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 complementation of lsr2 deletion mutants of Mycobacterium. smegmatis and M, tuberculosis. In M. smegmatis, none of the individual phosphoablative mutants (T8A, T22A, or T112A) complemented the defect in colony morphology. In M. tuberculosis, T8A, T22A, and T31A variants complemented the growth defect on solid medium, but T112A did not. Full-length Lsr2 showed non-specific binding to various DNA fragments, and this binding was completely abolished by PknB-mediated phosphorylation. Lsr2T112D showed reduced binding to DNA. Consequently, different DNA binding patterns in M. tuberculosis expressing Lsr2WT, and phosphoablative Lsr2T112A would result in alteration of gene expression of target genes.

Finally, Lsr2 from Mycobacterium marinum was also investigated in this project. I found that M. marinum lsr2 was essential for the growth of this bacterium since the gene could be only deleted from the strain containing a second copy of lsr2. M. marinum Lsr2 is missing the terminal threonine (T112) and serine 113 was not phosphorylated by PknB in vivo or in vitro, suggesting that DNA binding by M. marinum Lsr2 is regulated by a different mechanism. These findings suggest that PknB controls mycobacterial biology by phosphorylating the global transcriptional regulator Lsr2.