Cloning of the adenylate cyclase gene of Escherichia coli K-12

The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325. The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site. The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site. The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome. The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression. Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate.     

Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli

A 5.0-kilobase-pair HindIII fragment of Bradyrhizobium japonicum DNA containing the cya gene which encodes adenylate cyclase was isolated as an insert in pBR322, using marker rescue of the maltose-negative phenotype of an Escherichia coli cya mutant for identification. The isolated B. japonicum DNA fragment was capable of reversing the pleiotropic phenotype of cya mutations when inserted in either orientation in the HindIII site of pBR322. The complemented E. coli strains produced high levels of cyclic AMP. No sequence homology between the B. japonicum cya gene and that of E. coli was detected by hybridization analysis.     

Analysis of the cya locus of Escherichia coli

A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed. Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein. Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts. When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose. The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.     

Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica.

Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.     

High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system

The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.     

Mechanism of catabolite repression in Escherichia coli bacteria: interaction between transport proteins and adenylate cyclase

The mechanism of catabolite repression caused by sugar transported via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) and stipulated by the decrease of the adenylate cyclase activity was studied. It was demonstrated that the sensitivity of the adenylate cyclase and beta-galactosidase synthesis to methyl-L-D-glucoside (MeGlc) or sorbitol is correlated with the content and activity of glucose (EIIGlc) or mannitol enzyme II of the PTS, correspondingly. Under anaerobic conditions the cells become insensitive to catabolic repression caused by MeGlc and the adenylate cyclase activity does not decrease in the presence of the sugar despite the increased rate of MeGlc transport. The adenylate cyclase activity of the mutant with the Tn5 transposone inserted into the ptsG gene does not change in the presence of MeGlc, while the activity of adenylate cyclase and the differential rate of beta-galactosidase synthesis increase in these bacteria. The data obtained confirm the hypothesis on the "catabolite signal" which is generated when the substrate binds to its transporter, i. e. adenylate cyclase reacts to the conformational changes in the transporter being complexed with it. The strength of this complex depends on the affinity of adenylate cyclase for the transporter and on the value of the membrane potential, delta mu H+ A model is proposed, which explains the necessity of factor IIIGlc for EIIGlc binding to adenylate cyclase.     

Identification of the Escherichia coli cya gene product as authentic adenylate cyclase

The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase. Finally, the protein has been submitted to amino acid sequence analysis. It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .     

Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene.

From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively. The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis. They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression. Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background. Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose. The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex. All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region. The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex.     

A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli.

Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.     

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.     

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.     

Effect of catabolite repression on the mer operon

The plasmid-determined mer operon, which provides resistance to inorganic mercury compounds, was subject to a 2.5-fold decrease in expression when glucose was administered at the same time as the inducer HgCl2. This glucose-mediated transient repression of the operon was overcome by the addition of cyclic AMP. Permanent catabolite repression of the operon was observed in the 1.6- to 1.9-fold decrease in expression in mutants lacking either adenyl cyclase (cya) or the catabolite activator protein (crp). The effect of the cya mutation on mer expression could be overcome by the addition of cyclic AMP at the time of induction, In addition to these effects on the whole cells of a wild-type strains, we examined the effect of catabolite repression on the expression of the mercuric ion [Hg(II)] reductase enzyme, assayable in cell extracts, and on the Hg(II) uptake system, assayable in a mutant strain which lacked reductase activity. There was a two- to threefold effect of repression on the Hg(II) reductase enzyme assayable in vitro after induction under catabolite repressing conditions (either with glucose or in the crp and cya mutants). We did not find a similar repressing effect on the induction of the Hg(II) uptake system, which is also determined by the mer operon.     

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase

The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.     

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.     

Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: Sequence similarity to eukaryotic cyclases

Abstract A second adenylate cyclase ( cya2 ) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli Acya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similiar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cyal/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene.