P056

The Mycobacterium tuberculosis inhA operon encodes two mycolic acid synthesis genes, as well as hemZ, which encodes a ferrochelatase responsible for incorporating iron into heme. Mutations in the promoter region cause upregulation of this operon resulting in resistance to anti-tuberculosis drugs that target the mycobacterial cell wall. However, the role of hemZ as part of the operon is unknown. We hypothesize differential iron availability in response to upregulation of hemZ.

We introduced an inhA C-15T mutation into the wild-type M. tuberculosis H37Rv strain by homologous recombination. The genomic integrity was verified by Illumina whole genome sequencing (WGS); the transcriptional effect of the mutation was determined by RNAseq and FeRhoNox was used to visualize ferrous iron present within cells by super-resolution fluorescence microscopy.

WGS confirmed that two mutant clones were identical and had no off-target changes. RNAseq demonstrated that the only significant transcriptional effect of the mutation was upregulation of mabA, inhA and hemZ. FeRhoNox analysis revealed that absolute iron concentration in mutant strains was higher in long cells vs. short cells while the opposite was true in the wild-type.  This was interesting considering wild-type cells were on average longer compared to mutant cells. Short mutant cells had the highest iron concentration at the poles, whereas it was equally distributed in the wild-type and long mutant cells.

Our results demonstrate that the distribution of ferrous iron in mycobacterial cells is not uniform. Further, the inhA C-15T mutation affects iron distribution, which may play a role in cell division.