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The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein H1p, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture.

We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.