The GGDEF-EAL protein CdgB from Azospirillum baldaniorum Sp245, is a dual function enzyme with potential polar localization

Abstract

Azospirillum baldaniorum Sp245, a plant growth-promoting rhizobacterium, can form biofilms through a process controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP). A. baldaniorum has a variety of proteins potentially involved in controlling the turnover of c-di-GMP many of which are coupled to sensory domains that could be involved in establishing a mutualistic relationship with the host. Here, we present in silico analysis and experimental characterization of the function of CdgB (AZOBR_p410089), a predicted MHYT-PAS-GGDEF-EAL multidomain protein from A. baldaniorum Sp245. When overproduced, CdgB behaves predominantly as a c-di-GMP phosphodiesterase (PDE) in A. baldaniorum Sp245. It inhibits biofilm formation and extracellular polymeric substances production and promotes swimming motility. However, a CdgB variant with a degenerate PDE domain behaves as diguanylate cyclase (DGC). This strongly suggest that CdgB is capable of dual activity. Variants with alterations in the DGC domain and the MHYT domain negatively affects extracellular polymeric substances production and induction of swimming motility. Surprisingly, we observed that overproduction of CdgB results in increased c-di-GMP accumulation in the heterologous host Escherichia coli, suggesting under certain conditions, the WT CdgB variant can behave predominantly as a DGC. Furthermore, we also demonstrated that CdgB is anchored to the cell membrane and localizes potentially to the cell poles. This localization is dependent on the presence of the MHYT domain. In summary, our results suggest that CdgB can provide versatility to signaling modules that control motile and sessile lifestyles in response to key environmental signals in A. baldaniorum.

Fig 1. Structural architecture and homology modeling of CdgB protein of A. baldaniorum Sp245.

(A) Schematic representation of the conserved domains of CdgB determined by the SMART database (http://smart.embl-heidelberg.de). (B) Transmembranal regions of CdgB analyzed with the Protter server. (C) Schematic representation of the 3D structure of the CdgB, PAS-GGDEF-EAL domains. The MHYT transmembrane domain was not integrated into the homology model, however, it is represented schematically into the cytoplasmic membrane cartoon. The PAS domain is in beige, the GGDEF domain is in blue, and the EAL domain is shown in green.

Fig 2. CdgB GGDEF domain structure.

(A) Sequence alignment between CdgB GGDEF domain from A. baldaniorum Sp245 (GenBank access number CCD03171.1) and other GGDEF domains of known structure. PleD, C. crescentus (GenBank access number YP_002517919.1), and RbdA, P. aeruginosa (GenBank access number AAG04250.1). Secondary structure elements are displayed above 719 the sequences. The residues highlighted in blue are involved in the interaction with the substrate; in green, those that interact with the cofactor; the active site of the GGDEF motif is highlighted in red square, and the RXXD motif is in black square. (B) Structural model of the CdgB GGDEF monomer generated by homology modeling. The GGDEF motif is highlighted in red and the RXXD pattern is highlighted in orange. (C) Structural model of CdgB GGDEF monomer in complex with GTP. The GGDEF domain is shown in surface mode. (D) Model of the GGDEF/GTP interaction generated by molecular coupling with a ΔG = - 8.2 Kcal/mol and overlapped with the 5XGD crystal structure, corresponding to GGDEF domain from P. aeruginosa showed in light purple color with an RMSD of 0.539 of ligands. The Mg²⁺ ion is shown in green.

Fig 3. CdgB EAL domain structure.

