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

Production of the exopolysaccharide alginate by Azotobacter vinelandii, member of the Pseudomonadaceae family, is positively controlled by the second messenger c-di-GMP. This effect was solely attributed to the role of c-di-GMP in activating the alginate polymerase complex. In this study, the role of c-di-GMP in algD transcription, which encodes the key enzyme for alginate synthesis, was investigated. algD transcription correlated with artificially high or low levels of c-di-GMP. Moreover, FleQ, one of the best-characterized c-di-GMP effectors, was found to exert a negative effect on alginate production and algD transcription, as both increased in a Delta fleQ mutant relative to the wild-type strain or the Delta fleQ/fleQ+ complemented strain. Electrophoretic mobility shift assays (EMSAs) confirmed that FleQ directly binds to the regulatory region of algD, which was consistent with the presence of two FleQ binding sites in the vicinity of the algD RpoS-dependent promoter. In A. vinelandii, c-di-GMP is essential for the expression of alginate C-5 epimerases (AlgE1-6), which are necessary for structuring the envelope of differentiated cells, known as cysts. However, FleQ was not involved in this regulation. Collectively, our results support a model in which algD transcription is under the positive control of c-di-GMP, while FleQ may only partially mediate this effect. In contrast, our study revealed a FleQ-independent regulatory mechanism for the control of A. vinelandii encystment.