Adenylate Cyclase Synthesis Research Articles

The cyclic AMP (cAMP) phosphodiesterase CpdA required for growth, biofilm formation, motility and pathogenicity of Edwardsiella piscicida

Edwardsiella piscicida is a severe fish pathogen with wide host range, causing the huge economic losses in the aquaculture industry. Cyclic adenosine monophosphate (cAMP) as an important second messenger regulates the physiological and behavioral responses to environmental cues in eukaryotic and prokaryotic. The intracellular level of cAMP for effective activity is tightly controlled by the synthesis of adenylate cyclase, excretion and degradation of phosphodiesterase. In this study, we identified and characterized a class III cAMP phosphodiesterase, named as CpdA, in the E. piscicida. To investigate the role of CpdA in the physiology and pathogenicity, we constructed the in-frame deletion mutant of cpdA of E. piscicida, TX01ΔcpdA. The results showed that TX01ΔcpdA accumulated the higher intracellular cAMP concentration than TX01, indicating that CpdA exerted the hydrolysis of cAMP. In addition, compared to the TX01, the TX01ΔcpdA slowed growth rate, diminished biofilm formation and lost motility. More importantly, pathogenicity analysis confirmed that TX01ΔcpdA significantly impaired the ability of invading the epithelial cells, reproduction in macrophages, tissues dissemination and lethality for healthy tilapias. The most of lost properties of TX01ΔcpdA were restored partially or fully by the introduction of cpdA gene. These results suggest that cpdA is required for regulation of the physiology and virulence of E. piscicida.

Polyamine Enhancement of the Synthesis of Adenylate Cyclase at the Translational Level and the Consequential Stimulation of the Synthesis of the RNA Polymerase ς28 Subunit

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

Cyclic AMP in prokaryotes.

Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth.

Cyclic AMP in prokaryotes.

Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth.

Molecular mechanism of drug tolerance and dependence

Morphine and ethanol drugs known to develop tolerance and dependence, induce changes in the adenylate cyclase system. Morphine inhibits the adenylate cyclase activity in NG108-15 cells and causes increases in adenylate cyclase synthesis and the down-regulation of opiate receptors in cells treated for several days. Chronic exposure of NG108-15 cells to ethanol also causes a decrease in the mRNA of the GTP-binding protein (Gs). These observations suggest the possibility that a group of genes is expressed in response to morphine or ethanol during the acquisition of tolerance and dependence. Recently, it has been reported that cAMP regulates a number of genes through a cAMP response element (CRE) in their promotor regions and that nuclear CRE-binding proteins bind specifically to the CRE to stimulate the transcription of cAMP-responsive genes. The gel shift assay with a single stranded oligo-DNA of CRE in a somatostatin promotor region was employed to examine the possibility of transcriptional regulation of cAMP-inducible genes by chronic morphine or ethanol treatment of NG108-15 cells. When the nuclear proteins from the cells treated with morphine or ethanol for several days were provided for the assay, the amounts of DNA-protein complex were decreased. The decreased complexes were recovered by 1-2 days after morphine withdrawal. The nuclear proteins were purified partially by a combination of chromatography on Q-Sepharose, Sephacryl S-300 and DNA affinity-Sepharose. Changes in CRE-binding proteins from the cells treated chronically with morphine or ethanol suggest that these drugs can modulate the expression of cAMP-inducible genes through which tolerance and dependence may develop.

Aspects of the regulation of adenylate cyclase synthesis in Escherichia coli K12.

In Escherichia coli K12 expression of the adenylate cyclase gene is subject to multiple controls. In order to gain understanding of the regulation of adenylate cyclase synthesis, operon and protein fusions were constructed by in vitro recombination either into bacteriophage lambda or low-copy-number plasmids, or directly on the chromosome at the cya locus. The fusions were used in physiological experiments as probes to study transcriptional and translational controls of cya expression. It was found that adenylate cyclase synthesis was insensitive to glucose effects. As already described by other workers, the CAP-cAMP complex had a moderate negative control on cya expression. In addition it was observed that concomitant with a severe slackening of growth rate, specific to the growth of cya strains in rich medium, cya expression was considerably enhanced. This increase of adenylate cyclase synthesis did not appear to be directly dependent on the presence of a functional cAMP receptor (CAP), and seemed to be controlled at the level of transcription. Finally, translation of the cya message was very weak when compared to cya transcription (the mRNA level was the same in protein and operon fusions.

Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system.

The phosphoenolpyruvate:glucose phosphotransferase system (PTS) of Salmonella typhimurium is involved both in glucose transport and in the regulation and synthesis of adenylate cyclase and several transport systems. The crr gene has been implicated in this regulating mechanism. A 9.6-kb segment of the S. typhimurium chromosome containing the crr gene was cloned in pAT153. The cloned fragment also complemented cysA mutations but did not contain a functional pts operon which is closely linked to the crr gene and codes for two enzymes of the PTS. Although cysA and crr have been reported to be located on opposite sides of ptsHI, our results suggest that the correct gene order is cysK-ptsHI-crr-cysA. Expression of crr plasmids in a maxicell system yielded two proteins which reacted with specific anti-serum against IIIGlc. The apparent mol. wts. in SDS-polyacrylamide gels were 20 000 and 21 000, the former corresponding to the major band of purified IIIGlc. Both forms were also observed in bacterial extracts and purified IIIGlc. The crr gene was localized on a 1-kb EcoRI-EcoRV fragment of the 9.6-kb insert and sequenced. It codes for a single protein (18 556 D) containing 169 amino acid residues and identified as IIIGlc.

Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region.

The regulatory region of the cya gene from Escherichia coli has been characterized by nucleotide sequence analysis and genetic approaches. Two promoters, P1 and P2, organized in that order with respect to the beginning of the cya open reading frame, were identified. Using cya-lac operon and protein fusions, it was possible to show that both promoters are active in vivo. P1 activity seemed sensitive to catabolite repression whereas activity of the stronger promoter, P2, did not respond to inhibition by glucose. No effect of cAMP or its receptor, catabolite activator protein (CAP), could be found although the DNA sequence reveals a consensus CAP site downstream of P2. The 548 nucleotides situated at the 3' end of the sequence carry an open reading frame which can tentatively be assigned to the beginning of adenylate cyclase. Among noteworthy features of the corresponding sequence are an UUG codon as the putative start site of cyclase, and a long hydrophobic stretch of amino acids resembling leader peptides in secreted or membrane proteins.

Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains.

We have isolated cya-lac operon and protein fusions in Escherichia coli K-12, and we used these to study the regulation of cya, the structural gene for adenylate cyclase. Data obtained from these fusion strains suggest that neither cyclic AMP (cAMP) nor the cAMP receptor protein plays a major role in transcriptional or translational regulation of cya expression. Modulation of intracellular cAMP concentrations elicited only weak repression of cya-lac fusion activity under conditions of high intracellular cAMP, relative to fusion activity under conditions of low intracellular cAMP. The functional cAMP receptor protein was required for this effect. Incorporation of delta crp into cya-lac fusion strains did not affect fusion expression in glucose-grown cells as compared with similarly cultured isogenic crp+ strains. Furthermore, 20 independently obtained mutants derived from a cya-lacZ protein fusion strain exhibiting a weak Lac+ phenotype were isolated, and it was determined that the mutants had beta-galactosidase activities ranging from 2- to 77-fold greater than those of the parental strain. None of the mutations responsible for this increase in fusion activity map in the crp locus. We used these mutants to aid in the identification of a 160,000-dalton cya-lacZ hybrid protein. Finally, chromosome mobilization experiments, using cya-lac fusion strains, allowed us to infer a clockwise direction of transcription for the cya gene relative to the standard E. coli genetic map.

Multiple regulation of the activity of adenylate cyclase in Escherichia coli.

We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3',5' cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.

Cellular levels, excretion, and synthesis rates of cyclic AMP in Escherichia coli grown in continuous culture.

