Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tianning; Zhu, Deyu; Jin, Peng; Xia, Zhi‐Jie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

doi:10.1038/srep29499

Cited by 13 publications

(10 citation statements)

References 51 publications

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“…The GacA GGDEF domain has a βααββαβαβ global topology that positions one guanosine substrate above the signature [G/A/S]G[D/E]E[F/Y] motif and can be overlaid with a canonical GGDEF domain with an RMSD value of 1.152 Å (Figures 6B and 8). A region behind the two alpha helices that support substrate binding is modified from a beta sheet to a helical/loop motif, and varies considerably between GGDEF structures (Chen et al, 2016; Dahlstrom et al, 2015; Deepthi et al, 2014; Yang et al, 2011). Electron density for three guanine nucleotides was found in the GacA structure, two in nucleotide-interacting regions that are conserved in other GGDEF domains (Figure 7) (Chan et al, 2004).…”

Section: Resultsmentioning

confidence: 99%

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Hallberg

Chan

Wright

et al. 2019

eLife

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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 Å-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.

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Section: Resultsmentioning

confidence: 99%

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Hallberg

Chan

Wright

et al. 2019

eLife

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“…A high intracellular level of c-di-GMP induces P. aeruginosa pyoverdine synthesis, which is dependent on exopolysaccharides and DGC (16–19). Under iron-replete conditions, P.…”

Section: Introductionmentioning

confidence: 99%

Pleiotropic Effects of c-di-GMP Content in Pseudomonas syringae

Wang

Cai

Shao

et al. 2019

Appl Environ Microbiol

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Although the ubiquitous bacterial secondary messenger cyclic diguanylate (c-di-GMP) has important cellular functions in a wide range of bacteria, its function in the model plant pathogen Pseudomonas syringae remains largely elusive. To this end, we overexpressed Escherichia coli diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) in P. syringae, resulting in high and low in vivo levels of c-di-GMP, respectively. Via genome-wide RNA sequencing of these two strains, we found that c-di-GMP regulates (i) fliN, fliE, and flhA, which are associated with flagellar assembly; (ii) alg8 and alg44, which are related to the exopolysaccharide biosynthesis pathway; (iii) pvdE, pvdP, and pvsA, which are associated with the siderophore biosynthesis pathway; and (iv) sodA, which encodes a superoxide dismutase. In particular, we identified three promoters that are sensitive to elevated levels of c-di-GMP and inserted them into luciferase-based reporters that respond effectively to the c-di-GMP levels in P. syringae; these promoters could be useful in the measurement of in vivo levels of c-di-GMP in real time. Further phenotypic assays validated the RNA sequencing (RNA-seq) results and confirmed the effect on c-di-GMP-associated pathways, such as repressing the type III secretion system (T3SS) and motility while inducing biofilm production, siderophore production, and oxidative stress resistance. Taken together, these results demonstrate that c-di-GMP regulates the virulence and stress response in P. syringae, which suggests that tuning its level could be a new strategy to protect plants from attacks by this pathogen. IMPORTANCE The present work comprehensively analyzed the transcriptome and phenotypes that were regulated by c-di-GMP in P. syringae. Given that the majority of diguanylate cyclases and phosphodiesterases have not been characterized in P. syringae, this work provided a very useful database for the future study on regulatory mechanism (especially its relationship with T3SS) of c-di-GMP in P. syringae. In particular, we identified three promoters that were sensitive to elevated c-di-GMP levels and inserted them into luciferase-based reporters that effectively respond to intracellular levels of c-di-GMP in P. syringae, which could be used as an economic and efficient way to measure relative c-di-GMP levels in vivo in the future.

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“…In P. aeruginosa PAO1, these c-di-GMP modulating proteins are highly complex, with 17 GGDEF, 5 EAL, 16 GGDEF/EAL, and 3 HD-GYP domain-containing proteins (http://www.ncbi.nlm.nih.gov/Complete_Genomes/c-di-GMP.html). 7,8 To date, the biological functions and underlying mechanisms of a quantity of these proteins/signaling systems have been identified and investigated, including the well-studied WspR (PA3702), SadC (PA4332), FimX (PA4959), LapD (from P. ﬂuorescens Pf0–1), BdlA (PA1423), and the newly identified FcsR (PA2133), Dcsbis (PA2771) and HsbD (PA3343) et al9–16. The list is still in fast growth.…”

Section: Introductionmentioning

confidence: 99%

<p>Characterization and analysis of a novel diguanylate cyclase PA0847 from <em>Pseudomonas aeruginos</em>a PAO1</p>

Zhang

Guo

Zhang

et al. 2019

IDR

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Background: As a central signaling molecule, cyclic diguanylate (c-di-GMP) is found to regulate various bacterial phenotypes, especially those involved in pathogen infection and drug resistance. Noticeably, many microbes have up to dozens of proteins that are involved in c-di-GMP metabolism. This apparent redundancy and the relevant functional specificity have become the focus of research. While a number of these proteins have been identified and investigated, the functions of PA0847, a PAS and GGDEF domain-containing protein from Pseudomonas aeruginosa PAO1, remain unclear. Materials and methods: In the current study, microbiology, biochemistry and structural biology methods were applied to characterize the gene/protein of PA0847. Results: We showed that PA0847 affects bacterial motility but not biofilm formation. We recorded the phenotypic influences of amino acids and compounds, and found that PA0847 is involved in response to various environmental nutrients and factors, suggesting its possible role in sensing environmental cues. Both in-vitro and in-vivo studies showed that PA0847 is an active diguanylate cyclase (DGC), whose activity depends on the neighboring PAS domain. Interestingly, PA0847 demonstrates no significant product inhibition, though the key residues of two I-sites for c-di-GMP binding are conserved in its GGDEF domain. A local structural change imposed by an adjacent tyrosine residue was identified, which indicates the structural and functional diversities of the GGDEF family proteins. Conclusion: Our data provide evidence for understanding the signaling mechanism of the unique c-di-GMP metabolizing protein PA0847.

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Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

Cited by 13 publications

References 51 publications

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Pleiotropic Effects of c-di-GMP Content in Pseudomonas syringae

<p>Characterization and analysis of a novel diguanylate cyclase PA0847 from <em>Pseudomonas aeruginos</em>a PAO1</p>

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