Molecular Analysis of Two Bacterioferritin Genes, bfrα and bfrβ, in the Model Rhizobacterium Pseudomonas putida KT2440

The model rhizobacterium Pseudomonas putida KT2440 and other fluorescent pseudomonads possess two bacterioferritins, Bfr alpha and Bfr beta. However, the regulatory systems controlling the expression of these genes and the roles of these proteins in iron homeostasis are ill defined. Our studies show that both bfr alpha and bfr beta were monocistronic: promoter motifs and transcriptional start sites were identified, and Fur boxes and sigma(S)-dependent regulatory motifs were absent. The expressions of bfr alpha and bfr beta were enhanced by iron exposure and were maximal in cells rapidly growing in a high-iron environment. Both bfr alpha and bfr beta were positively regulated by Fur, and both were expressed independently of adjoining, functionally related genes. The loss of Bfr alpha or Bfr beta individually resulted in a significant reduction (ca. 17%) in cellular iron levels, and the deletion of both bfr alpha and bfr beta reduced cellular iron levels by 38% relative to those of the wild type. The mutants varied in their abilities to grow in low-iron medium; while growths (rate and final cell density) of single mutants and the wild type were similar, that of the double mutant was reduced significantly. Mutants lacking Bfr alpha and/or Bfr beta showed no change relative to the wild type in sensitivity to reactive oxygen species toxicity. Collectively, the data show that while Bfr alpha and Bfr beta could function independently of each other, an interaction-dependent function cannot be ruled out. Furthermore, regardless of the mechanism, a primary benefit of the bacterioferritins to P. putida KT2440 appears to be the enhancement of its survival in the environment by strengthening its tolerance to iron starvation.