The indicated proteins are pictured schematically with amino acid hydrophobicity as a color scale from blue (hydrophilic) to red (hydrophobic) according to the Kyte-Doolittle scale, with white being neutral. The predicted transmembrane domains (TM) are indicated and amino acid numbers are indicated at the top.

A. HA tagged anti-sigma factors used in Figure 2A-D were expressed either in wild type M. smegmatis (WT), or the ΔMSmegrip1 strain (M). Proteins were detected either with anti-HA or anti-DLAT (loading control).
B. C terminally truncated, HA tagged, anti-sigma factors accumulate in the absence of MSmegrip1
C terminally truncated anti-sigma factors were expressed in wild type M. smegmatis or the MSmegrip1 null mutant and monitored by anti HA western blotting.
C-D. Rip1 proteolytic activity is required for anti sigma factor processing. The MSmegrip1 null mutant bearing RskA160 (C) or RslA161 (D) was complemented with M. tuberculosis Rip1 or Rip1-H21A.

A. Quantitative RT-PCR of katG RNA in various Rip1 pathway mutants either treated with methanol control (white bar), or treated with 1mM phenanthroline for 30 minutes (grey bar). Y axis shows the level of katG transcript normalized to RNA encoding the housekeeping sigma factor SigA. Plotted values are means of biologic triplicates and error bars are standard deviation.
B. Western blot analysis of HA tagged anti-sigma factors (RskA/RslA) expressed in wild type M. tuberculosis (WT lanes), or the rip1 null mutant (ΔM lanes). Cells were grown in the presence of methanol (meth) and 1mM phenanthroline (phen) for 30min prior to analysis. Protein lysates were probed with either anti-HA or the loading control anti-DLAT.

Four anti-sigma factors RsdA (A), RskA (B), RslA (C), and RsmA (D) with N-terminal Hemagglutinin epitope tags were expressed in wild type M. tuberculosis (WT lanes), or the rip1 null mutant (ΔM lanes). HA tagged proteins were detected by immunoblotting using anti-HA antibodies in parallel with the loading control anti-DLAT. Panel E is a schematic representation of predicted cleavage products after the action of Site one (S1P) and site two proteases (S2P).
F. Rip1 constitutively degrades truncated RsmA and RskA, but not RsdA. C terminally truncated anti-Sigma factors with N terminal Hemagglutinin tags were expressed in wild type (WT) or rip1 mutant (ΔM) M. tuberculosis. Protein lysates were probed with either anti-HA or the loading control anti-DLAT.

A. Quantitative RT-PCR of rpfC RNA in Wild type (WT), Δrip1, ΔsigK, ΔsigL, and ΔsigM either treated with methanol (solvent control, white bar), or treated with 1mM phenanthroline for 30 minutes (grey bar). Y axis shows the level of katG transcript normalized to RNA encoding the housekeeping sigma factor SigA. Plotted values are means of biologic triplicates and error bars are standard deviation.
B. Quantitative RT-PCR of katG RNA in Wild type (WT), Δrip1, genetically complemented (Δrip1+rip1), ΔsigK, ΔsigL, and ΔsigM either treated with methanol (white bar), or treated with 1mM phenanthroline for 30 minutes (grey bar). Y axis shows the level of katG transcript normalized to RNA encoding the housekeeping sigma factor SigA. Plotted values are means of biologic triplicates and error bars are standard deviation.
C. Quantitative RT-PCR of bfrB RNA in Wild type (WT), Δrip1, Δrip1+rip1, ΔsigK, ΔsigL, and ΔsigM either treated with methanol control (white bar), or treated with 1mM phenanthroline for 30 minutes (grey bar). Y axis shows the level of bfrB transcript normalized to RNA encoding the housekeeping sigma factor SigA. Plotted values are means of biologic triplicates and error bars are standard deviation.
D. Quantitative RT-PCR of kasA RNA in phenanthroline treated cells with the same legend presented in A.
E. Quantitative RT-PCR of furA RNA in phenanthroline treated cells with the same legend presented in A.
F. SigK consensus at the 5′ proximal promoter of katG. The consensus was derived using computational analysis of known SigK target promoters (mpt70, mpt83, and rv0499) and the entire furA-katG genetic locus. Purple letters represent experimentally derived transcriptional start sites (see text), the FurA binding site is colored blue, −10 elements are colored red, and −35 elements are colored yellow.

The orange and red lines represent cleavage of three anti-Sigma factors, RskA, RslA, and RsmA, by unknown S1P(s) and Rip1 respectively. Rip1 cleavage of RskA and RslA releases SigK and SigL respectively, which activates transcription of katG. In addition, our data identifies Rip1 dependent regulation of kasA, bfrB, and rpfC which is independent of Sigma K, L, or M. A functional copy of Rip1 is required to repress bfrB transcription and activate kasAB and rpfC transcription. We have represented these regulatory interactions directly from Rip1, although we envision that they arise either through some combination of Sigma KLM, or through as yet unidentified additional substrates of Rip1 (pictured as the grey cylinder).