The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis . We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.

Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors.

Rice (Oryza sativa) produces ent-copalyl diphosphate for both gibberellin (GA) phytohormone and defensive phytoalexin biosynthesis, raising the question of how this initial biosynthetic step is carried out for these distinct metabolic processes. Here, a functional genomics approach has been utilized to identify two disparate ent-copalyl diphosphate synthases from rice (OsCPS1_ent and OsCPS2_ent). Notably, it was very recently demonstrated that only one of these (OsCPS1_ent) normally operates in GA biosynthesis as mutations in this gene result in severely impaired growth. Evidence is presented here strongly indicating that the other (OsCPS2_ent) is involved in related secondary metabolism producing defensive phytochemicals. In particular, under appropriate conditions, OsCPS2_ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins. Thus, transcriptional control of OsCPS2_ent seems to be an important means of regulating defensive phytochemical biosynthesis. Finally, OsCPS1_ent is significantly more similar to the likewise GA-specific gene An1/ZmCPS1_ent in maize (Zea mays) than its class II terpene synthase paralogs involved in rice secondary metabolism. Hence, we speculate that this cross-species conservation by biosynthetic process reflects derivation of related secondary metabolism from the GA primary biosynthetic pathway prior to the early divergence between the separate lineages within the cereal/grass family (Poaceae) resulting in modern rice and maize.