Fig 2.
Weakening the interaction between CarD and the RNAP compromises the growth of mycobacteria. (A) Table of mycobacterial strains engineered by replacing the CarDWT expression cassette with one that expresses the designated mutant and mutants that were unattainable by gene switching. The strength of RNAP interaction is based on the bacterial two-hybrid results shown in Fig. 1. (B and C) Representative growth curve from 5 replicates of M. smegmatis (B) and 3 replicates of M. tuberculosis (C) ΔcarD attB∷tet-carD strain expressing CarDWT, CarDR25E, or CarDR47E. (D) Immunoprecipitation experiments with a monoclonal antibody specific for HA in the M. smegmatis ΔcarD attB∷tet-carD strain expressing untagged CarDWT (lane 1, also expresses HA peptide), CarDWT-HA (lane 2), CarDR25E-HA (lane 3), or CarDR47E-HA (lane 4). Inputs (before immunoprecipitation) and eluates were analyzed by Western blotting with antibodies specific for either RNAP β or HA.