A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae

Abstract

The bacterial second messenger bis-(3′-5′)-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain–containing diguanylate cyclases and degraded by HD-GYP domain–containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain–containing PDE-As to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation. The bacterial second messenger bis-(3′-5′)-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain–containing diguanylate cyclases and degraded by HD-GYP domain–containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain–containing PDE-As to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation. Bis-(3′-5′)-cyclic diguanylate monophosphate (c-di-GMP) is a widely conserved bacterial second messenger found in all major bacterial phyla (1Romling U. Galperin M.Y. Gomelsky M. Cyclic di-GMP: The first 25 years of a universal bacterial second messenger.Microbiol. Mol. Biol. Rev. 2013; 77: 1-52Google Scholar). It was first discovered in 1987 as an activator of the cellulose synthase from Komagataeibacter xylinus (formerly known as Gluconacetobacter xylinus) by Ross et al. (2Ross P. Weinhouse H. Aloni Y. Michaeli D. Weinberger-Ohana P. Mayer R. Braun S. de Vroom E. van der Marel G.A. van Boom J.H. Benziman M. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid.Nature. 1987; 325: 279-281Google Scholar). c-di-GMP is associated with diverse cellular processes, such as bacterial growth, motility, virulence, biofilm formation, and cell cycle progression (3Jenal U. Reinders A. Lori C. Cyclic di-GMP: Second messenger extraordinaire.Nat. Rev. Microbiol. 2017; 15: 271-284Google Scholar). Low levels of cellular c-di-GMP upregulate motility by inducing flagellar expression, assembly, or motor function (4Hengge R. Principles of c-di-GMP signalling in bacteria.Nat. Rev. Microbiol. 2009; 7: 263-273Google Scholar) and are also required for the expression of acute virulence genes (5Tamayo R. Pratt J.T. Camilli A. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis.Annu. Rev. Microbiol. 2007; 61: 131-148Google Scholar). In contrast, high c-di-GMP levels stimulate the biosynthesis of fimbriae, adhesins, various matrix exopolysaccharides, and biofilm formation (4Hengge R. Principles of c-di-GMP signalling in bacteria.Nat. Rev. Microbiol. 2009; 7: 263-273Google Scholar, 6Jenal U. Malone J. Mechanisms of cyclic-di-GMP signaling in bacteria.Annu. Rev. Genet. 2006; 40: 385-407Google Scholar, 7Romling U. Amikam D. Cyclic di-GMP as a second messenger.Curr. Opin. Microbiol. 2006; 9: 218-228Google Scholar). Moreover, c-di-GMP–dependent spatiotemporal control of protein degradation is crucial in cell cycle progression in Caulobacter crescentus (8Duerig A. Abel S. Folcher M. Nicollier M. Schwede T. Amiot N. Giese B. Jenal U. Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression.Genes Dev. 2009; 23: 93-104Google Scholar). c-di-GMP is synthesized from two GTP molecules by diguanylate cyclases harboring the GGDEF domain, named after its conserved motif Gly-Gly-Asp-Glu-Phe (3Jenal U. Reinders A. Lori C. Cyclic di-GMP: Second messenger extraordinaire.Nat. Rev. Microbiol. 2017; 15: 271-284Google Scholar). The degradation of c-di-GMP is mediated by two types of phosphodiesterases (PDEs) containing either the HD-GYP or EAL domain (9Schirmer T. Jenal U. Structural and mechanistic determinants of c-di-GMP signalling.Nat. Rev. Microbiol. 2009; 7: 724-735Google Scholar). HD-GYP domain proteins hydrolyze c-di-GMP to two molecules of GMP in a one-step reaction (10Bellini D. Caly D.L. McCarthy Y. Bumann M. An S.Q. Dow J.M. Ryan R.P. Walsh M.A. Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre.Mol. Microbiol. 2014; 91: 26-38Google Scholar). In contrast, EAL domain-containing PDEs (PDE-As) degrade c-di-GMP into the linear product 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG) (11Schmidt A.J. Ryjenkov D.A. Gomelsky M. The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: Enzymatically active and inactive EAL domains.J. Bacteriol. 2005; 187: 4774-4781Google Scholar, 12Tamayo R. Tischler A.D. Camilli A. The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase.J. Biol. Chem. 2005; 280: 33324-33330Google Scholar), requiring a second PDE enzyme (PDE-B), which hydrolyzes pGpG into two GMP molecules, to complete the signaling process (13Cohen D. Mechold U. Nevenzal H. Yarmiyhu Y. Randall T.E. Bay D.C. Rich J.D. Parsek M.R. Kaever V. Harrison J.J. Banin E. