (caused by growth on 2% galactose) can be correlated with a lower and a higher level of cyclic 3',5'-adenosine monophosphate (cAMP), respectively. In cells of flaky mutant, growth on these carbon sources results in the same levels of cAMP as observed for the wild type. Consequently, in this mutant derepression in the presence of 2% glucose is not reflected in a higher level of cAMP.     

Antipain lethality to Escherichia coli: dependence upon cyclic adenosine 3'',5''-monophosphate and its receptor protein.

Antipain kills Escherichia coli K-12 cells in an exponential manner beginning 1 h after its addition. Mutant strains, delta cya and crp, which are unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) and the cAMP receptor protein, respectively, are not affected. Addition of cAMP (5 mM) to antipain-treated mutant strains causes killing of delta cya cells, but not crp cells. Thus the lethal effect of antipain is dependent upon cAMP and its receptor protein.     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.     

Amplification of the adenylate cyclase gene in Escherichia coli cells

Amplification of the cya gene of E. coli on the plasmid pBR325 leads to an increase of adenylate cyclase activity proportional to the gene dosage. In strains harboring hybrid plasmids with cya gene the intracellular level of cAMP and the rate of nucleotide secretion are also elevated. The adenylate cyclase activity in cells with truncated cya gene cloned on pBR322 remains sensitive to glucose inhibition. Amplification of the cya gene leads to considerable resistance of beta-galactosidase synthesis to transient repression by alpha-methylglucoside, but does not influence the permanent repression caused by glucose.     

Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli

We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.     

Agar plate screening procedure for cyclic adenosine 3',5'-monophosphate and inhibitors of cyclic nucleotide phosphodiesterase.

A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from barbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.     

Agar plate screening procedure for cyclic adenosine 3',5'-monophosphate and inhibitors of cyclic nucleotide phosphodiesterase.

A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from barbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.