Expression and function of the cdgD gene, encoding a CHASE-PAS-DGC-EAL domain protein, in Azospirillum brasilense

Abstract

The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO₃ or NH₄Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Figure 1

Structural architecture, domain organization, and homology modeling of the CdgD protein of A. brasilense Sp245. (a) CdgD domains determined by the SMART domain prediction server, with a sequence length of 946 amino acid residues. (b) A representation of the CdgD protein with its CHASE sensory domain, two transmembrane domains (TMD), PAS domain, GGDEF domain, EAL domain, the S-helix and H-helix connecting segments crucial for protein dynamics, and key structural features. Overlay of the structures of the periplasmic sensing domains and transmembrane domains of CdgD showing the CHASE domain of PcrK (histidine kinase from X. campestris pv campestris, PDB code 6K62) in gold and the CHASE domain of CdgD in light blue. cRbdA from P. aeruginosa (PDB code 5XGB), shown in gold, was compared with the cytoplasmic region of CdgD. The PAS domain is green, the GGDEF domain is blue, the EAL domain is yellow, and the S-helix and H-helix are cyan and magenta, respectively. The loops that are not included in the structural model are represented schematically by dashed lines. The N and C termini are labeled. Magnification of the CHASE domain showing the membrane-distal PAS subdomains and membrane-proximal PAS subdomains, in which the α-helices and ß-strands are indicated. CdgD and PcrK are light blue and gold, respectively.

Figure 2

Effects of the mutation of the cdgD gene on biofilm and EPS production determined in the A. brasilense WT and derivative strains. (a) Determination of biofilm formation in NFB* medium supplemented with KNO₃ via the quantification of CV/mg protein values. (b) Determination of biofilm formation in NFB* supplemented with NH₄Cl medium via the quantification of CV/mg protein values. (c,d) Determination of EPS production in the media described above via the quantification of CR/mg protein values in a staining assay. Wild type (Sp245), isogenic mutant (12-A), complemented mutant (C-56A), and empty vector mutant (C-40A). Error bars represent the standard deviations of three biological replicates, and statistically significant differences are indicated at *P < 0.05 according to Student’s t-test by SigmaPlot (Systat Software, San Jose, CA).

Figure 3

Effects of the mutation and overexpression of the cdgD gene on motility determined in A. brasilense WT and derivative strains. (a) The diameter of the swimming rings in the mutant and the complemented mutant relative to the wild-type strain was measured in minimal medium with the tested carbon sources at 10 mM after 48 h of incubation at 30 °C ( malate, fumarate, or proline). Error bars represent the standard deviations of three biological replicates, and the asterisks indicate values that are significantly different from those in the wild type (P < 0.05) according to Student’s T test by SigmaPlot (Systat Software, San Jose, CA). (b) Photograph of a typical agar plate containing minimal medium with 10 mM proline as the carbon source showing the swimming rings of each strain. Wild type (Sp245), isogenic mutant (12-A), complemented mutant (C-56A), and empty vector mutant (C-40A).

Figure 4

Determination of cyclic-di-GMP levels using a riboswitch biosensor. (a) The A. brasilense Sp245 , A. brasilense 12-A , and A. brasilense C-56A , and C-40A strains containing the c-di-GMP biosensor (pFY4535) were grown in NFB* + KNO₃ broth containing 30 μg/mL Gm with incubation for 24, 48, or 72 h at 30 °C. Then, Azospirillum cells were attached to the surface of a coverslip and sealed with a 1% agar plug. Cell images were collected at these time points after inoculation using a Nikon TE2000U microscope equipped with a 100× objective (oil immersion objective). Merge images represented the overlay of the fluorescence images AmCyan green, TurboRFP red and both yellow. (b) RFI represents the ratio between the TurboRFP and AmCyan fluorescence intensities and is directly proportional to c-di-GMP levels, as analysed 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 (Systat Software, San Jose, CA). The bar corresponds to 10 µm.

Figure 5

Expression analysis of the cdgD gene from planktonic and static cultures of the A. brasilense Sp245 strain. (a) Determination of cdgD gene transcript levels by RT-qPCR. The transcript level data were normalized to those of the reference gene glyA and determined relative to the control sample using Applied Biosystems StepOne software. (b) Fluorescence micrographs of the A. brasilense FPDm1 strain under different culture conditions. Left, bacteria grown in NFB* + KNO₃ media; right, bacteria grown in NFB* + NH₄Cl media. The fluorescence intensity is normalized to total protein values from the A. brasilense FPDm1 strain, as analysed using ImageJ. The presented data are the results of three independent experiments with two biological replicates, and the asterisks indicate significant differences from the wild type (P < 0.05) according to Student’s T test by SigmaPlot (Systat Software, San Jose, CA). The bar corresponds to 10 µm. NFB* + KNO₃, NFB* + NH₄Cl.

Figure 6

Expression of the pcdgD-mCherry transcriptional fusion from A. brasilense cells grown under hydroponic conditions and in wheat roots. For the visualization of mCherry-expressing A. brasilense, cells on wheat roots were observed at 24 h and 48 h postinoculation. Next, the freshly harvested seedling roots were visualized in a FluoroDish. Left, wheat seedling roots inoculated with the A. brasilense T7mCh control strain. Right, wheat seedling roots inoculated with the A. brasilense FPDm1 strain. The cells exhibiting mCherry fluorescence were visualized using a CLSM with an excitation wavelength of 561 nm, with fluorescence emission captured between 585 and 615 nm. Thereafter, the images were edited using the standard NIS Elements in Nikon software. The bar corresponds to 50 µm.

Figure 7

Rhizosphere colonization of A. brasilense Sp245, the 12-A mutant, the C-40A control mutant and the C-56A complemented mutant and visualization of tagged colonizing bacteria by CLSM. (a) The plants were grown in glass tubes with Hoagland hydroponic solution and examined at 7 days postinoculation with 5 × 10⁶–10⁷ CFU/mL of A. brasilense Sp245 (pMP2449-5), A. brasilense 12-A (pMP2449-5), A.brasilense C-40A (pMP2449-5) and A. brasilense C-56A (pMP2449-5) (the WT, mutant, and complemented mutant strains, respectively). Subsequently, the cells showing mCherry fluorescence were visualized using CLSM with an excitation wavelength of 561 nm, with fluorescence emission captured between 585 and 615 nm. Thereafter, the images were edited using the standard NIS elements in Nikon software. The bar corresponds to 10 µm. (b) The bacterial colonies recovered from the rhizosphere 1 week after inoculation under sterile soil conditions were tested for antibiotic resistance to distinguish the control and mutant strains and were counted. CFU/mL per gram of plant root values are presented on a logarithmic scale. Each experiment was repeated three times with five plants per experiment. Asterisks represent the statistical significance of the data (P < 0.05) according to Student’s t-test by SigmaPlot (Systat Software, San Jose, CA).