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11359-11364Google Scholar). In the two-step degradation mechanism via pGpG, the intermediate pGpG has been reported to competitively inhibit c-di-GMP-specific PDE-As catalyzing the first reaction by a feedback inhibition mechanism. The PDE activity of Escherichia coli YfgF, an EAL-containing PDE-A, has been found to be inhibited by high concentrations of pGpG (14Lacey M.M. Partridge J.D. Green J. Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response.Microbiology (Reading). 2010; 156: 2873-2886Google Scholar). Furthermore, pGpG acts as a competitive inhibitor of the EAL-containing response regulator RocR and inhibits c-di-GMP turnover in Pseudomonas aeruginosa (15Orr M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). These findings suggest that the appropriate degradation of pGpG by PDE-B is crucial for sustaining the c-di-GMP signaling pathway. Recently, a high-throughput DRaCALA-based screen on a Vibrio cholerae El Tor N16961 complete genome ORF library has revealed several pGpG-binding proteins including oligoribonuclease VC0341 (hereafter VcOrn), as well as EAL and HD-GYP domain proteins, and some proteins of unknown functions. which an domain was to pGpG PDE to the findings in P. aeruginosa novel oligoribonuclease of Escherichia and Biol. Chem. Scholar, Oligoribonuclease is by a conserved in the Bacteriol. Scholar, J.D. H. A. H. Lee V.T. a for the of Scholar). its in P. has been to be an M. C. J.J. The conserved bacterial is in Vibrio a in virulence, stress and 2014; Scholar), to that in E. which is for the degradation of from to S. Oligoribonuclease is an of the Natl. Acad. Sci. U. S. A. Scholar). the function of protein to be a of turnover J.D. H. A. H. Lee V.T. a for the of Scholar). In the intracellular concentration of c-di-GMP is by the specific activities of its to the (4Hengge R. Principles of c-di-GMP signalling in bacteria.Nat. Rev. Microbiol. 2009; 7: 263-273Google Scholar). we could degrade pGpG to diverse and thus the of a PDE-B which is for the c-di-GMP in V. in the of These to for a PDE-B that is for the degradation of pGpG in the c-di-GMP signaling Here, we that V. cholerae is a pGpG-specific PDE-B that is conserved the and which are in the of in various bacterial we the basis for the narrow substrate of and its as a of cellular c-di-GMP levels in V. cholerae. we the activity of using several including pGpG as a substrate to its in E. could hydrolyze including and a the and pGpG, which is with a J.D. H. A. H. Lee V.T. a for the of Scholar). the activity of be by found that the activity of was inhibited in the of excess of and and inhibition by was also we the activity of V. cholerae N16961 cell in the and of excess of we found that the of on the activity of the was that on the in These to the of pGpG-binding for the specific degradation of pGpG in V. of PggH and activity Orn, in a PggH, Orn, has also been reported to pGpG in V. cholerae El Tor N16961 (15Orr M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). An of the primary structure of revealed that protein and can be as a The are in the of in various bacterial R. R. S. C. of proteins in and stress in and J. Biol. Scholar), such as the PDE which hydrolyzes various linear M. Structural and of the phosphodiesterase from 2017; PDE which hydrolyzes cyclic and D. D.C. is a signaling protein that a cyclic phosphodiesterase domain and a GGDEF domain with Biol. Chem. 2010; the U. S. V. A. from has oligoribonuclease and 2007; Scholar) in and the which is conserved in bacterial and T. Y. Y. N. S. R. of its for Biol. Chem. 2010; Scholar). we that we the PDE activity of toward several known of c-di-GMP, and by the oligoribonuclease activity of toward the and and the and exhibited PDE activity toward pGpG, the the and degraded by and that hydrolyze linear as well as pGpG, is a PDE that hydrolyzes we named PggH PggH is conserved only of the Vibrio protein the protein that the of PggH was of the Vibrio and of a protein in Vibrio Bacteriol. Scholar). domain proteins function as the R. D. A. A. R. B. in to substrate in 2014; Scholar). revealed that PggH a and PggH the PDE-B activity in the of the in biochemical in the of The and turnover of PggH for pGpG by the of pGpG substrate of the activity of PggH and of and and Since has been previously that the concentrations of pGpG a of 2 to a inhibition of c-di-GMP degradation by PDEs (13Cohen D. Mechold U. Nevenzal H. Yarmiyhu Y. Randall T.E. Bay D.C. Rich J.D. Parsek M.R. Kaever V. Harrison J.J. Banin E. