Rv3839-Rv3840 links the endogenous heme biosynthesis pathway with <i>Mycobacterium tuberculosis</i> adaptation to nitric oxide and iron limitation stress

During infection, <i>Mycobacterium tuberculosis</i> (Mtb) encounters multiple environmental stressors, including nitric oxide (NO) and iron limitation, and an ability to mount an integrated response is essential for the bacterium's adaptation and continued survival. Iron-containing prosthetic groups in key enzymes are critical for Mtb sensing and detoxification of NO, and there is significant overlap between NO- and low iron-responsive genes. However, how Mtb adapts to these two stressors concurrently is largely unknown. Here, we find that exposure to NO globally augments expression of low iron-responsive genes and vice versa, with a two gene operon, <i>rv3839-rv3840,</i> among the most highly upregulated. Deletion of <i>rv3839-rv3840</i> resulted in increased growth under prolonged iron limitation and early exit of Mtb from an adaptive state of growth arrest induced upon exposure to NO/low iron. Δ<i>rv3839-rv3840</i> Mtb exhibited an elongated cell morphology compared to wild type Mtb in NO/low iron conditions, indicating effects of this operon on cell growth and division under stress conditions, with Rv3839 as the key driver of this phenotype. Coproporphyrin III tetramethyl ester (TMC), a modified precursor molecule in the endogenous Mtb heme biosynthesis pathway, was found to accumulate in Δ<i>rv3839-rv3840</i> Mtb under iron limiting conditions. Further, intrabacterial heme levels were increased in Δ<i>rv3839-rv3840</i> Mtb under NO stress and iron limitation. Together, these findings reveal Rv3839-Rv3840 as proteins involved in the downregulation of heme biosynthesis under NO stress and iron limitation, and highlight the link between Mtb growth control in response to NO/low iron and endogenous heme biosynthesis.