Metal homeostasis and SirR regulation during denitrification in Haloferax mediterranei.

Abstract Haloferax mediterranei is a haloarchaeon capable of complete denitrification, reducing nitrate to dinitrogen. While this enzymatic pathway is known, the regulatory mechanisms coordinating metal homeostasis during this process remain poorly understood. Previous transcriptomic analyses revealed increased expression of the gene encoding SirR (E6P09_18060), a DtxR-family transcriptional repressor, during the early stages of denitrification under oxygen-limited conditions. In this study, we investigated the functional role of SirR through the characterization of gene deletion and overexpression strains. Cell growth and intracellular metal concentrations (Fe, Mn, Zn, Cu, and Mo) were analysed using optical density measurements and ICP-MS. The results showed substantial metal accumulation in early growth stages, with higher accumulation in the overexpression strain under various conditions. qRT-PCR analysis of two genes (E6P09_00145 and E6P09_02480) encoding iron transporters indicated that SirR could regulate E6P09_00145, which encodes a putative iron-binding protein. This regulation is significantly modulated by the interplay between iron and oxygen availability. Furthermore, bioinformatic analysis of iron-uptake gene promoters identified a conserved regulatory motif, and gel filtration chromatography of the purified protein confirmed that SirR functions as a homodimer. These findings establish direct regulatory links among SirR, metal homeostasis, and oxygen levels providing new insights into the metabolic adaptations required for haloarchaeal denitrification under changing environmental conditions.