The Experts below are selected from a list of 1116 Experts worldwide ranked by ideXlab platform

Zhao-xun Liang - One of the best experts on this subject based on the ideXlab platform.

  • Structure of a Diguanylate Cyclase from Thermotoga maritima: Insights into Activation, Feedback Inhibition and Thermostability
    PLoS ONE, 2014
    Co-Authors: Angeline Deepthi, Zhao-xun Liang, Chong Wai Liew, Kunchithapadam Swaminathan, Julien Lescar
    Abstract:

    Large-scale production of bis-39-59-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF Domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

  • yybt is a signaling protein that contains a cyclic dinucleotide phosphodiesterase Domain and a GGDEF Domain with atpase activity
    Journal of Biological Chemistry, 2010
    Co-Authors: Feng Rao, Rui Yin See, Dongwei Zhang, Delon Chengxu Toh, Zhao-xun Liang
    Abstract:

    The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase Domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 Domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase Domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 Domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (Km) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 Domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF Domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF Domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.

  • A Flavin Cofactor-Binding PAS Domain Regulates c-di-GMP Synthesis in AxDGC2 from Acetobacter xylinum
    Biochemistry, 2009
    Co-Authors: Feng Rao, Zhen Luo, Zhao-xun Liang
    Abstract:

    The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the obligate aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) Domain and two putative catalytic Domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS Domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF Domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential and unexpected modification of FAD in one of the mutants, none of the single mutations was able to completely disrupt the transmission of the signal to the GGDEF Domain, indicating that the change in the FAD redox state can still trigger structural changes in the PAS Domain probably by using substituted hydrogen-bonded water networks. Meanwhile, although the EAL Domain of AxDGC2 was found to be catalytically inactive toward c-di-GMP, it was capable of hydrolyzing some phosphodiester bond-containing nonphysiological substrates. Together with the previously reported oxygen-dependent activity of the homologous AxPDEA1, the results provided new insight into relationships among oxygen level, c-di-GMP concentration, and cellulose synthesis in A. xylinum.

  • a flavin cofactor binding pas Domain regulates c di gmp synthesis in axdgc2 from acetobacter xylinum
    Biochemistry, 2009
    Co-Authors: Yaning Qi, Zhao-xun Liang
    Abstract:

    The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the obligate aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) Domain and two putative catalytic Domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS Domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF Domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential...

  • catalytic mechanism of cyclic di gmp specific phosphodiesterase a study of the eal Domain containing rocr from pseudomonas aeruginosa
    Journal of Bacteriology, 2008
    Co-Authors: Ye Yang, Yaning Qi, Zhao-xun Liang
    Abstract:

