Defining mechanisms of gene regulation is critical for understanding how pathogenic mycobacteria survive in diverse environments and for identifying novel drug targets. For the human pathogen Mycobacterium tuberculosis, resuscitation factors are believed to play an important role in primary infections as well as for reactivation of latent infections. The main goal of the project is to identify the inter-species conservation and function of an important resuscitation-promoting factor (Rpf) from recently identified potential fish pathogens Mycobacterium shottsii and Mycobacterium pseudoshottsii. A significant impediment in mycobacterial research is the slow growth of these microbes in vitro, necessitating very long incubations that sometimes take months for appreciable growth. This research investigates if RpfB proteins accelerate growth in vitro and if Rpf proteins from one Mycobacterium species impact the growth of others. As proteins from this class are secreted into culture media, culture filtrates from aged bacterial cultures are tested for growth stimulation of low-density mycobacterial cultures. Recently, RpfB from M. tuberculosis H37Rv was over-expressed in E. coli strain CH3DE3 and purified using Ni+2- NTA chromatography. Further tests for growth stimulation are in progress. This research is beneficial in that Rpf proteins could potentially be used as a diagnostic marker for M. tuberculosis infection and may be especially helpful for diagnosing latent infections. Also, use of purified Rpf proteins may help accelerate culture of M. tuberculosis in sputum, thereby aiding in more-rapid diagnosis and treatment.