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11359-11364Google Scholar), the of PggH suggest that enzyme a in the c-di-GMP in that in the activity of PggH was by and a in the of an excess of To the basis for the pGpG-specific PDE activity of PggH, we the structure of the protein in the of The two protein molecules that with with the in a The two in the a the was found in the a active the and The structure that is by and the active and are from the conserved motif In and are in the of in a with and are conserved PggH in Vibrio and that the with is for its activity as PDEs R. R. S. C. of proteins in and stress in and J. Biol. Scholar). the two and also the in the of the The domain of PggH a conserved which is known to be for the of in several of the M. Structural and of the phosphodiesterase from 2017; Scholar, Y. N. T. S. R. Crystal structure of the of from a of the of 2013; Scholar). To the of the motif in PggH, we to The pGpG activity of PggH, the of the motif in the with domain–containing proteins, we found several of all proteins including with an by the M. Structural and of the phosphodiesterase from 2017; Scholar, R. D. A. A. R. B. in to substrate in 2014; Scholar, Y. N. T. S. R. Crystal structure of the of from a of the of 2013; Scholar, Wang S. D. H. B. Wang H. J. Structural and biochemical into the mechanism of from as a phosphodiesterase.J. Biol. Chem. Scholar), PggH a the two The and in are by the domain of the in the domain the the two which also to the of the in response to the substrate Structural was found the The and in PggH are via a which is also from the in which a the two M. Structural and of the phosphodiesterase from 2017; Scholar, Y. N. T. S. R. Crystal structure of the of from a of the of 2013; Scholar). The of in the and in with the substrate M. Structural and of the phosphodiesterase from 2017; Scholar). and which was by was a of the and enzyme Scholar). to the domain of proteins, the domain of PggH a the of a findings suggest that PggH could of substrate and its which could the basis the narrow substrate specificity. the of PggH for linear and using to the mechanism of substrate in the of PggH with pGpG and to PggH and These that the in the substrate is crucial for the to PggH, the is for the formation of the active for with either or PggH that PggH has a narrow substrate specificity. we to of to PggH, we could To the crucial required for substrate we PggH in which the in the active and of for pGpG and of to to a for pGpG, a for to that by the These the the pGpG activity that is for the of for the In contrast, the of with the for that for pGpG, that is for to the substrate These on the and suggest that pGpG has a to by of to which the catalytic activity of Moreover, we found two in the of the The a for pGpG and the exhibited to the two that two be in the of and thus is for the catalytic in the for pGpG and that is in the substrate on we the of the PggH structure to pGpG and using a and J. M. C. that the Chem. 40: Scholar). In of pGpG and are in a narrow by and The of with and the of with of to the the of with the and the of is and is in with such as be by for the substrate of PggH the of the we with we to of pGpG and to PggH, the The substrate is by several of with the of and of and was by the which that the the for pGpG and of the the to be by of the The formation of of and the and of a in the pGpG the to could with in a In the and was to a by a that with is for the appropriate of the substrate that PggH is a pGpG-specific PDE with its we pGpG activities the cell from and a to the pGpG degradation by cellular PggH of the degraded pGpG into that proteins including and some M.W. Severin G.B. H. A. Waters C.M. Lee V.T. of as degradative for pGpG in c-di-GMP Bacteriol. Scholar). the of the activity of cell that PggH a in pGpG in V. cholerae biochemical revealed that the accumulation of pGpG could inhibit the c-di-GMP PDE activity of PDE-As (13Cohen D. Mechold U. Nevenzal H. Yarmiyhu Y. Randall T.E. Bay D.C. Rich J.D. Parsek M.R. Kaever V. Harrison J.J. Banin E. