A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

Abstract

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.

FIG 1

Complementation of increased biofilm formation in the A. tumefaciens dcpA mutant requires an intact DcpA EAL catalytic motif. (A) Diagram of PruR-DcpA genetic locus. Atu3497 is a conserved hypothetical protein with no annotated domains. (B) Protein topology of DcpA. Protein domains predicted by the BLAST database (NCBI) are shown. Domains are drawn to scale. TM, transmembrane; DGC, diguanylate cyclase; PDE, phosphodiesterase. (C) A. tumefaciens quantitative biofilm formation on PVC coverslips after 48 h of static growth at 28°C. Adherent biomass was quantified by staining with crystal violet (CV). CV absorbance was quantified by absorbance at 600 nm (A₆₀₀). In parallel, the optical density at 600 nm (OD₆₀₀) of planktonic culture was determined. CV absorbance was normalized to culture growth by calculating the A₆₀₀/OD₆₀₀ ratio. IPTG (400 µM) was added to all strains. The wild-type (WT) strain, ΔdcpA mutant with no plasmid inserted (-), and ΔdcpA mutant strain with P_lac-dcpA derivatives are shown. A wild-type copy of dcpA provided on a plasmid expressed from the lacZ promoter (P_lac-dcpA; DGC⁺PDE⁺) was introduced into a ΔdcpA mutant. The P_lac-dcpA derivatives include a DcpA variant containing catalytic site GGDEF→GGDAF mutation (DGC−) and a DcpA variant containing catalytic site EAL→AAL mutation (PDE−). Values are results of three independent biological replicates consisting of three technical replicates each. The error bars show 1 standard deviation (SD).

FIG 2

Genetic analysis reveals DGC activity of DcpA. (A) Biofilms grown for 48 h on PVC coverslips were quantified as described in the legend to Fig. 1C. A wild-type strain with no plasmid inserted (-) is shown. The error bars show 1 standard deviation (SD). (B) Congo red colony phenotypes of A. tumefaciens strains after 48 h of growth at 28°C (vertically aligned with strain designations in panel A). (C) Quantification of intracellular levels of c-di-GMP in the indicated A. tumefaciens or E. coli strains. A DcpA variant containing catalytic site GGDEF→GGDAF mutation (DGC−) and a DcpA variant containing catalytic site EAL→AAL mutation (PDE−) were tested. c-di-GMP was measured by LC-MS/MS as described in Text S1 in the supplemental material. Values are results of two (A. tumefaciens) or three (E. coli) independent biological replicates consisting of three technical replicates each. The error bars show 1 SD.

FIG 3

Enzymatic activity of PruA required for control of attachment. (A) Biofilms grown on PVC coverslips for 48 h were quantified as described in the legend to Fig. 1C. A wild-type strain with no plasmid inserted (-) and the pruA mutant strain with no plasmid inserted (-) are indicated. The error bars show 1 SD. (B) Congo red colony phenotypes of the indicated A. tumefaciens strains. The bacteria were grown for 48 h at 28°C. (C) Unipolar polysaccharide (UPP) production (red) visualized by staining with Alexa Fluor 594-labeled wheat germ agglutinin (afWGA). Exponential-phase planktonic cultures were incubated with afWGA (10 µg/ml) and spotted (1 µl) onto 1% agarose pad. Bacteria were viewed at a magnification of ×100 on a Nikon E800 epifluorescence microscope (excitation, 510 to 560 nm; emission, >610 nm). The images shown are overlays of phase-contrast and fluorescence microscopy images. The images were exposed for 40 and 400 ms for phase-contrast and fluorescence microscopy, respectively. IPTG (400 µM) was added to each culture. Bars, 5 µm.

FIG 4

PruA enzymatic activity required for pterin synthesis. (A) HPLC traces of pterin extracts from the indicated A. tumefaciens strains. Lyophilized cells were resuspended, and pterins were extracted, purified, and analyzed by HPLC as described in Materials and Methods. (B) Pterin chemical structures (ring positions shown).

FIG 5

Regulatory connections between PruA and DcpA. (A) Biofilms grown for 48 h on PVC coverslips (gray bars) were quantified as described in the legend to Fig. 1C. The error bars show 1 SD. c-di-GMP levels were also quantified (black bars). Strains were grown to stationary phase, nucleotides were extracted, and c-di-GMP was measured as described in Materials and Methods. A DcpA variant with catalytic site GGDEF→GGDAF mutation (DGC−), DcpA variant containing catalytic site EAL->AAL mutation (PDE−), and mutant with no plasmid inserted (-) were used. The Δcel background was utilized to reduce excess clumping. Values are results of three (biofilm) or two (c-di-GMP) independent biological replicates consisting of three technical replicates each. The error bars show 1 SD. (B) UPP production visualized by staining with afWGA, as described in the legend to Fig. 3C. IPTG (400 µM) was added to each culture. Bars, 5 µm. (C) Adherent biomass grown for 48 h was quantified as described above for panel A, with IPTG omitted.

FIG 6

PruR negatively regulates biofilm formation. (A) Biofilms grown for 48 h on PVC coverslips were quantified as described in the legend to Fig. 1C. A ΔpruR mutant with no plasmid inserted is indicated (-). The error bars show 1 SD. (B) UPP production visualized by staining with afWGA, as described in the legend to Fig. 3C. Bars, 5 µm. (C) Adherent biomass grown for 48 h was quantified as described above for panel A, with IPTG omitted. The Δcel background was utilized to reduce excess clumping. (D) Quantification of c-di-GMP levels in the indicated E. coli strains. The E. coli strains were grown to late exponential phase, and nucleotides were extracted and quantified as described in Text S1 in the supplemental material. Values are results of two independent biological replicates each with three technical replicates. The error bars show 1 SD.

FIG 7

Model of pterin-dependent DcpA regulation. A tentative model for DcpA regulation is shown. The model is based on the findings reported here, with details provided in the text. Solid black arrows are enzymatic reactions, dashed arrows are regulatory interactions, and the squiggly arrow is a speculative environmental influence on the putative PruR-pterin (Pt) complex. Red text indicates essential genes in A. tumefaciens. IM, inner membrane; OM, outer membrane; GTs, glycosyl transferases; MoPT, molybdopterin; P-ase, phosphatase.