The c-di-GMP effector FleQ controls alginate production by repressing transcription of the algD gene in Azotobacter vinelandii

Azotobacter vinelandii , belonging to the Psedomonadaceae family, produces the exopolysaccharide alginate during its vegetative growth and also during its differentiation process to form desiccation resistant cysts. The second messenger c-di-GMP regulates a vast array of cellular processes. It is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). In A. vinelandii the absence of the AvgReg DGC impairs alginate synthesis while the absence of the PDE MucG increased alginate production. The effect of the different c-di-GMP levels was solely attributed to its essential role in activating the alginate polymerase complex. Here in we investigated the possible role of c-di-GMP in the control of algD transcription, encoding the key enzyme of the alginate biosynthetic pathway. At artificially high or reduced levels of c-di-GMP algD transcription was increased, or almost abrogated, respectively. Therefore, the role of the transcriptional regulator FleQ, one of the best characterized c-di-GMP effectors, was investigated. Alginate production increased in the Δ fleQ relative to the wt strain, which agreed with increased algD transcription. These phenotypes were rescued in the Δ fleQ / fleQ + complemented strain indicating a FleQ repressing effect. EMSA assay showed that FleQ was able to directly bind to the regulatory region of algD which agreed with the presence of a FleQ binding site overlapping its RpoS-dependent promoter. In A. vinelandii the c-di-GMP is also necessary for expression of alginate C-5 epimerase genes, essential for structuring mature cysts. Out data revealed that FleQ is not an intermediary in this regulation since its absence did not impair mature cyst formation nor affected expression of algE1-6 genes. Collectively, our results support a model in which FleQ is the intermediary in the regulation of algD by c-di-GMP, exerting a direct repressing effect and reveal the existence of a FleQ-independent regulatory mechanism for the control of A. vinelandii encystment.