In vitro and in vivo studies of cyclic di-GMP signaling proteins

Cyclic di-GMP is a bacterial secondary messenger important for the regulation of biofilm formation and bacterial pathogenesis. Although much progress has been made in recent years, many questions about the hierarchical and complex cyclic di-GMP signaling networks remain to be answered. With only a relatively small number of cyclic di-GMP signaling proteins, Bacillus subtilis serves as a simple and genetically tractable model organism for us to study cyclic di-GMP signaling. We identified two catalytically active diguanylate cyclases (DGC), one phosphodiesterase (PDE) and one bifunctional enzyme. Phenotypic analysis revealed the regulatory roles of these proteins in bacterial motility, sporulation and biofilm formation. In addition, a few representative GGDEF-EAL didomain proteins with known input signals were nvestigated to understand how the GGDEF or EAL domain is regulated by its associated N-terminal regulatory domain. The catalytic activities of the AxDGC2 and AxPDEA2 proteins from Gluconacetobacter xylinus were found to be modulated by the flavin and heme-associated PAS domain respectively; while the Tbd_1269 protein from the Thiobacillus denitrificans was found to possess both DGC and PDE activities that are likely to be regulated by phosphorylation of the N-terminal regulatory domain.​Doctor of Philosophy (SBS

Crystallization and preliminary X-ray diffraction studies of Xanthomonas campestris PNPase in the presence of c-di-GMP

Bacterial polynucleotide phosphorylase (PNPase) is a 3'-5' processive exoribonuclease that participates in mRNA turnover and quality control of rRNA precursors in many bacterial species. It also associates with the RNase E scaffold and other components to form a multi-enzyme RNA degradasome machinery that performs a wider regulatory role in degradation, quality control and maturation of mRNA and noncoding RNA. Several crystal structures of bacterial PNPases, as well as some biological activity studies, have been published. However, how the enzymatic activity of PNPase is regulated is less well understood. Recently, Escherichia coli PNPase was found to be a direct c-di-GMP binding target, raising the possibility that c-di-GMP may participate in the regulation of RNA processing. Here, the successful cloning, purification and crystallization of S1-domain-truncated Xanthomonas campestris PNPase (XcPNPaseΔS1) in the presence of c-di-GMP are reported. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 132.76, b = 128.38, c = 133.01 Å, γ = 93.3°, and diffracted to a resolution of 2.00 Å.Published versio

Crystallization and preliminary X-ray diffraction characterization of the XccFimXEAL-c-di-GMP and XccFimXEAL-c-di-GMP-XccPilZ complexes from Xanthomonas campestris

c-di-GMP is a major secondary-messenger molecule in regulation of bacterial pathogenesis. Therefore, the c-di-GMP-mediated signal transduction network is of considerable interest. The PilZ domain was the first c-di-GMP receptor to be predicted and identified. However, every PilZ domain binds c-di-GMP with a different binding affinity. Intriguingly, a noncanonical PilZ domain has recently been found to serve as a mediator to link FimXEAL to the PilB or PilT ATPase to control the function of type IV pili (T4P). It is thus essential to determine the structure of the FimXEAL-PilZ complex in order to determine how the binding of c-di-GMP to the FimXEAL domain induces conformational change of the adjoining noncanonical PilZ domain, which may transmit information to PilB or PilT to control T4P function. Here, the preparation and preliminary X-ray diffraction studies of the XccFimXEAL-c-di-GMP and XccFimXEAL-c-di-GMP-XccPilZ complexes from Xcc (Xanthomonas campestris pv. campesteris) are reported. Detailed studies of these complexes may allow a more thorough understanding of how c-di-GMP transmits its effects through the degenerate EAL domain and the noncanonical PilZ domain.Published versio

Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells

Cyclic di-GMP (c-di-GMP) has emerged as a prominent intracellular messenger that coordinates biofilm formation and pathogenicity in many bacterial species. Developing genetically encoded biosensors for c-di-GMP will help us understand how bacterial cells respond to environmental changes via the modulation of cellular c-di-GMP levels. Here we report the design of two genetically encoded c-di-GMP fluorescent biosensors with complementary dynamic ranges. By using the biosensors, we found that several compounds known to promote biofilm dispersal trigger a decline in c-di-GMP levels in Escherichia coli cells. In contrast, cellular c-di-GMP levels were elevated when the bacterial cells were treated with subinhibitory concentrations of biofilm-promoting antibiotics. The biosensors also revealed that E. coli cells engulfed by macrophages exhibit lower c-di-GMP levels, most likely as a response to the enormous pressures of survival during phagocytosis.Accepted versio

Crystallization studies of the murine c-di-GMP sensor protein STING

The innate immune response is the first defence system against pathogenic microorganisms, and cytosolic detection of pathogen-derived DNA is believed to be one of the major mechanisms of interferon production. Recently, the mammalian ER membrane protein STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) has been found to be the master regulator linking the detection of cytosolic DNA to TANK-binding kinase 1 (TBK1) and its downstream transcription factor IFN regulatory factor 3 (IRF3). In addition, STING itself was soon discovered to be a direct sensor of bacterial cyclic dinucleotides such as c-di-GMP or c-di-AMP. However, structural studies of apo STING and its complexes with these cyclic dinucleotides and with other cognate binding proteins are essential in order to fully understand the roles played by STING in these crucial signalling pathways. In this manuscript, the successful crystallization of the C-terminal domain of murine STING (STING-CTD; residues 138-344) is reported. Native and SeMet-labelled crystals were obtained and diffracted to moderate resolutions of 2.39 and 2.2 Å, respectively.Published versio

Visualizing the Perturbation of Cellular Cyclic di-GMP Levels in Bacterial Cells

Cyclic di-GMP (c-di-GMP) has emerged as a prominent intracellular messenger that coordinates biofilm formation and pathogenicity in many bacterial species. Developing genetically encoded biosensors for c-di-GMP will help us understand how bacterial cells respond to environmental changes via the modulation of cellular c-di-GMP levels. Here we report the design of two genetically encoded c-di-GMP fluorescent biosensors with complementary dynamic ranges. By using the biosensors, we found that several compounds known to promote biofilm dispersal trigger a decline in c-di-GMP levels in Escherichia coli cells. In contrast, cellular c-di-GMP levels were elevated when the bacterial cells were treated with subinhibitory concentrations of biofilm-promoting antibiotics. The biosensors also revealed that E. coli cells engulfed by macrophages exhibit lower c-di-GMP levels, most likely as a response to the enormous pressures of survival during phagocytosis

An analysis of uncertainty in structural design

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Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains