Increased c-di-GMP Levels Lead to the Production of Alginates of High Molecular Mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.

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

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.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.