The role of Lsr2 phosphorylation in regulation of mycobacterial growth and gene expression

One-fourth of the world population is estimated to have latent tuberculosis infection associated with dormant Mycobacterium tuberculosis bacilli, which are more resistant to antimicrobial treatment and can resuscitate, resulting in the development of active tuberculosis. M. tuberculosis has a wide range of regulatory proteins that control growth, including the protein kinase B (PknB) that controls peptidoglycan biosynthesis and growth by phosphorylating several substrates. Lsr2 is one of the substrates of PknB that is highly conserved in mycobacteria. Lsr2 is DNA-binding protein that controls gene transcription and protects the nucleoid from damage. This project was focused on the investigation of Lsr2 phosphorylation and the role of Lsr2 in DNA binding, and regulation of gene expression in mycobacterial growth and dormancy. Recombinant M. tuberculosis Lsr2 and its phosphomimetic form (Lsr2T112D) were purified and used for kinase assay, DNA binding experiments, and generation of polyclonal antibodies. Mass-spectrometry confirmed that recombinant PknB phosphorylated recombinant Lsr2 in vitro at threonine 8, threonine 21, threonine 31 and threonine 112. Phosphoablative forms of Lsr2 were generated and used for ... (continues)