CLONING AND FUNCTIONAL-CHARACTERIZATION OF NEISSERIA-GONORRHOEAE TONB, EXBB AND EXBD GENES

Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of s ources including transferrin (TF) and lactoferrin (LF). To gain insigh t into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild-type DNA that repaired t he defect in flu mutants, which are unable to take up Fe from either T F or LF despite exhibiting apparently normal ligand binding to the rec eptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged i n tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26-32% identity with TonB proteins of n ine other bacteria. The 663bp, ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32-38% and 24-36% identity, respectively, with Exb B and ExbD proteins of three other bacteria. Thus, the sequence compar ison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of th e E. coil tonS, exbS and exbD genes. An insertional mutation in the to nS homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to t ake up Fe-55 from TF and LF. The tonS mutation did not prevent the uti lization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid-phase binding assay was largely unaffect ed by the tonS mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on th e TonB system.