Construction and phenotypic characterization of fur-deleted mutant of Pseudomonas aeruginosa

Abstract:

Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence in Pseudomonas aeruginosa. Many research groups have failed to construct the fur-deleted mutant of P. aeruginosa, so fur has always been considered to be an essential gene in P. aeruginosa, and the knowledge of its biological function is limited. 【Objective】 This study aims to construct a fur-deleted mutant of P. aeruginosa and analyze its phenotypes. 【Methods】 With P. aeruginosa PAO1 as the parental strain, the fur-deleted mutant was constructed by homologous recombination. After that, we studied the effects of fur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence of P. aeruginosa. In addition, we explored the cause of the growth defect phenotype of the fur-deleted mutant by genetic analysis. 【Results】 The fur-deleted mutant of P. aeruginosa was successfully constructed. The deletion of fur greatly limited the growth of P. aeruginosa and reduced the growth adaptability of P. aeruginosa to the iron-limited environment, while it did not affect the growth adaptability of P. aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologous fur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur of P. aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype of P. aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype of P. aeruginosa, the deletion of fur also made P. aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence to Galleria mellonella larvae. Moreover, the deletion of fur increased the intracellular cyclic diguanylate (c-di-GMP) level of P. aeruginosa to induce the expression of pelF and pslA, thereby promoting the biofilm formation of P. aeruginosa. 【Conclusion】 fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence of P. aeruginosa, which lays a foundation for the development of vaccines and agents against P. aeruginosa.