Neisseria meningitidis and Neisseira gonorrhoeae are pathogens that only colonise humans. Pathogenic Neisseria are able to use nitrite as a terminal electron acceptor to grow anaerobically. In this pathway, an outer membrane copper-containing nitrite reductase, AniA, converts nitrite( NO2 -) to nitric oxide ( NO. ) Nitric oxide is then reduced to nitrous oxide (N2O) by nitric oxide reductase (NorB). Nitrite reductases in other Gram-negative bacteria are localized in the periplasm but in contrast, AniA of pathogenic Neisseria is expressed on the surface. The accessory proteins required for AniA function (i.e. with roles in folding, localization, electron transport, and etc.) are not known.
In the presence of nitrite in growth media, NorB mutant strains of pathogenic Neisseria accumulate nitric oxide leading to bacterial death. This lethal phenotype of the NorB mutant can be suppressed by loss of AniA function. This observation allowed development of a genetic screen in which cells could only survive if they lost AniA activity. Using random deletion mutagenesis, selection for growth in the presence of nitrite enabled identification of genes required for AniA function. Via this genetic screening method, we identified a copper chaperone that is essential for AniA activity and is named AniA Copper Chaperone A (accA). The role of AccA in AniA function was characterized by in vivo nitrite reductase assays and in vitro bathocuproine disulfonate (BCS) assays and surface plasmon resonance (SPR) experiments. Our study will help us in understanding the molecular mechanisms of copper homeostasis in pathogenic Neisseria.