The Eukaryotic-Like Ser/Thr Kinase PrkC Regulates the Essential WalRK Two-Component System in Bacillus subtilis

Abstract

Most bacteria contain both eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs). Their role in bacterial physiology is not currently well understood in large part because the conditions where the eSTKs are active are generally not known. However, all sequenced Gram-positive bacteria have a highly conserved eSTK with extracellular PASTA repeats that bind cell wall derived muropeptides. Here, we report that in the Gram-positive bacterium Bacillus subtilis, the PASTA-containing eSTK PrkC and its cognate eSTP PrpC converge with the essential WalRK two-component system to regulate WalR regulon genes involved in cell wall metabolism. By continuously monitoring gene expression throughout growth, we consistently find a large PrkC-dependent effect on expression of several different WalR regulon genes in early stationary phase, including both those that are activated by WalR (yocH) as well as those that are repressed (iseA, pdaC). We demonstrate that PrkC phosphorylates WalR in vitro and in vivo on a single Thr residue located in the receiver domain. Although the phosphorylated region of the receiver domain is highly conserved among several B. subtilis response regulators, PrkC displays specificity for WalR in vitro. Consistently, strains expressing a nonphosphorylatable WalR point mutant strongly reduce both PrkC dependent activation and repression of yocH, iseA, and pdaC. This suggests a model where the eSTK PrkC regulates the essential WalRK two-component signaling system by direct phosphorylation of WalR Thr101, resulting in the regulation of WalR regulon genes involved in cell wall metabolism in stationary phase. As both the eSTK PrkC and the essential WalRK two-component system are highly conserved in Gram-positive bacteria, these results may be applicable to further understanding the role of eSTKs in Gram-positive physiology and cell wall metabolism.

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

“…Consistent with this observation, the suppressor mutations we identified in the FliM cluster in regions predicted to regulate interactions with FliG and other motility regulators (25,26) and potentially the ability for FliM to self-assemble (27). Similarly, mutations in the homolog of the B. subtilis Ser/Thr kinase PrkC, which is linked to cell-wall metabolism (28), also emerged as common suppressors. Additional suppressors of ea2619 included mutations in a lipoteichoic acid biosynthetic protein (LtaS2) and MprF, a phosphatidyl lysil transferase, which are involved in maintenance of the cell envelope (29,30).…”

Molecular Mechanisms of Two-Component Signal Transduction

“…Initial in vitro identification of potential phosphorylation sites of Staphylococcus aureus GraR [114] and VraR [115], Mycobacterium tuberculosis DosR and Rv2175c [116–118] , Streptococcus pneumoniae RitR [119] and RR06 [120] and Group A and B Streptococci CovR [121] have been followed by physiological studies of RR06 [120], CovR [122] and B. subtilis WalR [123]. The molecular mechanism of these reactions remains poorly understood.…”

“…Some molecular insights stem from studies of WalR. This essential RR involved in cell wall homeostasis regulation was shown to be subject to phosphorylation on threonine residue 101 by the S/T kinase PrkC [123]. Position T101 is a moderately conserved residue position among RR proteins.…”

Two-component signal transduction systems in oral bacteria

“…In addition to potential cross-talk between TCS, studies on streptococci and other Gram-positive bacteria indicate that VicKR can interact with a transmembrane eukaryotic-like serine/threonine kinase (STK) [144,145] that functions in concert with cognate cytoplasmic eukaryotic-like serine/threonine phosphatases (STP) present in Gram-positive bacteria as STK–STP operons [146,147]. STK are transmembrane enzymes with extracellular domains containing one to five PASTA (penicillin-binding protein and serine/threonine kinase associated domain) repeats and a cytoplasmic kinase domain [146,148].…”

“…STK is conserved among Gram-positive bacteria, including oropharyngeal streptococci, e.g. S. pneumoniae , S. gordonii , S. sanguinis , and S. mutans , and analysis of these orthologues might help decipher the VicRK network [147]. Analysis of the STK-STP system in S. mutans was investigated using an isogenic mutant in pknB , the STK-encoding gene [144,150,151].…”