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11359-11364Google Scholar) and that appropriate degradation of pGpG is required for the of the c-di-GMP we PggH could the c-di-GMP signaling in V. cholerae. To the of PggH on the regulation of the c-di-GMP we intracellular concentrations of pGpG and c-di-GMP the and a using revealed that an concentration of pGpG that PggH the pGpG PDE activity in V. cholerae also found a of c-di-GMP in to that in to the that PggH is in the c-di-GMP To the of PggH in signaling we biofilm formation, which a high of c-di-GMP D. J.H. H. The and of cyclic di-GMP signaling in Vibrio Opin. Microbiol. 2017; Scholar, A.D. Camilli A. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm Microbiol. Scholar). that exhibited a activity that PggH regulates the c-di-GMP signaling in the The by the PggH expression to the V. cholerae exhibited a of biofilm formation with that of the the the inactive phenotypic These the of PggH in c-di-GMP signaling its activity was in the the expression could be that PggH can hydrolyze pGpG to the feedback inhibition of c-di-GMP It has been that c-di-GMP turnover by PDE-As is inhibited by pGpG (14Lacey M.M. Partridge J.D. Green J. Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response.Microbiology (Reading). 2010; 156: 2873-2886Google Scholar, M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). To that the biofilm formation of the is associated with the inhibition of PDE-As by the accumulation of pGpG and thus the concentration of c-di-GMP, the c-di-GMP PDE activity of PDE-A was in the or of PggH in Recently, we identified a novel PDE-A, named in V. cholerae Lee J. Lee regulation of a c-di-GMP phosphodiesterase in Vibrio Scholar). the expression of in the that of PggH also and we that was to the of PggH on the activity of PDE-As and the c-di-GMP the PDE activity of was by the of PggH that the product pGpG inhibits the activity of PDE-A by a feedback mechanism and that PggH as a of PDE-A by the pGpG a the c-di-GMP PDE and biofilm formation, using which an of of a to the exhibited an in biofilm formation to a to that of the harboring which an inactive of Lee J. Lee regulation of a c-di-GMP phosphodiesterase in Vibrio Scholar), the that the The biofilm formation of the was also we the with an expression for a PDE R. Tischler A.D. Camilli A. The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase.J. Biol. Chem. 2005; 280: 33324-33330Google Scholar). on we that PggH regulates c-di-GMP signaling by the activities of PDE-As in V. cholerae. The degradation of by is a process for cell in of and the of cyclic In the only protein previously identified to degrade is the oligoribonuclease Orn, and its roles in c-di-GMP signaling been well in P. aeruginosa (15Orr M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar, J.D. H. A. H. Lee V.T. a for the of Scholar). in bacterial in which is for cell growth, such as E. coli and V. is in cyclic signaling has been In we two proteins that been reported to pGpG in the V. cholerae from the DRaCALA-based screen (15Orr M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). Biochemical that and PggH to the cellular pGpG and the of the c-di-GMP as a high of pGpG inhibits the c-di-GMP PDE activity of PDE-As in V. cholerae (13Cohen D. Mechold U. Nevenzal H. Yarmiyhu Y. Randall T.E. Bay D.C. Rich J.D. Parsek M.R. Kaever V. Harrison J.J. Banin E. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11359-11364Google Scholar, M.M. Partridge J.D. Green J. Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response.Microbiology (Reading). 2010; 156: 2873-2886Google Scholar, M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). PggH a Orn, in to a Orn, and and that has a of from to levels in the pGpG pGpG and its activity is in the of can be that pGpG activity of the two proteins be in the cell pGpG the major in the of pGpG as well as PggH a in the degradation of pGpG pGpG concentrations to for a of the c-di-GMP signaling is with that the expression of either PggH or in the biofilm formation by and These suggest that PggH could a in the degradation of pGpG and the of c-di-GMP signaling in specific in V. cholerae. the structure of PggH high to into the pGpG-specific activity of PDEs substrate to various which could be by an by a substrate M. Structural and of the phosphodiesterase from 2017; Scholar, Y. N. T. S. R. Crystal structure of the of from a of the of 