    Cyclic di-GMP was first discovered as a regulator of cellulose synthesis in Glucoacetobacter xylinus and is emerging as a major bacterial second messenger (14, 27, 28, 37). The intracellular concentration of cyclic di-GMP is controlled by the GGDEF Domain proteins with diguanylate cyclase (DGC) activity and the EAL Domain proteins with cyclic di-GMP-specific phosphodiesterase activity (27). GGDEF Domains catalyze the condensation of two molecules of GTP to generate cyclic di-GMP, while the EAL Domains catalyze the hydrolysis of cyclic di-GMP to generate the dinucleotide 5′-pGpG (Fig. ​(Fig.1).1). A family of HD-GYP Domain proteins is also able to hydrolyze cyclic di-GMP to produce GMP (30), but the overwhelmingly large number of genes encoding the EAL Domains in bacterial genomes suggests that they are the major phosphodiesterases responsible for maintaining the cellular cyclic di-GMP concentration. FIG. 1. Phosphodiesterase A (PDE-A) catalyzes hydrolysis of cyclic-di-GMP to generate the linear diguanylic acid 5′-pGpG. Accumulating evidence suggests that EAL Domain-containing proteins, including a large number of proteins that contain both the EAL and GGDEF Domains, regulate a variety of cellular functions and phenotypes associated with bacterial infection. The regulation of virulence gene transcription, biofilm formation, motility, and adhesion has been reported in various pathogenic bacteria. Several proteins in the human pathogen Vibrio cholerae, including VieA and CdgC, have been implicated in biofilm formation, motility, and virulence factor production (15, 23, 40). An EAL Domain protein was found to control lateral flagellar-gene expression and swarming behavior in Vibrio parahaemolyticus (17). In Salmonella enterica, the disruption of the EAL Domain protein CdgR weakens bacterial resistance to hydrogen peroxide and accelerates bacterial killing by macrophages (12). In the opportunistic pathogen Pseudomonas aeruginosa, the EAL Domain-containing protein FimX controls twitching motility and biofilm formation (13, 16) and the BifA protein controls biofilm formation and swarming (19). A systematic analysis of the GGDEF and EAL Domain proteins in P. aeruginosa identified several other EAL Domain proteins as being involved in virulence expression and biofilm formation (16, 20). Therefore, although the EAL Domain proteins are not essential for the in vitro viability of pathogenic bacteria, they may be critical for the in vivo survival of the pathogens in host organisms, considering their roles in virulence expression and biofilm formation. This makes them potential targets for developing antibacterial agents that aim to neutralize virulence functions. The genomes of the bacteria that contain cyclic di-GMP signaling networks generally encode multiple EAL Domain proteins. For example, the genomes of P. aeruginosa PAO-1 and V. cholerae contain 21 and 32 open reading frames, respectively, for EAL Domain proteins. In previous biochemical studies of EAL Domain proteins, it was shown that Mg2+, or Mn2+, is required for the enzymes to hydrolyze cyclic di-GMP (31, 36, 38). It was also found that Zn2+ and Ca2+ can strongly inhibit the enzymatic activity, presumably by dislodging the Mg2+ ion. The Glu in the EAL (or EXL) signature motif seems to be essential for the enzymatic activity, because the E→A mutations in two EAL Domain proteins abolished their phosphodiesterase activities (6, 38). Additionally, Schmidt and coworkers suggested that other conserved motifs, including a DDFGTG motif, may be essential for the catalytic activity (31). The phosphodiesterase activity of the EAL Domain of CC3396 from Caulobacter crescentus can be stimulated by the binding of GTP to the adjacent enzymatically inactive GGDEF Domain (6). A similar observation was reported for the protein FimX, involved in the regulation of twitching motion in P. aeruginosa (16). The utilization of the GGDEF Domain could be a major strategy for regulating the catalytic activity of EAL Domains, considering the large number of proteins that contain the GGDEF-EAL diDomain. Meanwhile, the widespread occurrence of GGDEF-EAL Domains also raises the possibility that some EAL Domains may function as regulatory rather than catalytic Domains. However, due to limited information about the catalytic mechanism of EAL Domains, such a distinction has remained speculative. RocR was identified as a response regulator in the RocSAR (or SadARS) two-component signaling system in P. aeruginosa (18, 21, 29). RocSAR consists of the histidine kinase RocS1 and two response regulators, RocA1 and RocR. The RocSAR system controls bacterial biofilm formation and virulence gene expression by regulating the transcription of various genes, including the cup fimbrial-gene clusters and type III secretion system genes (18, 20, 21). Deduced from the protein sequence, RocR contains an N-terminal CheY-like phosphoryl receiver Domain and a C-terminal EAL Domain. It was postulated that RocR negatively regulates the expression of cup genes by antagonizing the activity of RocA1, which is a typical response regulator with a DNA-binding Domain (21). The detailed molecular mechanism for this antagonism is not known at present, though it has been speculated that the EAL Domain may function as a regulatory Domain lacking phosphodiesterase activity. Here, we present biochemical data to demonstrate that the EAL Domain of RocR is catalytically active, with cyclic di-GMP-specific phosphodiesterase activity. Using RocR as a model system, we carried out systematic mutagenesis in the EAL Domain to probe the roles of 14 conserved polar residues in catalysis. Based on the biochemical data and aided by the crystal structure of a homologous EAL Domain protein, we assigned functions to the conserved residues and proposed a general base-catalyzed mechanism with the assistance of the Mg2+ ion. In the context of the proposed catalytic mechanism, we rationalize the inactivity of some characterized EAL Domains and discuss the possibility of predicting the phosphodiesterase activities of EAL Domains based on protein sequences.

