| In order to understand ftsZ regulation in Mycobacterium tuberculosis, we generated three recombinant M. tuberculosis strains. These strains were chromosomal null for ftsZ but carried an integrated copy of ftsZ expressed from vectors carrying different combinations of putative ftsZ promoters. We examined the effect of transcription from four different ftsZ promoters on M. tuberculosis by conducting growth rate analysis, viability studies, western blot analysis, quantitative real time PCR analysis and cell length measurement, under different growth conditions. We found that M. tuberculosis does not require multiple ftsZ promoters for its growth and survival. We also examined the functional regulation of M. tuberculosis cell division by a putative negative regulator of FtsZ assembly and expression, named crgA. We measured crgA transcript levels using quantitative real time PCR analysis under various growth conditions that inhibit M. tuberculosis cell division. We found that the crgA expression is upregulated in M. tuberculosis grown in macrophages. Finally, we developed a technique using immunofluorescence microscopy to visualize FtsZ structures in M. tuberculosis. We were able to visualize FtsZ rings and other FtsZ structures in M. smegmatis and M. tuberculosis. Further studies will address the reproducibility of this protocol and its potential use in quantitating FtsZ rings in M. tuberculosis. Our results suggest that FtsZ is regulated transcriptionally as well as functionally in M. tuberculosis. |
