GUZMAN, J. | bioRxiv Science

bioRxiv2025

Azotobacter vinelandii AmrZ is a global regulator linking alginate production and c-di-GMP homeostasis

Azotobacter vinelandii, a member of the Pseudomonadaceae, produces the exopolysaccharide alginate during vegetative growth; however, the circuitry linking alginate biosynthesis to lifestyle transitions remains poorly defined. Here we show that the Ribbon-Helix-Helix (RHH) transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels, and motility. Deletion of amrZ abolished alginate synthesis, whereas chromosomal complementation restored it. A PalgD-gusA fusion and RT-qPCR demonstrated that algD, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of algD, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: PamrZ-gusA activity decreased in {Delta}amrZ, and purified AmrZ bound the amrZ promoter in EMSA. Moreover, PamrZ activity required the sigma factor AlgU, consistent with the presence of an AlgU promoter; this positive, AlgU-dependent feedback may stabilize AmrZ under alginate-inducing conditions. To probe AmrZ control of c-di-GMP, we implemented a riboswitch-based biosensor in A. vinelandii. The {Delta}amrZ strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant; whereas a phosphodiesterase (PDE) mutant displayed elevated output, validating the assay. RNA-seq and RT-qPCR identified two DGC genes, AVAEIV_RS11610 and AVAEIV_RS18795, as AmrZ-activated targets; EMSA verified direct binding at the RS11610 regulatory region. By contrast, transcription of the principal vegetative DGC AvGreg was not AmrZ-regulated. Lower c-di-GMP in {Delta}amrZ correlated with larger swimming halos. Collectively, these genetic, biochemical, and transcriptomic data support a model in which AmrZ directly activates algD and elevates c-di-GMP via selected DGCs, thereby promoting alginate synthesis while reducing motility. RNA-seq data also indicate that AmrZ influences broader cellular programs, including metabolism and ion homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii.

microbiology↗

bioRxiv2024

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 {Delta}fleQ relative to the wt strain, which agreed with increased algD transcription. These phenotypes were rescued in the {Delta}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.

microbiology↗