Neisseria gonorrhoeae bacterioferritin: structural heterogeneity, involvement in iron storage and protection against...

The iron-storage protein bacterioferritin (Bfr) from Neisseria gonorrhoeae strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Escherichia coli Bfr. The DNA sequence of bfrA was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial fur sequence) was located within the 5' flanking region of bfrA in front of a putative -35 hexamer. The homology between the DNA sequences of bfrA and bfrB was 55.7%; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 39.7% identity, and showed 41.3% and 56.1% identity, respectively, to E. coli Bfr. Expression of recombinant BfrA and BfrB in E. coli strain DH5alpha was detected on Western blots probed with polyclonal anti-E. coli Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the omega fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress.