Neisseria Meningitidis TonB, ExbB, and ExbD Genes: Ton-dependent Utilization of Protein-bound Iron in Neisseriae

We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism.