A Signaling Pathway Involving the Diguanylate Cyclase CelR and the Response Regulator DivK Controls Cellulose Synthesis in Agrobacterium tumefaciens

ABSTRACTThe motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogenAgrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP inA. tumefaciensare controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.IMPORTANCEPathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogenAgrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

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Faculty Opinions recommendation of A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725607237.793522871 ◽

2016 ◽

Author(s):

Alain Filloux

Keyword(s):

Agrobacterium Tumefaciens ◽

Signaling Pathway ◽

Dual Function ◽

Diguanylate Cyclase ◽

Surface Attachment ◽

Dependent Signaling Pathway

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CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens

Applied and Environmental Microbiology ◽

10.1128/aem.02148-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7188-7202 ◽

Cited By ~ 23

Author(s):

D. Michael Barnhart ◽

Shengchang Su ◽

Brenna E. Baccaro ◽

Lois M. Banta ◽

Stephen K. Farrand

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Bacterial Species ◽

Cellulose Synthesis ◽

Diguanylate Cyclase ◽

Cellulose Biosynthesis ◽

Plant Host ◽

Cellulose Production ◽

Signal Molecule ◽

Content Type

ABSTRACTCellulose fibrils play a role in attachment ofAgrobacterium tumefaciensto its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, includingA. tumefaciens. In this study, we identified two putative diguanylate cyclase genes,celR(atu1297) andatu1060, that influence production of cellulose inA. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion ofcelR, but notatu1060, resulted in decreased cellulose biosynthesis.celRoverexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis ofcelRand Δcelmutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing bothcelRandcelA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lackcelAuse CelR, called PleD, to regulate specific processes associated with polar localization and cell division.

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Faculty Opinions recommendation of Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1093811.548733 ◽

2007 ◽

Author(s):

Michael Y Galperin

Keyword(s):

Feedback Inhibition ◽

Response Regulator ◽

Diguanylate Cyclase ◽

Cyclase Activation

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Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB,attD, and attR Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.64.7.2341-2345.1998 ◽

1998 ◽

Vol 64 (7) ◽

pp. 2341-2345 ◽

Cited By ~ 52

Author(s):

Ann G. Matthysse ◽

Susan McMahan

Keyword(s):

Agrobacterium Tumefaciens ◽

Root Length ◽

Root Colonization ◽

Bacterial Pathogenesis ◽

Bacterial Suspension ◽

Cellulose Synthesis ◽

Wild Type ◽

Tomato Roots ◽

Extensive Growth ◽

Number Of Bacteria

ABSTRACT Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 103 bacteria/cm of root length at the time of inoculation to more than 107 bacteria/cm after 10 days. The numbers of cellulose-minus and nonattachingattB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thalianaecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel andatt mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.

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Mapping of Agrobacterium tumefaciens chromosomal genes affecting cellulose synthesis and bacterial attachment to host cells.

Journal of Bacteriology ◽

10.1128/jb.170.3.1408-1411.1988 ◽

1988 ◽

Vol 170 (3) ◽

pp. 1408-1411 ◽

Cited By ~ 20

Author(s):

J L Robertson ◽

T Holliday ◽

A G Matthysse

Keyword(s):

Agrobacterium Tumefaciens ◽

Bacterial Attachment ◽

Host Cells ◽

Cellulose Synthesis ◽

Chromosomal Genes

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The SagS sensory protein modulates biofilm formation and c-di-GMP levels by Pseudomonas aeruginosa in response to glucose-6-phosphate.

10.5194/biofilms9-122 ◽

2020 ◽

Author(s):

Soyoung Park ◽

Jozef Dingemans ◽

Madison Gowett ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Genetic Analysis ◽

Signaling Pathway ◽

Biofilm Formation ◽

Phosphate Concentration ◽

Dependent Manner ◽

Diguanylate Cyclase ◽

Swarming Motility ◽

Two Component ◽

Insight Into

<p>In <em>Pseudomonas aeruginosa</em>, the orphan two-component sensor SagS contributes to both, the transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment as glucose-6-phosphate likewise enhanced biofilm formation. We show that exposure to glucose-6-phosphate results in decreased swarming motility but increased cellular c-di-GMP levels in biofilms. Genetic analysis indicated that the diguanylate cyclase NicD is an activator of biofilm formation and is not only required for enhanced biofilm formation in response to glucose-6-phosphate but also interacts with SagS. Our findings indicate glucose-6-phosphate to likely mimic a signal or conditions sensed by SagS to activate its motile-sessile switch function. Additionally, our findings provide new insight into the interfaces between the ligand-mediated TCS signaling pathway and c-di-GMP levels.</p>

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Constitutive Activation of Two-Component Response Regulators: Characterization of VirG Activation in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00387-06 ◽

2006 ◽

Vol 188 (14) ◽

pp. 5204-5211 ◽

Cited By ~ 15

Author(s):

Rong Gao ◽

Aindrila Mukhopadhyay ◽

Fang Fang ◽

David G. Lynn

Keyword(s):

Agrobacterium Tumefaciens ◽

Conformational Changes ◽

Response Regulator ◽

Structural Features ◽

Response Regulators ◽

Two Component System ◽

Plant Host ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.

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Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽

10.1128/mbio.00242-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 69

Author(s):

Varisa Huangyutitham ◽

Zehra Tüzün Güvener ◽

Caroline S. Harwood

Keyword(s):

Signal Transduction ◽

Biofilm Formation ◽

Response Regulator ◽

Cluster Formation ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Regulator Protein ◽

Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

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