Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

Abstract

The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

“…1B). As observed previously (15), the induction of sigF in both H37Rv::pMYsigF and H37Rv(⌬rsbW/ sigF)::pMYsigF brought about a decrease in growth rate after 24 h, whereas control strains were not affected.…”

“…To identify the SigF promoters for the SigF targets validated above, 40-bp sequences upstream of the identified TSPs were in-spected and, in all cases, the consensus sequence, GGTTT-N (15)(16)(17) -GGGTA, was identified (Fig. 5).…”

“…P-IA was purified from pyostatin tablets as described previously (15). Rabbit anti-SigF polyclonal antibodies were kindly provided by Ida Rosenkrands (Statens Serum Institut, Copenhagen, Denmark), and a mouse anti-HbhA polyclonal antibody was provided by Giovanni Delogu (Catholic University of the Sacred Heart, Rome, Italy).…”

“…All experiments were performed in either M. tuberculosis strain H37Rv or its isogenic mutant, H37Rv(⌬rsbW/sigF), lacking the rsbW and sigF operon, as described previously (15). These strains were transformed with either pMY769, an empty pristinamycin IA (P-IA)-inducible integrating vector, or pMYsigF, a pMY769 derivative with a P-IA-inducible sigF gene (15) to create H37Rv::pMY769, H37Rv::pMYsigF, H37Rv(⌬rsbW/sigF)::pMY769, and H37Rv(⌬rsbW/sigF)::pMYsigF. The strains were routinely grown in Middlebrook 7H9 medium supplemented with 10% albumin-dextrosecatalase (ADC), 0.2% glycerol, and 0.05% Tween 80.…”

“…To analyze the total protein content, bacteria were harvested, and the total cell extract was subjected to SDS-PAGE for Simply Blue staining or Western blot analysis as described previously (15). Protein bands were excised from the gel and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis following trypsin or chymotrypsin digestion.…”