(A) Sequence alignment between CdgB EAL domain from A. baldaniorum Sp245 (GenBank access number CCD03171.1) and other EAL domains of known structure. RocR, (GenBank access number NP_252636.1), and MucR (GenBank access number AAG05116.1) from P. aeruginosa. Secondary structure elements are displayed above the sequences. The residues highlighted in blue are involved in the interaction with the substrate. Those that interact with cofactor are marked in green. The residue that stabilizes structurally the loop 6 is shown in yellow. The catalytic residue is in red. The EAL motif is marked with a red box, and loop 6 is inside a box with black outline. (B) Structural model of the CdgB EAL monomer generated by homology modeling. The EAL motif is highlighted in red and loop 6 is highlighted in orange. (C) Structural model of the CdgB EAL monomer in complex with c-di-GMP. The EAL domain is presented in surface mode. (D) Model of the EAL domain in complex with c-di-GMP generated by molecular coupling with a ΔG = -8.58 Kcal/mol and overlapped with the 5M1T crystal structure, corresponding 744 to EAL domain from P. aeruginosa showed in light purple color with an RMSD of 0.757 of ligands. The Mg²⁺ ions are shown in green.

Fig 4. CdgB alters biofilm formation in A. baldaniorum Sp245.

Graph representing the mean and standard deviation of biofilm quantification from 5 days of growth in statical conditions at 30°C. Data represent results of three independent experiments with three biological replicates. Significant differences relative to WT (P < 0.05) are indicated by “*”and to +cdgB (P < 0.005) by “**” according to Student´s t-test.

Fig 5. CdgB alters EPS accumulation in A. baldaniorum Sp245.

Graph representing the mean and standard deviation of EPS quantification after 5 days of growth in statical conditions at 30°C. Data represent results of three independent experiments with three biological replicates. Significant differences relative to WT (P < 0.05) are indicated by “*”and to +cdgB (P < 0.005) by “**” according to Student´s t-test.

Fig 6. Biofilm formation analysis with confocal laser scanner microscopy.

Images represent Three-dimensional reconstruction of 5-day old static biofilms; the blue fluorescence signal represents exopolysaccharides staining. Gray images are Intensity surface plots corresponding to the emitted fluorescence by CWC. The biofilm formed was directly observed using Nikon confocal microcopy C2+ with a 20X objective (CFI Plan ApVC 20X numerical aperture of 1.2). Exciting laser intensity, background level contrast, and electronic zoom were maintained at the same level.

Fig 7. CdgB alters swimming motility in A. baldaniorum Sp245.

Graph representing the mean of motility halos, in centimeters (cm), of the indicated strains on K-media plates with agar 0.25%, supplemented with (A) malate, (B) succinate, or (C) proline (10mM) at 30°C for 48 h. Error bars indicate standard deviations of 6 replicates per strain. (D) Representative images of motility halos from three independent experiments with two biological determinations. Significant differences relative to WT (P < 0.005) according to Student´s t-test are indicated by “*”.

Fig 8. CdgB promotes c-di-GMP accumulation in a heterologous host.

Representative images of macroscopic and microscopic observations of c-di-GMP levels judged by green and red coloring of colony spots and liquid concentrates as well as green and red fluorescence of individual cells from strains harboring the genetic biosensor in pDZ-119 and (A) pGEX-CdgA (DGC positive control), (B) pGEX-4T1 (negative control), (C) pGEX-CdgB, (D) pGEX-CdgB_SGDEF-SGKEF, or (E) pGEX-CdgB_EAL-AAL. (F) The relative fluorescence intensity represents the ratio between the TurboRFP and AmCyan fluorescence intensities and is directly proportional to c-di-GMP levels, as analyzed using ImageJ software. The RFI values represent the standard deviations of three biological replicates, and significant differences are indicated at *(P < 0.05) according to Student´s t-test by SigmaPlot. The white bar corresponds to 10 μm.

Fig 9. CdgB is anchored to the cytoplasmic membrane and polarly localized.

The first two columns (left to right) show representative images acquired to detect fluorescence of the FM4-64FX fluorophore (red) that bind to lipidic membranes, and fluorescence from the eGFP protein (green). The third column shows images where the two fluorescent signals were merged. The white arrowheads indicate polar localization of CdgB protein. The white bar corresponds to 10 μm.