Ute Römling - One of the best experts on this subject based on the ideXlab platform.

  • gil a new c di gmp binding protein Domain involved in regulation of cellulose synthesis in enterobacteria
    Molecular Microbiology, 2014
    Co-Authors: Xin Fang, Ute Römling, Michael Y Galperin, Irfan Ahmad, Andrea Blanka, Marco Schottkowski, Annika Cimdins, Mark Gomelsky
    Abstract:

    Summary In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the Domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like Domain. The RxGD motif of the GIL Domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF Domain. Thus, GIL is the second protein Domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ Domain containing glycosyltransferase BcsA.

  • pseudomonas aeruginosa cupa encoded fimbriae expression is regulated by a GGDEF and eal Domain dependent modulation of the intracellular level of cyclic diguanylate
    Environmental Microbiology, 2007
    Co-Authors: Andree Meissner, Ute Römling, Verena Wild, Roger Simm, Manfred Rohde, Christian Erck, Florian Bredenbruch, Michael Morr, Susanne Häussler
    Abstract:

    Summary Cyclic-diguanylate (c-di-GMP) is a widespread bacterial signal molecule that plays a major role in the modulation of cellular surface components, such as exopolysaccharides and fimbriae, and in the establishment of a sessile life style. Here, we report that intracellular c-di-GMP levels influence cupA-encoded fimbriae expression in Pseudomonas aeruginosa. In an autoaggregative P. aeruginosa small colony variant (SCV) CupA fimbriae and the intracellular c-di-GMP concentration were found to be enhanced as compared with the clonal wild-type. The SCV morphology and the expression of CupA fimbriae were dependent on a functional PA1120 and morA gene both encoding a GGDEF Domain. Overexpression of the GGDEF Domain protein PA1120 complemented the PA1120 and the morA mutant with respect to CupA fimbriae expression. In agreement with these findings, overexpression of the EAL Domain containing phenotypic variance regulator (PvrR) in the SCV resulted in a decreased intracellular level of c-di-GMP, a reduced cupA fimbriae expression and a switch to wild-type colony morphology.

  • The role of c-di-GMP signaling in an Aeromonas veronii biovar sobria strain
    FEMS Microbiology Letters, 2007
    Co-Authors: Mokhlasur Rahman, Abdul Kader, Ute Römling, Roger Simm, Eugénie Bassères, Roland Möllby
    Abstract:

    Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria. To investigate the impact of c-di-GMP signaling, we introduced heterologous GGDEF and EAL Domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF Domain protein AdrA increased c-di-GMP concentration and biofilm formation and reduced motility. Production of the quorum-sensing signaling molecule C4-homoserine lactone and adhesion to aquatic plant duckweed and amoeba surfaces were enhanced. On the other hand, overexpression of the EAL Domain protein YhjH decreased biofilm formation and increased motility.

  • Hierarchical involvement of various GGDEF Domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium.
    Molecular Microbiology, 2006
    Co-Authors: Abdul Kader, Roger Simm, Michael Morr, Ulrich Gerstel, Ute Römling
    Abstract:

