The two genes encoding DNA gyrase in Mycobacterium tuberculosis are present next to each other in the genome, with gyrB upstream of gyrA. We show that the primary transcript is dicistronic. However, in addition to the principal promoter, there are multiple weaker promoters that appear to fine-tune transcription. With these and other mycobacterial promoters, we propose consensus promoter sequences for two distinct sigma factors. In addition to this, the gyr genes in M. tuberculosis, as in other species, are subject to autoregulation, albeit with slower kinetics, probably reflecting the slower metabolism of the organism.Most of our understanding of prokaryotic transcription initiation is based on extensive analysis of promoter architecture in Escherichia coli. Since the regions in 70 involved in contacting the promoter show extensive conservation across the prokaryotic world (13), a similar picture for transcription initiation is expected in all bacteria. However, this does not appear always to be the case. For instance, results of earlier random promoter screens indicate that only a small fraction of mycobacterial promoters are recognized by the E. coli machinery (6, 26). Furthermore, a random promoter screen in Mycobacterium paratuberculosis detected only promoters that were highly GC-rich in both their Ϫ10 and Ϫ35 regions (2). Thus, the features that define species-specific promoters are not clear.Here we present the analysis of the transcription of the DNA gyrase genes in Mycobacterium tuberculosis. As the sole supercoiling activity in the cell, DNA gyrase faces the daunting task of opposing the relaxing activities of both topoisomerases I and IV (29). As a result, DNA gyrase is essential in all eubacterial cells that have been tested so far, and the final topology of DNA is maintained by the equilibrium achieved by these divergent forces. Since DNA gyrase needs to oppose the relaxation induced by other topoisomerases, it regulates its own synthesis by a unique mechanism. In general, transcription of most genes is induced by increased negative supercoiling. In contrast, negative supercoiling represses transcription of the gyrase genes in E. coli (15). This phenomenon, referred to as relaxation-stimulated transcription, is believed to be the cell's strategy to homeostatically maintain the topology of DNA (15). Thus, increased gyrase levels lead to an increase in supercoiling, which, in turn, repress the expression of gyrase and allow other topoisomerases to bring the topology of the DNA back to its optimum state. Relaxation-stimulated transcription appears to be conserved in all organisms tested so far (14,23,25,28); however, the underlying mechanism appears to vary (27).Therefore, there are multiple reasons to analyze the transcription of the gyr genes in M. tuberculosis, especially since the genome lacks both topoisomerases III and IV (5). In addition, since the expression of many virulence genes is dependent on the topology of DNA in many pathogenic bacteria (8), understanding the regulation of DNA gyrase in...