Bacillus cereus Fur regulates iron metabolism and is required for full virulence

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a beta-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Deltafur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.