Abstract
Mycobacterial persistence is a major hindrance in the treatment of tuberculosis. Persistence is thought to result from bacterial adaptation to the prevailing environment within tuberculous lesions and granulomas that are believed to be deficient in oxygen and nutrient supply (Wayne & Sohaskey, 2001). In vitro models of mycobacterial persistence have provided us with valuable insights into the physiological, structural, metabolic and molecular changes that accompany adaptation to oxygen limitation (Wayne & Sohaskey, 2001). A recent global analysis of expression revealed that the expression of more than 100 genes was altered when Mycobacterium tuberculosis was subjected briefly to defined hypoxic conditions (Sherman et al., 2001). Among the induced genes was the devRdevS two-component regulatory system. The genes encoding the DevRDevS two-component system were first identified and partially characterized in M. tuberculosis (Dasgupta et al., 2000) and found to be well conserved in several mycobacteria including Mycobacterium smegmatis (Zahrt & Deretic, 2001; Tyagi & Sharma, 2002). M. smegmatis exhibits an adaptation response to oxygen limitation that is similar to that of M. tuberculosis (Dick et al., 1998). In fact, the molecular mechanisms involved in the dormancy response are likely conserved in the two species in so far as the involvement of the DevRDevS genetic system is concerned (Mayuri et al., 2002). The slow growth rate and pathogenicity of M. tuberculosis often pose a hurdle for the study of its physiology. This has prompted the use of a fast-growing, non-pathogenic species, namely M. smegmatis, as a surrogate host. In view of the conservation between the two species of several aspects of the in vitro hypoxia response (Wayne & Sohaskey, 2001; Dick et al., 1998), the transcription machinery (Bashyam et al., 1996), several sigma factor genes (Tyagi & Sharma, 2002) and hypoxic induction of Rv3134c, devR and devS (Sherman et al., 2001; Mayuri et al., 2002), we assessed the suitability of M. smegmatis for studying the regulation of the M. tuberculosis devRdevS promoter.
Rv3134c, devR and devS are co-transcribed from the complementary strand of the M. tuberculosis genome. The coding regions of devR and devS overlap by 1 bp, and a 27 bp segment separates Rv3134c from devR (Dasgupta et al., 2000). We reasoned that sequences mapping upstream of devR and Rv3134c were likely to contain promoter(s) driving transcription of these genes. The promoter activity of this region was assessed by lacZ reporter assays in M. smegmatis cultures. Briefly, Rv3134c- and devR-upstream sequences were PCR-amplified and cloned in the integration-proficient vector pDK16 (Jain et al., 1997; A. K. Tyagi & D. Kaushal, unpublished data), to generate pDP3 and pDP2 (Fig. 1a). Both constructs supported a low β-galactosidase activity that was consistently higher than that of the vector control in aerated cultures. Subsequently, the activity of putative PRv3134c and PRv3134cdevR promoters (in pDP3 and pDP2, respectively) was assessed in cultures grown in a sealed, static system where bacteria slowly settled to the bottom of the tube during growth causing oxygen depletion (Mayuri et al., 2002). Oxygen depletion was monitored via the fading and decolorization of methylene blue, which occurred by 24 and 48 h, respectively. Under these conditions, the various cultures grew in an exponential manner to a cell density of 2x107 cells ml-1 and then entered a plateau phase by day 6 or 7, which extended till the experiment was terminated on day 15. In contrast, under aerobic conditions, cells grew exponentially to an initial cell density of 3x108 to 1x109 cells ml-1 and shifted thereafter to stationary phase. In cultures carrying pDP3 (PRv3134c promoter construct), a 3·3-fold induction in lacZ activity was noted just 24 h after the initiation of static culture. A decline in activity was noted thereafter till day 4; lacZ activity was then stably maintained at 1·5-fold of basal level till the end of the experiment. In cultures carrying pDP2 (PRv3134cdevR promoter construct), a 5·7-fold induction in lacZ activity was noted at 24 h, which was followed by a steady decline in activity till day 4; thereafter lacZ activity was maintained at 2·2-fold of basal activity till the end of the experiment (Fig. 1b). Comparison of the β-galactosidase activities on day 1 of hypoxic cultures carrying pDP2 versus pDP3 pointed to the dual promoter (PRv3134cdevR) being 2·6-fold more active than the PRv3134c promoter. This indicated that this locus was driven by at least two hypoxia-responsive promoters: one mapping upstream of Rv3134c and the other upstream of devR. Induction of PRv3134c promoter activity was also confirmed by Reverse Transcriptase-PCR (RT-PCR) performed using RNA isolated from aerated and hypoxic cultures (day 2). RNA input in the reaction was first normalized by RT-PCR targeting 23S rDNA. M. tuberculosis-specific primers R8 and R9c, targeting the PRv3134c promoter region, were used to amplify a 263 bp fragment from M. smegmatis cultures carrying pDP2. An 3-fold increase in transcript levels from this region was noted in hypoxic cultures versus aerated cultures (Fig. 1c).