Deciphering of a bioactive molecule with a three-domain inhibitory activity from a halophilic bacterium Arhodomonas sp. AD133

Pyoluteorin, a biological pesticide, is mainly produced by growth promoting rhizosphere bacteria such as Pseudomonas species showing strong antifungal and antibacterial activities. Little is known about pyoluteorin from halophiles living in hypersaline environments. The halophilic bacterium Arhodomonas sp. strain AD133, which was isolated from a hypersaline environment, demonstrated clear inhibitory activity against Bacteria, Eukaryotes and Archaea. Strain AD133 not only can inhibit halophilic archaea and bacteria that live in the same niche, but also can suppress other microorganisms, such as the opportunistic pathogen Staphylococcus aureus ATCC 6538, and plant pathogen Dickeya zeae WH1, Pyricularia oryzae and Valsa spp. It can utilize various carbon sources—including xylose, glucose, fructose, sorbitol, sucrose, maltose, glycerol, and acetate—for pyoluteorin production. Crucially, the pyoluteorin biosynthetic gene cluster was identified on the genome of AD133, and its functionality was supported by transcriptional data, leading to a proposal of pyoluteorin biosynthesis pathway. Consequently, this work establishes a comprehensive pipeline spanning from strain isolation through bioactive compound identification to biosynthetic gene cluster characterization. Therefore, it provides an instructive reference case for research on natural bioactive compounds derived from halophiles. Importance This study identifies the halophilic bacterium Arhodomonas sp. strain AD133, isolated from a hypersaline environment, as a novel producer of the antifungal and antibacterial compound pyoluteorin. The bacterium exhibits broad-spectrum inhibitory activity against diverse microorganisms, including archaea, bacteria, eukaryotes, and pathogens such as Staphylococcus aureus and Dickeya zeae. The pyoluteorin biosynthetic gene cluster was functionally characterized in its genome, enabling the proposal of its biosynthesis pathway. This work provides a complete research framework for discovering bioactive compounds from halophiles, from isolation and activity testing to genetic validation.