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A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

ABSTRACT: The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK.

SUBMITTER: Barnhart DM

PROVIDER: S-EPMC3957726 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

Barnhart D Michael DM Su Shengchang S Farrand Stephen K SK

Journal of bacteriology 20140117 6

The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as t ...[more]

PMID: 24443526

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Project description:UNLABELLED:The 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 pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are 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. IMPORTANCE:Pathogenic 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 pathogen Agrobacterium 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.

Project description:UnlabelledWspR 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.ImportanceBacterial 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.

Dataset Information

A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

Publications

A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

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