A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria

Abstract

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.

Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

“…A striking finding was the isolation of several transductants with insertions in pbpA and rodA. These genes are involved in PGN synthesis and cell shape control and are part of the operon containing pknA and pknB, genes encoding essential Ser/Thr kinases that regulate these processes (5,6,8,9,35). These suppressors thus indicate a link between the ⌬cuvA growth phenotype and cell wall synthesis and morphology and are reminiscent of the suppression of the growth and morphology defects of the B. subtilis yvcK mutant by deletion Cell shape/peptidoglycan synthesis, Ser/Thr phosphorylation pbpA ϩ142 (1r), ϩ232 (1r), ϩ250 (1f), ϩ270 (1f), ϩ541 (1f) Peptidoglycan synthesis/cell shape, Ser/Thr phosphorylation Rv2683 Ϫ1 (3f), ϩ191 (1f) Conserved hypothetical regulatory protein/transmembrane transport trcR ϩ681 (1f), ϩ732 (1r) Response regulator/TrcRS two-component system a Genes in which two or more independent insertions were obtained are included in the table.…”

“…One is our previous finding that it is phosphorylated in M. tuberculosis cells by the Ser/Thr protein kinases PknA and PknB. These kinases are essential for growth and regulate cell division, peptidoglycan (PGN) synthesis, and cell morphology (5)(6)(7)(8)(9), suggesting that the Rv1422 protein may play a role in one or more of these processes. Second, Rv1422 was identified in a genome-wide transposon screen for genetic interactions with the mce1 and mce4 loci.…”

Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis*

“…The cellular location of several kinases is also consistent with their functions. PknA and PknB are essential for cell growth and are found exclusively at the pole and septum, sites of new cell wall synthesis for elongation and division (13). It is at these sites where the peptidoglycan-binding extracellular domain of PknB is likely to receive its activating signal (32).…”

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

“…In many Gram-positive bacteria, the ortholog of the mycobacterial Ser/Thr protein kinase PknB regulates cell division and cell-wall synthesis in response to the state of the PG (26). The PknB extracellular sensor domain binds a PG fragment and activates the intracellular kinase domain (27).…”