    Summary GGDEF and EAL Domain proteins are involved in the turnover of the novel secondary messenger cyclic-di(3′5′)-guanylic acid (c-di-GMP) in many bacteria. In this work the role of the 12 GGDEF Domain proteins encoded by the Salmonella enterica serovar Typhimurium (S. Typhimurium) chromosome in rdar morphotype development was investigated. Previously, it was shown that the GGDEF Domain protein AdrA activated the biosynthesis of cellulose by production of c-di-GMP. Enhancement of the c-di-GMP levels by overexpression of the GGDEF Domain protein AdrA did lead to the activation of curli fimbriae biosynthesis through the elevated expression of CsgD and CsgA. Although knock-out of the chromosomal copy of adrA influenced CsgA expression, CsgD expression was not altered, although more than half of the total cellular c-di-GMP was produced by AdrA at 16 h of growth. On the other hand, chromosomally encoded GGDEF–EAL Domain proteins STM2123 and STM3388 were required to additively activate CsgD expression on a transcriptional and post-transcriptional level. Enhanced c-di-GMP levels did overcome temperature regulation of rdar morphotype expression by activation of curli fimbriae as well as cellulose biosynthesis through CsgD expression. Thus in the regulatory cascade leading to rdar morphotype expression c-di-GMP activates several subsequent steps in the network.

  • phenotypic convergence mediated by GGDEF Domain containing proteins
    Journal of Bacteriology, 2005
    Co-Authors: Roger Simm, Abdul Kader, Ute Römling, Jacqueline D Fetherston, Robert D. Perry
    Abstract:

    GGDEF Domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF Domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.

Chinghong Yang - One of the best experts on this subject based on the ideXlab platform.

  • the GGDEF Domain protein gdpx1 attenuates motility exopolysaccharide production and virulence in xanthomonas oryzae pv oryzae
    Journal of Applied Microbiology, 2016
    Co-Authors: Fenghuan Yang, Shanshan Qian, Fang Tian, Huamin Chen, William Hutchins, Chinghong Yang
    Abstract:

    Aims Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-Domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although eleven genes encoding GGDEF-Domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A, the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-di-GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-Domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis. Methods and results GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-Domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production, and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in ∆gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity. Conclusion GdpX1 is the first GGDEF-Domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription. Significance and impact of study Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression. This article is protected by copyright. All rights reserved.

  • the degenerate eal GGDEF Domain protein filp functions as a cyclic di gmp receptor and specifically interacts with the pilz Domain protein pxo_02715 to regulate virulence in xanthomonas oryzae pv oryzae
    Molecular Plant-microbe Interactions, 2014
    Co-Authors: Fenghuan Yang, Fang Tian, Huamin Chen, Susu Fan, Chinghong Yang
    Abstract:

    Degenerate GGDEF and EAL Domain proteins represent major types of cyclic diguanylic acid (c-di-GMP) receptors in pathogenic bacteria. Here, we characterized a FimX-like protein (Filp) which possesses both GGDEF and EAL Domains in Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Both in silico analysis and enzyme assays indicated that the GGDEF and EAL Domains of Filp were degenerate and enzymatically inactive. However, Filp bound to c-di-GMP efficiently within the EAL Domain, where Q(477), E(653), and F(654) residues were crucial for the binding. Deletion of the filp gene in X. oryzae pv. oryzae resulted in attenuated virulence in rice and reduced type III secretion system (T3SS) gene expression. Complementation analysis with different truncated proteins indicated that REC, PAS, and EAL Domains but not the GGDEF Domain were required for the full activity of Filp in vivo. In addition, a PilZ-Domain protein (PXO_02715) was identified as a Filp interactor by yeast two-hybrid and glutathione-S-transferase pull-down assays. Deletion of the PXO_02715 gene demonstrated changes in bacterial virulence and T3SS gene expression similar to Δfilp. Moreover, both mutants were impaired in their ability to induce hypersensitive response in nonhost plants. Thus, we concluded that Filp was a novel c-di-GMP receptor of X. oryzae pv. oryzae, and its function to regulate bacterial virulence expression might be via the interaction with PXO_02715.

Mark Gomelsky - One of the best experts on this subject based on the ideXlab platform.

  • gil a new c di gmp binding protein Domain involved in regulation of cellulose synthesis in enterobacteria
    Molecular Microbiology, 2014
    Co-Authors: Xin Fang, Ute Römling, Michael Y Galperin, Irfan Ahmad, Andrea Blanka, Marco Schottkowski, Annika Cimdins, Mark Gomelsky
    Abstract:

    Summary In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the Domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like Domain. The RxGD motif of the GIL Domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF Domain. Thus, GIL is the second protein Domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ Domain containing glycosyltransferase BcsA.