Changes in dilution rate did not elicit large and systematic changes in cellular cyclic AMP levels in Escherichia coli grown in a chemostat under carbon or phosphate limitation. However, the technical difficulties of measuring low levels of cellular cyclic AMP in the presence of a large background of extracellular cyclic AMP precluded firm conclusions in this point. The net rate of cyclic AMP synthesis increased exponentially with increasing dilution rate through either the entire range of dilution rates examined (phosphate limitation) or a substantial part of the range (lactose and glucose limitations). Thus, it is probable that growth rate regulates the synthesis of adenylate cyclase. The maximum rate of net cyclic AMP synthesis was greater under lactose than under glucose limitation, which is consistent with the notion that the uptake of phosphotransferase sugars is more inhibitory to adenylate cyclase than the uptake of other carbon substrates. Phosphate-limited cultures exhibited the lowest rate of net cyclic AMP synthesis, which could be due to the role of phosphorylated metabolites in the regulation of adenylate cyclase activity. Under all growth conditions examined, greater than 99.9% of the cyclic AMP synthesized was found in the culture medium. The function of this excretion, which consumed up to 9% of the total energy available to the cell and which evidently resulted from elaborate regulatory mechanisms, remains entirely unknown.

Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains.

Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains.

Isolation and characterization of an Escherichia coli mutant affected in the regulation of adenylate cyclase.

A mutant, cyaR1, affecting regulation of adenylate cyclase expression or activity is described. It was obtained as a thermoresistant revertant of a strain harboring a thermosensitive transcription termination factor, rho (rho-15). This mutant failed to synthesize adenosine 3',5'-phosphate and exhibited a carbohydrate-negative phenotype. A secondary mutation at the crp locus (crpC) restored the ability of the mutant to synthesize adenosine 3',5'-phosphate, enabled the expression of catabolite-sensitive operons, and conferred on the strain an extreme sensitivity to catabolite repression. In addition, we showed that the crpC mutation restored the pleiotropic carbohydrate-positive phenotype even in a delta cya background. We interpret this to mean that the adenosine 3',5'-phosphate receptor protein regulates negatively either the activity or synthesis of adenylate cyclase and that the cyaR1 mutation is either in a regulatory protein or a regulatory site of adenylate cyclase.

Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex.

The synthesis of the adenylate cyclase [ATP pyrophosphatelyase-(cyclizing), E.C. 4.6.1.1.] of Escherichia coli, appears to be regulated negatively by the cAMP receptor protein, CRP. This conclusion is based on a comparison of adenylate cyclase activities measured in vitro with the rates of cAMP synthesis by intact bacteria. The activity of adenylate cyclase, depending on conditions of growth, is also regulated by CRP; this effect, however, is indirect insofar as it is mediated by a protein or proteins under CRP control.

Variations du taux d'amp cyclique et des activités spécifiques de l'adénylate cyclase et de l'amp cyclique-phosphodiestérase pendant le cycle cellulaire d'un actinomycète

The variations in the concentrations of intra- and extracellular cyclic AMP and in the specific activities of adenylate cyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) have been monitored in synchronized culture of Nocardia restricta, a prokaryote belonging to the group of Actinomycetes. At the beginning of the cell cycel, during a first period of RNA and protein synthesis, there is an increasing synthesis of adenylate cyclase which can be suppressed in the presence of chloramphenicol or rifampicin. Simultaneously, the specific activity of cyclic AMP phosphodiesterase decreases and the concentrations of intra- and extracellular cyclic AMP rise. After the end of DNA replication, during a second period of RNA and protein synthesis, the specific activity of cyclic AMP phosphodiesterase increases; during the same time, the specific activity of adenylate cyclase and the level of intracellular cyclic AMP drop. It appears that the overall metabolism of cyclic AMP is coordinated so that the cyclic AMP level will be high at the beginning of DNA replication and will fall thereafter. The results are discussed in comparison with known data about the variations of cyclic AMP during the cell cycle of mammalian cells in cultures.

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum

Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.