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

## TL;DR

The AmrZ protein in Azotobacter vinelandii controls alginate production and cell movement by regulating c-di-GMP levels and gene expression.

## Contribution

This study identifies AmrZ as a global regulator linking alginate biosynthesis and c-di-GMP homeostasis in Azotobacter vinelandii.

## Key findings

- AmrZ activates algD transcription and increases c-di-GMP, promoting alginate synthesis.
- AmrZ positively autoregulates its expression via an AlgU-dependent feedback loop.
- AmrZ influences motility and broader cellular processes like metabolism and iron homeostasis.

## Abstract

Azotobacter vinelandii AmrZ positively regulates alginate production by directly activating algD transcription and enhancing intracellular c-di-GMP accumulation, which, in turn, activates the alginate polymerization complex. Two diguanylate cyclases (DGCs) were identified as AmrZ targets, likely accounting for the reduced c-di-GMP levels observed in the absence of AmrZ. Transcription of avGReg, which encodes the main DGC during vegetative growth, was not under AmrZ control; however, post-translational regulation of AvGReg activity by AmrZ cannot be ruled out. AmrZ also positively autoregulates its own expression through an AlgU-dependent feedback loop. Finally, AmrZ positively modulates swimming motility, and our transcriptomic data suggest that this effect is indirect, occurring via its positive influence on c-di-GMP levels.

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 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 ΔamrZ, 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 ΔamrZ 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 ΔamrZ 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 programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the Pseudomonas genus and reveals important differences in its targets and regulatory mechanisms.