Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii

Abstract

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Keywords: Azotobacter vinelandii; MucR; alginate; c-di-GMP; cyst capsule; encystment; mannuronan C-5 epimerases.

FIG 1

MucR contributes to the c-di-GMP pool under encysting-inducing conditions. c-di-GMP was quantified in the WT strain and in the ΔmucR mutant during encystment induction. Vegetative cells were harvested after 48 h of growth in Burk’s-sucrose medium (0 h), washed three times to remove traces of sucrose, and transferred to Burk’s-butanol medium for the indicated time. The bars for standard deviations from three independent experiments are shown. Significant differences were analyzed by t test. Statistical significance is indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001.

FIG 2

MucR is necessary for the formation of mature cysts resistant to desiccation but not for the production of alginate. (A) Mature cyst formation assay, based on the percentage of cells resistant to a 5-day period of desiccation. The bars for standard deviations from three independent experiments are shown. Significant differences were analyzed by t test. Statistical significance is indicated. **, P < 0.01; ***, P < 0.0001. (B) Electron micrographs of A. vinelandii cysts after a 5-day period of differentiation. The cells were cultivated on plates of Burk’s-butanol medium for encystment induction. In the WT strain, the central body (CB) and the PHB granules are indicated, as is the cyst capsule, which is composed of two laminated alginate layers, the exine (EX) and the intine (IN). (C) Alginate quantification in A. vinelandii cells grown on plates of Burk’s-butanol medium after 48 h of encystment induction.

FIG 3

MucR is necessary for the production of extracellular C-5 epimerases. (A) Determination of the relative abundance of algE1 to algE6 mRNA by qPCR in encystment-induced cells at 48 h. The bars for standard deviations from three independent experiments are shown. Significant differences were analyzed by t test. Statistical significance is indicated. **, P < 0.01; ****, P < 0.00001. (B) Detection of AlgE C-5 epimerases associated with the central body of A. vinelandii cells undergoing differentiation in Burk’s-butanol medium for 5 days. Anti-AlgE4 antibody was used as the primary antibody.

FIG 4

Identification of the transcription start sites of mucR. (A) Transcription start sites were identified by primer extension with total RNA extracted from the WT (lane 1) and ΔalgR (lane 2) strains grown in Burk´s-sucrose medium for 48 h and a primer complementary to mucR. The cDNA obtained was resolved in a denaturing polyacrylamide gel, along with DNA sequence ladders generated by chemical sequencing of mucR. The transcription start sites (P1 and P2) are indicated. (B) Genomic context of the mucR gene. The mucR flanking genes encode hypothetical conserved proteins. In the lower panel, the location of the two mucR transcription start sites (P1 and P2) identified in panel A are indicated, along with putative AlgR binding sites (BS1 and BS2).

FIG 5

Transcriptional regulation of mucR assessed by qPCR analysis. (A) Effect of AlgR on mucR transcription. The mucR mRNA levels in the ΔalgR strain were determined under vegetative (Burk’s-sucrose medium for 48 h) or encysting (Burk’s-butanol medium for 48 h) conditions and were compared to those in the WT strain. (B) mRNA levels of mucR under encysting conditions, relative to those under vegetative conditions. The bars for standard deviations from three independent experiments are shown. Significant differences were analyzed by t test. Statistical significance is indicated. ***, P < 0.0001; ****, P < 0.00001.

FIG 6

The response regulator AlgR binds directly to PmucR. (A) Schematic representation of the putative AlgR binding sites (BS1 and BS2) identified in PmucR. The DNA fragments used in panels B (438 bp), C (114 bp), and D (211 bp) are shown. (B, C, and D) EMSAs were performed in order to analyze the AlgR binding to a PmucR fragment carrying BS1 and BS2 (B), BS1 only (C), or BS2 only (D). The DNA fragments were incubated with increasing concentrations of AlgR. As a negative control, a fragment carrying the regulatory region of Avin_05510 was included in each DNA binding reaction. The migration of the DNA fragments was visualized by staining with ethidium bromide.