Table 1.
Species specific occurrence of nucleases relevant for this study.
Fig 1.
A: Comparison of DrrS accumulation between H37Rv and ΔdosR over two weeks shown by Northern blotting. Total RNA (5 μg) from the two strains was separated on a single 8% denaturing acrylamide gel and probed for DrrS. The 5S loading control also shows trace amounts of DrrS, particularly in H37Rv, demonstrating that DrrS is slightly smaller than 5S. OD600 measurements after day 3 were unreliable due to clumping. B: Induction of DrrS expression with DETA-NO. Cells were grown to mid-log phase and exposed to 0.1 mM and 1.0 mM DETA-NO for two hours, respectively. RNA was analysed by Northern blotting.
Fig 2.
. Transcription start sites (TSS) are indicated with red asterisks, putative -10 boxes for TSS2 are shown in green; previously identified DosR binding site (DBS) is shown as a black box, while a putative DBS is shown as a black dashed box. Regions cloned as lacZ fusions are shown as blue arrows above the promoter sequence. Red lines indicate ATCCT repeats in 3’ region (see text).
Table 2.
Sequences of -10 regions in selected DosR regulated genes.
Fig 3.
A: ß-galactosidase assays of DrrS promoter-reporter fusions containing either core or ‘full-length’ promoter, including DBS (Fig 2) were transformed into M. bovis BCG. Stationary phase cultures (Sta) were grown one week past OD600 = 1.0 before harvest and ß-galactosidase assays performed on cell extracts. Results represent mean ± standard deviation of three biological replicates normalised to total protein and values for empty vector subtracted; p<0.05. B: ß-galactosidase assays of DrrS promoter-reporter fusions expressed in M. smegmatis mc2155 or C: ΔdosRsm [45] with and without dosRtb. The assays were performed on exponential phase cultures and the Results represent mean ± standard deviation of three biological replicates normalised to total protein and values for empty vector subtracted; p<0.05.
Table 3.
β-gal activity of DrrS promoter constructs assayed in M. smegmatis.
Fig 4.
Predicted structure of the 109 nucleotide DrrS.
The 109 nucleotide DrrS sequence from TSS2 to the mapped CU(C) 3’ end was used to predict the structure in mfold.
Fig 5.
Mapping of 3’ termini of DrrS variants.
The panels illustrate how the DrrS 3’ termini vary as the 3’ region is modified. The top panel shows the 3’ RACE results of wildtype DrrS sequence with two ATCCTC repeats, which in M. tuberculosis leads to the majority of transcripts ending in U or C within the first repeat (indicated with red, vertical lines). Panels 2–4 show the results of DrrS variants expressed in M. smegmatis; panel 2 represents 3’ RACE results of DrrS cloned with two ATCCTC repeats before the SynB terminator, similar to wildtype DrrS; panel 3 shows the results when an extra repeat has been inserted, and panel 4 the results when only one repeat is included.
Fig 6.
Growth phase dependent accumulation of DrrS+.
A: Time course of expression from log- to stationary phase (as in Fig 1), but with more RNA and probed with the DrrS+ probe complementary to RNA downstream of position C109. Arrow indicates prominent transcript around 160 nucleotides described in the text. B: total RNA from M. tuberculosis H37Rv and M. tuberculosis ΔdrrS also probed for DrrS+ showing a range of transcripts similar to A in H37Rv but no detectable transcripts in ΔdrrS. Marker: Century marker (Ambion).
Fig 7.
Alignment of DrrS 3’ termini in WebLogo.
All the 3’ termini obtained with extended 3’ RACE (i.e. beyond C109) were aligned in WebLogo, which resulted in a GC|G consensus cleavage site.
Fig 8.
Turnover of DrrS in M. tuberculosis.
A stationary phase culture of M. tuberculosis OD600 ~6 was diluted into fresh, pre-warmed medium to OD600 = 0.4 either without rifampicin (panel A and C) or with 200 μg/ml rifampicin (panel B and D) and samples removed for RNA extraction and Northern blotting at the indicated time points. A and B: Northerns probed for DrrS; C and D: Northerns re-probed for MTS2823.
Fig 9.
Turnover of DrrS and its 5’ derivatives in M. smegmatis.
A: Northern blot of total RNA from M. smegmatis expressing DrrS from a heterologous promoter. Rifampicin (200 μg/ml rifampicin) was added at time zero and RNA was isolated at the indicated time point and probed for DrrS as previously described. B: Northern blot of 1–20 μg of total RNA from M. smegmatis expressing wildtype and 5’ variants of DrrS. RNA from the strain expressing wildtype DrrS was loaded in the first and last wells to ensure proper alignment. C: Turnover of DrrS 5’ variants. Each DrrS 5’ variant was expressed in M. smegmatis, cultures were grown to OD600 = 0.6 and rifampicin added to a final concentration of 200 μg/ml. Samples were withdrawn at the indicated time points and subjected to Northern blotting. As the relative amount of DrrS varied significantly between the wildtype and the derivatives (see panel B), different amounts of total RNA were loaded on the gels, i.e. 1 μg each wildtype and A1DrrS; 2 μg of A2DrrS; 10 μg A3DrrS and 20 μg A4DrrS. Cartoon on the right illustrate the 5’ additions (red asterisks) to the DrrS structure.
Fig 10.
PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.
Fig 11.
Cartoon showing the likely order of events in DrrS expression and function.
Host-associated stress such as hypoxia or NO stress induces DosR expression and activation, which in turn leads to increased expression of DrrS+. This transcript is rapidly processed in multiple places to obtain the highly stable DrrS, which is able to regulate target genes for extended periods of time.