Neisseria gonowhoeae has evolved a repertoire of high-affinity iron acquisition systems to facilitate essential iron uptake in the human host. Acquisition of iron requires both the energy-harnessing cytoplasmic membrane protein, TonB, as well as specific outer membrane TonB-dependent transporters (TdTs). The overall goal of this study was to investigate the extra- and intracellular iron acquisition mechanisms of N. gonorrhoeae and determine the role of the TonB and TdTs in this process.The ability of gonococci to acquire potential exogenous iron sources was determined by plate bioassay. Gonococcal growth was promoted by various catecholate and hydromate siderophores; however, growth was not dependent upon TonB expression.As all previously characterized siderophore-iron uptake is dependent upon this protein, apotential TonB-bypass mechanism is suggested.The role of the Ton system and TdTs in gonococcal survival within human cervicalepithelial cells was also determined for two gonococcal strains, FA1090 and MS 1 1. Wedemonstrate that intracellular survival of both strains was dependent upon host cell ironacquisition, yet the expression of the Ton system was only critical to the survival ofFA1090. One characterized difference between these two strains is possession of thegonococcal genetic island (GGI) which is present in approximately 80% of gonococcalstrains. This study demonstrates that the GGI provides a mechanism to bypassintracellular TonB-dependent iron acquisition.In the strain lacking the genetic island, none of the characterized TdTs provided abenefit to the gonococcus when grown intracellularly. However, expression of oneuncharacterized TdT, TdfF, was necessary for successful intracellular survival. To ourknowledge, this is the first demonstration of a specific requirement for a single irontransporter in the survival of a bacterial pathogen within host epithelial cells.In the GGI-containing strain, TonB function was not critical to survival withincervical epithelial calls. The presence of the GGI was associated with the ability to bypass TonB-dependant uptake. Specifically, this bypass mechanism was mediated bycomponents of the T4S machinery encoded by the GGI, and replication was directlyrelated to iron acquisition. To our knowledge, this study provides the first direct linkbetween bacterial iron acquisition and a type IV secretion system.