Analysis of the transcriptional profile of the DosR mutant over a hypoxic time-course showed that the EHR is NVP-BEZ235 largely independent of the DosR regulon. Additional study of the EHR may provide important clues to MTB mechanisms of survival during bacteriostasis. In the defined hypoxic model, a constant flow of low oxygen gas over the surface of a stirred, early log phase culture is used to deplete the oxygen in a rapid and highly reproducible way. This model was used initially to characterize the MTB transcriptional response to hypoxia; the DosR regulon is induced within two hours and bacteriostasis is evident within 24 hours, with less than a single doubling occurring after the initial exposure to low oxygen conditions. In this system, the dosR mutant and wild-type strains showed no survival difference over a one-week period. Longer time points are not feasible in this system, due to complications from evaporation. To test survival following exposure to prolonged hypoxia, we employed a standing culture model. Wild-type and mutant bacilli were cultured in competition in small cryovials with no head space for up to 1 year. By 90 days, survival of the dosR mutant was about one log lower than that of wild-type. This difference was still evident after one year in standing culture. The most frequently used experimental approach to hypoxiainduced MTB dormancy is the defined headspace model of nonreplicating persistence described by Lawrence Wayne and colleagues. In this model MTB is grown in stirred, airtight tubes with a defined LY294002 headspace-to-culture ratio. The oxygen in the tube is depleted gradually over the course of days by the growing bacilli, with induction of the DosR regulon seen as early as 5 days. At 17 days, well after the DosR regulon is induced and bacteriostasis is firmly established, the dosR mutant showed a modest survival defect. The drop in relative viability increased to nearly fifty fold after 26 days, and after 35 days in the Wayne model survival of the mutant was,75-fold less than the wild-type strain. This result is consistent with the,2 log drop shown earlier with a related MTB mutant in which DosR expression is disrupted, though significantly less than the 1000-fold drop in viability reported in a dosR deletion in a Mycobacterium bovis BCG vaccine strain. To assess the link between DosR regulon expression and virulence or persistence in vivo, the dosR mutant was used to infect C57BL/6 mice. The bacterial burden as measured by colony forming units and histopathology of the mutant was indistinguishable from the parent strain H37Rv. Additional experiments with more susceptible DBA2 and C3He/ FEJ mice confirmed that DosR is dispensable for persistence and virulence in these models.