Increased c-di-GMP levels lead to the production of alginates of high molecular mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exo-polysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa draws several similarities but is regulated somewhat differently in the two microbes. Here we show that the second messenger cyclic dimeric guanosine monophosphate (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 the 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 for alginate production. Overproduction of AvGReg in the parental strain, phenocopied a ΔmucG strain with regards 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 (OTR). In this work, we show that cultures exposed to reduced OTR 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 exo-polysaccharides 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 oxygen transfer rates. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.