Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

Abstract

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

Figure 1.

TopoI expression is sensitive to gyrase inhibition. M. smegmatis cells were grown to exponential phase and treated with the different concentrations of novobiocin to induce chromosome relaxation. The cells were lysed and subjected to immunoblot analysis using specific antibodies against the proteins of interest (as indicated in the panel). (A) M. smegmatis cells treated with 200 μg/ml novobiocin for 4–6 h and the expression of TopoI, GyrA and SigA was monitored. (B) Dose dependent repression of TopoI expression by novobiocin. The M. smegmatis cells were exposed to various concentrations of novobiocin for different time and TopoI expression was monitored. (C) Transcript analysis of TopoI expression. The exponential phase cells of M. smegmatis and M. tuberculosis treated with the novobiocin (100 μg/ml) for 3 h–6 h and 12–24 h, respectively. Total RNA was isolated from treated and untreated cells and cDNA was prepared using random hexamer primers. Abundance of topoI transcript was measured by qRT PCR analysis using gene specific primers. Fold change in topoI expression compared to the untreated culture. sigA was used as a reference gene for the expression analysis. The error bars represent the SD (standard deviation) obtained from three independent experiments. *P < 0.01, **P < 0.001.

Figure 2.

Reduced expression of TopoI upon chromosome relaxation. The chromosome relaxation was carried out by ectopic overexpression of M. smegmatis TopoI using tetracycline inducible system. The exponential phase M. smegmatis cells were treated with the tetracycline (25 ng/ml) for 6 h and the expression of TopoI and DNA gyrase was monitored both at protein as well as RNA level. (A) Schematic for the discriminatory qRT PCR. The 5′ UTR of the ectopically expressed topoI (etopoI) and genomic copy of topoI (gtopoI) were different. The primers specific for gtopoI were used to determine the alteration in the expression of gtopoI upon TopoI overexpression (to cause chromosome relaxation). (B) Immunoblot analysis of TopoI and gyrase expression upon induction with the tetracycline. The increased overexpression of GyrA upon TopoI overexpression indicates the chromosome relaxation (RST). (C) Measurement of the genomic topoI transcript abundance in the cells upon overexpression of TopoI by discriminatory qRT PCR analysis. The fold change is expression represents the expression of topoI transcripts upon tetracycline induction normalized with the uninduced RNA samples. topoI (ORF) indicates the expression of total topoI transcripts; gtopoI and etopoI represents the expression of genomic copy and plasmid copy of topoI transcripts, respectively. The error bars represents the SD obtained from three independent experiments. *P < 0.01, **P < 0.001, ns: not significant (P > 0.1).

Figure 3.

Contribution of upstream elements on topoI promoter activity. (A) Schematic representation of the constructs generated for the study. (B) Measurement of the activity of various constructs harboring topoI promoter(s) by β-galactosidase assay. (C) Determination of gyrase binding on upstream region of topoI by ChIP-qRT PCR using the primers specific to the topoI upstream region. The enrichment values represent the enrichment of DNA fragment of interest (topoI promoter region and ORF) in immunoprecipitated (IP) sample over the mock. The error bars represent the SD obtained from three independent experiments.*P < 0.01, **P < 0.001, ***P < 0.0001, ns: not significant (P > 0.1).

Figure 4.

Identification of Transcription Start Sites of topoI gene from M. smegmatis. (A) Primer extension analysis was carried out to map the TSS upstream of topoI as described in Materials and Methods. The primer extension products corresponding to the transcription start site for each promoter is indicated (arrows). E: exponential phase culture without novobiocin treatment, N: exponential phase culture treated novobiocin (100 μg/ml) for 3 h, S: stationary phase culture without novobiocin. (B) Table representing the 5′ UTR length and first nucleotide of the each transcripts.

Figure 5.

Supercoiling sensitive expression of topoI is specific to native topoI promoter(s). (A) Promoter elements upstream to the start sites. Arrows indicates the transcription start sites. (B) Schematic showing the replacement of native promoter(s) of topoI with a ptr promoter (having optimally spaced −35 and −10 elements—17 bp spacer). (C) Immunoblot analysis of the TopoI and gyrase expression in native M. smegmatis cells and mutant strain with ptr promoter. The exponential phase recombinant and WT M. smegmatis cells were treated with novobiocin (100 μg/ml) for 6 h and processed for the immuno-detection of TopoI.

Figure 6.

Effect of chromosome relaxation on RNAP binding on TopoI promoter(s). (A) Schematic of the experimental design. Negative supercoiling may bring the −35 and −10 elements of topoI promoter(s) on the same phase of DNA thus enhancing the RNAP-promoter interaction. (B) Exponentially grown M. smegmatis cells were treated with novobiocin (100 μg/ml) for 6 h. The treated cells were subjected to ChIP using anti-RpoB antibody. The immunoprecipitated DNA was analyzed for the enrichment of promoter region of various genes by qRT-PCR using primers flanking the promoter region. (C) Model representing the SST of TopoI. Sub-optimal spacer length between −10 and −35 elements align them out of phase affecting the optimal RNAP-promoter interaction. Additionally upstream gene transcription affects the DNA topology restricting the topoI activity. The negative supercoiling activity of DNA gyrase maintains the optimal topology of the topoI promoter aligning the −35 and −10 elements in the appropriate orientation required for the optimal transcription. Conditions leading to the chromosome relaxation reduce the topoI expression by altering optimal orientation of −35 and −10 elements. The error bars represent the SD obtained from three independent experiments.*P < 0.01, ns: not significant (P > 0.1).