Abstract
Bacterial phosphosignalling has been synonymous with two-component systems and their histidine kinases, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignalling enzymes in eukaryotes but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is untested. Here we explored the global signalling capacity of the STPKs in Mtb using a panel of STPK loss-of-function and overexpression strains combined with mass spectrometry-based phosphoproteomics. A deep phosphoproteome with >14,000 unique phosphosites shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >80% of the proteome and extensively interfaces with the transcriptional machinery. Mtb O-phosphorylation gives rise to an expansive, distributed and cooperative network of a complexity that has not previously been seen in bacteria and that is on par with eukaryotic phosphosignalling networks. A resource of >3,700 high-confidence direct substrate-STPK interactions and their transcriptional effects provides signalling context for >80% of Mtb proteins and allows the prediction and assembly of signalling pathways for mycobacterial physiology.