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

Abstract

<i>Azotobacter vinelandii</i> produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in <i>A. vinelandii</i> and <i>Pseudomonas aeruginosa</i> 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 <i>A. vinelandii</i> 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 <i>A. vinelandii</i>, <i>Av</i>GReg, 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 <i>Av</i>GReg in the parental strain phenocopied a Δ<i>mucG</i> 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 <i>Av</i>GReg.<b>IMPORTANCE</b> c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by <i>Pseudomonas</i> and <i>Azotobacter</i> 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.