M. tuberculosis Ser/Thr Protein Kinase D Phosphorylates an Anti-Anti–Sigma Factor Homolog

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Abstract
Receptor Ser/Thr protein kinases are candidates for sensors that govern developmental changes and disease processes of Mycobacterium tuberculosis (Mtb), but the functions of these kinases are not established. Here, we show that Mtb protein kinase (Pkn) D overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-anti–sigma factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c, but no other sigma factor regulator, in vitro. In contrast, the purified PknB and PknE kinase domains phosphorylated distinct sigma regulators. Rather than modifying a consensus site, PknD phosphorylated Rv0516c in vitro and in vivo on Thr2 in a unique N-terminal extension. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-anti–sigma factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma factor regulator at an unprecedented control site. Many bacteria, including Mycobacterium tuberculosis (Mtb), sense the environment using a family of signaling proteins called Ser/Thr protein kinases (STPKs), but the functions of these sensors are not well understood. This study shows that the Mtb protein kinase (Pkn) D STPK attaches a phosphate group to one and only one member of a family of regulators of “alternative” sigma factors, which activate sets of genes in numerous bacteria. Phosphorylation of the regulator at an unprecedented position abolished binding in vitro to a putative partner. Remarkably, increasing PknD activity in Mtb not only strongly activated the gene encoding the specific regulatory protein phosphorylated by PknD, but also altered the expression of genes controlled by an alternative sigma factor. By providing evidence for a mechanistic link between PknD and gene regulation, this work supports a new model in which STPKs in numerous microorganisms transduce environmental signals by controlling expression of specific groups of genes. Thus, certain bacterial STPKs may orchestrate aspects of the coordinate control of gene expression essential for adaptation in the environment and in host infections.