M. tuberculosis AlkX Encoded by rv3249c Regulates a Conserved Alkane Hydroxylase System That Is Important for Replication in Macrophages and Biofilm Formation

Mycobacterium tuberculosis is a highly specialized human pathogen. The success of M. tuberculosis is due to its ability to replicate within host macrophages, resist host immune responses, and ultimately enter a persistent state during a latent tuberculosis infection. Understanding how M. tuberculosis adapts to and replicates in the intracellular environment of the host is crucial for the development of novel, targeted therapeutics. We report the characterization of an M. tuberculosis mutant lacking Rv3249c, a TetR transcriptional regulator. We show that Rv3249c directly represses the adjacent alkB-rubA-rubB operon encoding an alkane hydroxylase/rubredoxin system. For consistency with related systems, we have named the rv3249c gene alkX. The alkX mutant survived better than wild-type M. tuberculosis inside macrophages. This could be phenocopied by overexpression of the alkB-rubA-rubB locus. We hypothesized that the improved intracellular survival phenotype is a result of increased fitness of the mutant; however, we found that the alkX mutant had a defect when grown on some host-associated carbon sources in vitro. We also found that the alkX mutant had a defect in biofilm formation, also linked to the overexpression of the alkB-rubAB genes. Combined, these results define the primary role of AlkX as a transcriptional repressor of the alkB-rubAB operon and suggest the operon contributes to intracellular survival of the pathogen. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the leading cause of death worldwide due to a single infectious agent. It is important to understand how M. tuberculosis adapts to and replicates in the intracellular environment of the host. In this study, we characterized the TetR transcriptional regulator Rv3249c and show that it regulates a highly conserved alkane hydroxylase/rubredoxin system. Our data demonstrate that the AlkBRubAB system contributes to the success of the bacterium in host macrophages.