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Title: Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis
Authors: Roy, Sougata
Advisors: Ajithkumar, Parthasarathi
Keywords: Gene Transcription
Gene Regulation
Mycobacterium Tuberculosis
Mycobacterium Smegmatis
Cell Division Gene
Submitted Date: Jun-2006
Series/Report no.: G20328
Abstract: The success of Mycobacterium tuberculosis as a pathogen is due to its remarkable ability to: (i). adapt to and survive inside activated macrophages under nonproliferating condition, (ii). put up drug resistance and (iii). enter into hypoxia-induced dormancy and remain in nonproliferating condition, be resistant to drugs, and get reactivated into proliferation when favourable conditions arise. Thus, regulation of cell division (arrest and resumption) is an obligatory event that is critical to the pathogen for the establishment of successful infection, latency and reactivation process in human host. Therefore, in order to understand and combat the successful survival strategy of the bacterium inside the host macrophages or in granuloma, a basic knowledge of the regulation of cell division in tubercle bacillus is essential. Bacterial cytokinetic protein FtsZ (a tubulin homologue) is the key regulatory molecule for cell division and its intracellular level is critical for initiation of cell division in bacteria. Therefore, in order to understand the regulation cell division by expression and maintenance of ftsZ mRNA and protein, we initiated studies on the transcriptional regulation of ftsZ gene in the slow growing pathogen, M. tuberculosis, and in the fast-growing saprophyte M. smegmatis. Identification of regions containing ftsZMt promoter activity In order to identify promoter activity-containing regions of ftsZ gene of M. tuberculosis H37Rv (ftsZMt) in vivo, different regions upstream of ftsZMt namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were cloned in a gfp reporter plasmid and analyzed for gfp expression in M. smegmatis mc2155 cells. Flow cytometric analysis of exponentially grown M. smegmatis mc2155 cells containing these transcription fusion constructs revealed GFP expression in the cells harbouring ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467 bp and 3’ 217 bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc2155 cell transformants carrying the entire ftsQ ORF-ftsQ-ftsZ intergenic region containing construct, as well as on total RNA from M. tuberculosis confirmed that the regions identified indeed elicit promoter activity. RT-PCR analysis on M. tuberculosis RNA as well as semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed that about 70% of the total promoter activity comes from ftsQ ORF and there is co-transcription of ftsQ-ftsZ genes. Multiple transcripts code for ftsZMt Primer extension analysis, using primers annealing at different positions in the ftsQ-ftsZ chromosomal region, on RNA from M. tuberculosis as well as from M. smegmatis transformants containing 1.117 kb ftsZMtpromoter region in a promoter probe vector, identified origin of six different transcripts (T1-T6) for the gene. Among them, five transcripts (T1, T2, T3, T4, and T6) were detected in M. tuberculosis cells at exponential phase of growth. T5 could be detected only in M. smegmatis transformants containing 1.117 kb ftsZMt promoter upstream of mycgfp2+ reporter gene. Transcript T1 and T2 originate in the ftsQ-ftsZ intergenic region, while T3, T4, and T6 start in the ftsQ ORF. Analysis of sequence in the –10 and –35 regions of the corresponding promoters for the individual transcripts identified high GC content of the regions, which is characteristic of promoters of M. tuberculosis. All of the individual promoter sequences were independently cloned in a promoter probe vector and confirmed that they are true promoters, active in M. smegmatis cells, and that the T1-T6 transcripts were not products of RNA processing. Differential expression from the multiple ftsZMt promoters In order to study the activity and regulation of ftsZMt promoters in M. tuberculosis cells, which is a slow grower and also asymptomatically survives as dormant bacteria for decades in human granuloma, a stably genome-integrated plasmid was required where activity of the promoters can be studied by means of stable and enhanced gfp expression. For that purpose, an L5-mycobacteriophage attP (attachment site)-specific integration proficient promoter probe vector, which contains a stable gfp gene (mycgfp2+) whose codon has been optimized for mycobacterial expression, was generated. Using the vector, all the six promoter regions (P1-P6) were studied in M. smegmatis