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

Production of the exopolysaccharide alginate by <i>Azotobacter vinelandii</i>, member of the <i>Pseudomonadaceae</i> 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 <i>algD</i> transcription, which encodes the key enzyme for alginate synthesis, was investigated. <i>algD</i> 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 <i>algD</i> transcription, as both increased in a Δ<i>fleQ</i> mutant relative to the wild-type strain or the Δ<i>fleQ</i>/<i>fleQ</i>+ complemented strain. Electrophoretic mobility shift assays (EMSAs) confirmed that FleQ directly binds to the regulatory region of <i>algD</i>, which was consistent with the presence of two FleQ binding sites in the vicinity of the <i>algD</i> RpoS-dependent promoter. In <i>A. vinelandii</i>, 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 <i>algD</i> 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 <i>A. vinelandii</i> encystment.