2013; Scholar, Wang S. D. H. B. Wang H. J. Structural and biochemical into the mechanism of from as a phosphodiesterase.J. Biol. Chem. Scholar). to the active of in the of substrate PggH has a and narrow active we the of active of PggH and using with a of we found that PggH has an active In a the and and the of PggH the These lead to an narrow substrate of PggH to that by and can a on cyclic Here, we found that the degradation of pGpG by PggH is inhibited in the of excess of that can be the degradation of J. J. M. S. H. B. and of phosphodiesterases that degrade 2015; Scholar). been reported to activity that or degrades c-di-GMP and Wang B. J. GGDEF cyclic Natl. Acad. Sci. U. S. A. Scholar, J.J. B. Second and HD-GYP phosphodiesterases Microbiol. Scholar, M. U. of phosphodiesterase of a novel and its J. Scholar), that the concentration of two cyclic are In is that PggH could as a in the the c-di-GMP and signaling pathway. Moreover, to the of PggH is the first identified enzyme that degrades that on the regulation of the pGpG activity of PggH to the c-di-GMP signaling in V. cholerae. The bacterial and in are in and in using the reaction and by To a of the V. cholerae N16961 the and of the by using the and of the two the and into the of the to the The E. coli was with V. cholerae and the was by and the in containing of in the was by protein regulates activity in response to in Vibrio Microbiol. 2015; Scholar, D. Vibrio cholerae of to the in the of Scholar). To and the was by using the and using the the ORF by from E. coli using the and was with and and into the of S. Lee of a flagellar biosynthesis protein from the Microbiol. Scholar, S. J. Lee Lee Lee protein protein in Vibrio Microbiol. 112: Scholar). To and the ORF was by using the and using the proteins and in the E. coli by and for The proteins using to the with and containing proteins using To the of proteins and the to using a with M. H. Structural into of the 9: Scholar, M. Lee The the for 2017; 7: Scholar). activity was in a reaction containing and concentrations of pGpG and for the the reaction was by the of and was to a concentration of The reaction product from pGpG was by using a to an reaction was to a with and in and using a linear of to containing and a of for The by the activity of in Escherichia Natl. Acad. Sci. U. S. A. Scholar). The activities of PggH and in was by with the and the substrate and product for V. cholerae in an of by for The was and the was in PDE which and by two a cell pGpG was to the to a concentration of and the was into for the the reaction was by the of and was to a concentration of The reaction product from pGpG was by (15Orr M.W. Donaldson G.P. Severin G.B. Wang J. Sintim H.O. Waters C.M. Lee V.T. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5048-E5057Google Scholar). The of the protein as previously J. P. J. P. S. H. and of with a c-di-GMP activity in Vibrio 2017; Scholar). the by the protein and the containing and 2 by the The to the with for and in for To the was in of several The of on a in of of a of and The of PggH to the with cell of a and The was to and the of in Scholar). are in from found in and the resulting was to an using the and N. J.J. A.J. R. D.C. et for structure Biol. 2010; Scholar, P. for Biol. Scholar). The structure was by using the the experiments 25 using experiments of a of by of 2 pGpG, and in 25 and into the reaction cell in the The using with a in with and in a for and with and the using several using the biofilm was with and Lee J. Lee regulation of a c-di-GMP phosphodiesterase in Vibrio Scholar). J. M. C. that the Chem. 40: Scholar) was to the of pGpG and to the PggH To for protein changes by substrate for the the using a H. Lee C. using a function and its to in 2014; Scholar). of pGpG and on the protein by and the with and structure been in the Lee J. Lee regulation of a c-di-GMP phosphodiesterase in Vibrio Scholar, J.J. novel and its in of of proteins and virulence determinants in Vibrio cholerae Bacteriol. Scholar, D.L. R. P. H. is for the virulence of Vibrio Bacteriol. Scholar, Y. of the Vibrio and regulation of its expression by the regulator in response to oxidative stress and iron Biol. Chem. 2014; Scholar). The that of with the of was by the and by the of and and by the H. and H. and Y. S. and J. H. C. and S. and H. and S. S. with

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