and M. tuberculosis cells. Flow cytometric and semi-quantitative RT-PCR analyses showed that promoter P5 is unable to elicit activity in M. tuberculosis cells, unlike in M. smegmatis transformants. Semi-quantitative RT-PCR analyses showed that expression of P3 is only 10-20% of the total promoter activity. Promoters P1, P2, P4 and P6 showed 50-80% activity of the total promoter activity and their activity were comparable in M. smegmatis and M. tuberculosis. The presence of multiple promoters reflects the requirement to maintain high basal level of, or to differentially regulate a critical level of, FtsZ expression during different pathogenic stages of tubercle bacilli. In order to investigate the role of multiple promoters, we verified the levels of expression of the five transcripts from the five ftsZ promoters in M. tuberculosis cells under conditions of growth inside mouse macrophage cell line and also under various stress conditions mimicking those that exist in the granuloma environment, like conditions of nonreplicating persistence, gradual nutrient depletion stress, oxidative stress, surface tension stress, acidic stress, heat shock, DNA damaging conditions and osmotic stress. For this purpose, individual promoter regions were cloned into a stably inheritable gfp reporter plasmid vector, and into an L5 mycobacteriophage attP (attachment site)-specific integration-proficient variant of the same vector, for the expression of the promoters from the chromosomal locus in M. smegmatis and M. tuberculosis cells. Quantitative primer extension analyses, semiquantitative RT-PCR analyses on RNA from M. tuberculosis cells grown under these different conditions, and quantitative GFP fluorescence analyses in these cells showed differential activation of the five promoters under different conditions of growth. Under hypoxic and nutrient-depleted stationary phase of growth, two new promoters, Tdor and Ts, in the ftsQ ORF were identified, and these promoters showed maximal activity only under those specific conditions of growth. None of the ftsZ promoters were found to be responsive to stringent response mediated by overexpression of M. tuberculosis RelA. None of the promoters were also found to be responsive of overexpression of heat-shock sigma factor SigH in M. tuberculosis, implicating new pathway of regulation of ftsZ promoters. Multiple promoters driving expression of ftsZ gene of M. smegmatis Similar studies, which were carried out on the identification, structural and functional characterization, regulation of the promoters of cell division gene ftsZ in the fast growing saprophyte M. smegmatis cells, showed the presence of four ftsZ promoters, three of which originates from the 249 bp ftsQ-ftsZ intergenic region and one from the ftsQ ORF. RT-PCR analysis showed that both ftsQ and ftsZ are co-transcribed. Cloning and expression analysis of the individual promoters mapped by primer extension in a GFP based reporter plasmid showed that all the four putative regions are true promoters. Quantitative primer extension on RNA from a synchronously grown culture identified P2 promoter to be responsive to either initiation of cell division or stress, although expression of P1, P3, and P4 did not vary with respect to synchronous division. Quantitative primer extension analysis and semi-quantitative RT-PCR analysis on the RNA from M. smegmatis cells showed that under various stress conditions, P2 activity goes down significantly. Under nutrient depleted stationary phase and hypoxic nonreplicating persistence stage-2, the levels of P2 and P3 activity could hardly be detected, whereas, expression from P1 and P4 goes down only in hypoxia. Level of total ftsZ mRNA remains almost the same under various stress conditions, although upon hypoxia and stationary phase the level goes down almost two fold. Thus, in fast growing M. smegmatis too, multiple ftsZ promoters are differentially regulated under various stress conditions and a critical level of ftsZ mRNA is maintained. Taken together, the study of ftsZ promoters of a slow-growing pathogenic mycobacterium and a fast growing non-pathogenic mycobacterium indicate that differential expression of the multiple promoters, along with conditional activation of stage specific promoters like Pdor or Ps, is one of the mechanisms through which the bacilli probably maintain required levels of FtsZ protein that are crucial for the cell survival, probably through cytoskeletal maintenance, and cell division.
URI: http://hdl.handle.net/2005/400
Appears in Collections:Microbiology and Cell Biology (mcbl)

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