Three cdg Operons Control Cellular Turnover of Cyclic Di-GMP in Acetobacter xylinum : Genetic Organization and Occurrence of Conserved Domains in Isoenzymes
| العنوان: | Three cdg Operons Control Cellular Turnover of Cyclic Di-GMP in Acetobacter xylinum : Genetic Organization and Occurrence of Conserved Domains in Isoenzymes |
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| المؤلفون: | Peter Ross, Roger D. Calhoon, Hing Cheung Wong, David H. Gelfand, Anna Lisa Fear, Haim Weinhouse, Patricia Ohana, Raphael Mayer, Dorit Amikam, Avital Cohen, Shai Sapir, Gail Volman, Moshe Benziman, Rony Tal |
| المصدر: | Journal of Bacteriology. 180:4416-4425 |
| بيانات النشر: | American Society for Microbiology, 1998. |
| سنة النشر: | 1998 |
| مصطلحات موضوعية: | Cyclic di-GMP, Diguanylate cyclase activity, Protein subunit, Molecular Sequence Data, Regulatory site, Biology, Microbiology, Conserved sequence, Open Reading Frames, chemistry.chemical_compound, Operon, EAL domain, Amino Acid Sequence, Cyclic GMP, Molecular Biology, DNA Primers, Recombination, Genetic, Base Sequence, Sequence Homology, Amino Acid, Gluconacetobacter xylinus, Phosphoric Diester Hydrolases, GGDEF domain, Enzymes and Proteins, Isoenzymes, Oxygen, Biochemistry, chemistry, biology.protein, Diguanylate cyclase |
| الوصف: | Cyclic di-GMP (c-di-GMP) acts as a reversible and highly specific positive effector of cellulose (1,4-β-d-glucan) synthase in the cellulose-producing bacterium Acetobacter xylinum (34, 35; for a review, see reference 36). This unique nucleotide stimulates the enzyme reaction rate up to 200-fold (activation constant [Kact] = 0.35 μM), such that the in vitro rate of cellulose synthesis may reach 50% of that in the intact cell (2, 32). c-di-GMP activation of purified cellulose synthase is kinetically independent of the concentration of the substrate UDP-glucose (UDP-glc), and its effect is exerted by binding directly to the enzyme at a regulatory site distinct from the catalytic site (35). The intracellular concentration of c-di-GMP is maintained by the opposing action of the enzymes diguanylate cyclase (DGC), which catalyzes its formation from two molecules of GTP, and Ca2+-sensitive phosphodiesterase A (PDEA), which in conjunction with phosphodiesterase B (PDEB) catalyzes its degradation to 5′-GMP (34). The pathway to c-di-GMP synthesis occurs via the linear dinucleotide pppGpG in two distinct PPi-releasing steps; the degradative pathway is initiated by PDEA, which cleaves a single phosphodiester bond in the cyclic structure, yielding the inactive linear dimer pGpG, which is converted to 5′-GMP by PDEB. Within the cell, c-di-GMP is tightly associated with the c-di-GMP binding protein (CDGBP), a membrane protein which exhibits saturable and reversible c-di-GMP binding with high affinity (46). The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+, such that the intracellular concentration of free c-di-GMP is only 10% of the overall intracellular c-di-GMP concentration (5 to 10 μM), consistent with the high K+ level in the cell. CDGBP is apparently structurally associated with the cellulose synthase, since both activities reside within highly purified cellulose synthase preparations and comigrate in gel filtration of solubilized membrane preparations. This structural association and implied physical proximity within the membrane may enable CDGBP to functionally regulate synthase activity in vivo by modulating the intracellular level of free c-di-GMP. Four proteins essential for bacterial cellulose synthesis are encoded by the bcs operon in A. xylinum (47). The first gene in the operon, bcsA, encodes the subunit of the cellulose synthase which binds the substrate UDP-glc and presumably catalyzes the polymerization of 1,4-β-d-glucan from glucose units (23). The second gene, bcsB, encodes the subunit which binds the activator c-di-GMP (25). Examination of a series of synthetic cyclic dimer and trimer analogs of c-di-GMP for the ability to interact with cellulose synthase and PDEA revealed that although the two enzymes have a similar high degree of specificity for the c-di-GMP structure, their cyclic dinucleotide sites are not identical (35). c-di-GMP may also be produced and active in other cellulose-producing systems. In Agrobacterium tumefaciens, the cellular occurrence of c-di-GMP and DGC and PDEA activities have been demonstrated, and addition of exogenous c-di-GMP to cell extracts enhances cellulose synthesis (4, 42). Cotton fiber extracts contain peptides which specifically bind c-di-GMP with high affinity in photolabeling studies (5). Labeling is most pronounced in fibers harvested at the developmental phase of maximal cellulose production, suggesting a physiological role for c-di-GMP in this process. Additional evidence that the plant kingdom utilizes c-di-GMP-dependent pathways for cellulose synthesis has been provided by the discovery of a unique saponin in both Pisum sativum and A. xylinum which acts as a specific inhibitor of the DGC reaction (29, 30). The intracellular abundance of this compound in numerous plant systems may be a significant factor which has thus far prevented in vitro detection of DGC activity in plants. In another significant finding, plant homologs of A. xylinum bcsA genes (39, 47) and of A. tumefaciens celA genes (24) encoding catalytic subunits of cellulose synthase have been isolated from cotton and rice cDNA libraries (31). These plant celA genes encode proteins containing three regions of conserved sequence with respect to the bacterial gene products, within which are found highly conserved subdomains proposed to be critical for catalysis and/or UDP-glc binding. Here we report the isolation of the cdg1, cdg2, and cdg3 operons, which encode homologous isoforms of DGC and PDEA. Each of these operons is organized with a pdeA gene upstream of a dgc gene, yet genetic disruption analyses indicate that they contribute differentially to cellular PDEA and DGC enzymatic activities. cdg1 is responsible for 80% of each activity and contains two flanking genes of as-yet-unknown function, cdg1a and cdg1d. The cdg1a gene product is similar in sequence to known prokaryotic transcriptional activators, suggesting that it has a regulatory role. The proteins encoded by the dgc and pdeA genes display a high degree of identity within each isoenzyme set, and significant structural conservation is also apparent between the two isoenzyme sets. In their N termini, all six isoenzymes contain domains similar to those found in various oxygen-sensing proteins, suggesting an oxygen-mediated mechanism of signal transduction for c-di-GMP metabolism and, ultimately, cellulose production. Further downstream, the DGC and PDEA sequences share a lengthy consensus motif, consisting of two adjacent domains termed GGDEF (16) and EAL. The organization of the cdg operons is distinguished by the juxtaposition of genes encoding enzymes of opposing action on the same genetic unit and by its multiplicity. The coordinate expression of pdeA and dgc provides a requisite balance of PDEA and DGC for achieving the optimal concentration of c-di-GMP, which is essential for a rate of cellulose synthesis in tune with environmental conditions. The presence of homologs in noncellulose-producing bacteria raises the possibility that c-di-GMP may be involved in additional cellular functions. |
| تدمد: | 1098-5530 0021-9193 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8f78e20d5553130ee253850af371bd60 https://doi.org/10.1128/jb.180.17.4416-4425.1998 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....8f78e20d5553130ee253850af371bd60 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10985530 00219193 |
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