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

<i>Azotobacter vinelandii,</i> 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 transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels and motility. Deletion of <i>amrZ</i> abolished alginate synthesis, whereas chromosomal complementation restored it. A P<i>algD-gusA</i> fusion and RT-qPCR demonstrated that <i>algD</i>, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of <i>algD</i>, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: P<i>amrZ-gusA</i> activity decreased in Δ<i>amrZ</i>, and purified AmrZ bound the <i>amrZ</i> promoter in EMSA. Moreover, P<i>amrZ</i> 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 <i>A. vinelandii</i>. The Δ<i>amrZ</i> strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant, whereas a phosphodiesterase 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 Δ<i>amrZ</i> correlated with larger swimming halos. Collectively, these genetic, biochemical and transcriptomic data support a model in which AmrZ directly activates <i>algD</i> 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 programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in <i>A. vinelandii</i>. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the <i>Pseudomonas</i> genus and reveals important differences in its targets and regulatory mechanisms.