Functional and Proteomic Analyses of a Putative Carbamoyl Phosphate Synthase Large Subunit in Relation to Virulence, Arginine and Pyrimidine Biosynthesis, and Siderophore Production in Erwinia amylovora

Abstract

The apple is a significant global fruit cultivated extensively worldwide. Fire blight, caused by Erwinia amylovora (Ea), poses a significant threat to global apple production. To control this disease, characterizing the virulence mechanisms/factors is imperative. Carbamoyl phosphate synthase is an essential enzyme in the biosynthesis of arginine and pyrimidine. However, the functions of this protein in Ea remains poorly understood. This study aimed to investigate the functions of the carbamoyl phosphate synthase large subunit in Ea (CarBEa). In a virulence assay using fruitlets, an Ea strain lacking CarBEa exhibited significantly reduced virulence on fruitlets. In the auxotrophy assay, this mutant failed to grow in minimal media lacking both arginine and pyrimidine, but growth was restored when both compounds were supplemented. The comparative proteomic analysis suggests that CarBEa is involved in diverse biological processes, including amino acid and nucleotide metabolism, and inorganic ion transport. Finally, we demonstrated that CarBEa is related to siderophore secretion/production by the chrome azurol S agar plate assay. This report provides valuable insights into the functions of carbamoyl phosphate synthase large subunit, which serves as a potential target for developing efficient anti-virulence substances to control fire blight.