FIG 3.
Mutations in MAB_4384, encoding a TetR repressor, results in upregulation of MAB_4383c (mmpS5) and MAB_4382c (mmpL5) genes. (A) Mutations in MAB_4384 resulting in resistance to TAC analogues. The red box indicates the putative HTH DNA-binding domain. Broken lines indicate alterations in the primary amino acid sequence as a result of the frameshifts caused by indel mutations. The M1A mutations in R1 and R2 result in no translation. (B) Three putative mmpS5-mmpL5 operons are found in M. abscessus. Boxed in red is the MAB_4383c-MAB_4382c operon, which bears the highest homology to the mmpS5-mmpL5 locus from M. tuberculosis, as well as the upstream regulator MAB_4384. While both the M. tuberculosis and M. abscessus operons are preceded by putative regulators, these regulators share very little amino acid sequence identity. (C) Exponential-growth-phase cultures in Mueller-Hinton broth were used for total RNA isolation in order to determine relative gene expression by qRT-PCR and the ΔΔCT method. sigA-normalized gene expression in several spontaneous D15-resistant mutants reared in the S background relative to WT M. abscessus S. (D) sigA-normalized gene expression in several spontaneous D15-resistant mutants reared in the R background relative to WT M. abscessus R. (E) sigA-normalized gene expression in the WT and the D15_S4 mutant overexpressing MAB_4384 (pMV261-MAB_4384) relative to M. abscessus carrying the empty pMV261 vector. (F) sigA-normalized gene expression in the MmpS5-MmpL5-overexpressing (pMV261-mmpS5/mmpL5) strains relative to M. abscessus carrying the empty pMV261 vector. Histograms and error bars shown in panels C to F depict median fold change and the interquartile range, respectively, and were calculated from at least three independent qRT-PCR experiments, each in which fresh RNA was reverse transcribed prior to the qRT-PCR. Data are representative of two independently repeated RNA extractions.