  • a zinc lock on GGDEF Domain dimerization inhibits e coli biofilms
    Structure, 2013
    Co-Authors: Mark Gomelsky
    Abstract:

    In this issue of Structure, Zahringer and colleagues present crystal structures of an important signal transduction enzyme, diguanylate cyclase DgcZ from E. coli. The authors show that zinc ions bound to the CZB Domain inhibit enzyme activity and reveal how zinc availability affects biofilm formation.

  • a staphylococcal GGDEF Domain protein regulates biofilm formation independently of cyclic dimeric gmp
    Journal of Bacteriology, 2008
    Co-Authors: Linda M Holland, Dmitri A Ryjenkov, Mark Gomelsky, Sinead T Odonnell, Larissa Gomelsky, Shawn R Slater, Paul D Fey, James P Ogara
    Abstract:

    Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF Domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF Domain protein, GdpS (for GGDEF Domain protein from Staphylococcus), and a second protein with a highly modified GGDEF Domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF Domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF Domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

  • cyclic diguanylate is a ubiquitous signaling molecule in bacteria insights into biochemistry of the GGDEF protein Domain
    Journal of Bacteriology, 2005
    Co-Authors: Dmitri A Ryjenkov, Marina Tarutina, Oleg V Moskvin, Mark Gomelsky
    Abstract:

    Proteins containing GGDEF Domains are encoded in the majority of sequenced bacterial genomes. In several species, these proteins have been implicated in biosynthesis of exopolysaccharides, formation of biofilms, establishment of a sessile lifestyle, surface motility, and regulation of gene expression. However, biochemical activities of only a few GGDEF Domain proteins have been tested. These proteins were shown to be involved in either synthesis or hydrolysis of cyclic-bis(3′→5′) dimeric GMP (c-di-GMP) or in hydrolysis of cyclic AMP. To investigate specificity of the GGDEF Domains in Bacteria, six GGDEF Domain-encoding genes from randomly chosen representatives of diverse branches of the bacterial phylogenetic tree, i.e., Thermotoga, Deinococcus-Thermus, Cyanobacteria, spirochetes, and α and γ divisions of the Proteobacteria, were cloned and overexpressed. All recombinant proteins were purified and found to possess diguanylate cyclase (DGC) activity involved in c-di-GMP synthesis. The individual GGDEF Domains from two proteins were overexpressed, purified, and shown to possess a low level of DGC activity. The oligomeric states of full-length proteins and individual GGDEF Domains were similar. This suggests that GGDEF Domains are sufficient to encode DGC activity; however, enzymatic activity is highly regulated by the adjacent sensory protein Domains. It is shown that DGC activity of the GGDEF Domain protein Rrp1 from Borrelia burgdorferi is strictly dependent on phosphorylation status of its input receiver Domain. This study establishes that majority of GGDEF Domain proteins are c-di-GMP specific, that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria, and that it is highly regulated.

Ming C Hammond - One of the best experts on this subject based on the ideXlab platform.

  • structure and mechanism of a hypr GGDEF enzyme that activates cgamp signaling to control extracellular metal respiration
    eLife, 2019
    Co-Authors: Zachary F Hallberg, Chi Ho Chan, Todd A Wright, Philip J Kranzusch, Kevin W Doxzen, James J Park, Daniel R Bond, Ming C Hammond
    Abstract:

    A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3',3'-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 A-resolution crystal structure of the Geobacter Hypr GGDEF Domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

  • hybrid promiscuous hypr GGDEF enzymes produce cyclic amp gmp 3 3 cgamp
    Proceedings of the National Academy of Sciences of the United States of America, 2016
    Co-Authors: Zachary F Hallberg, Todd A Wright, Xin C Wang, Beiyan Nan, Jongchan Yeo, Ming C Hammond
    Abstract:

    Over 30 years ago, GGDEF Domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.