The WalKR two-component regulator coordinates cell wall homeostasis with DNA replication in Staphylococcus aureus

Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon poorly defined. Here, we use a panel of isogenic walKR mutants with a spectrum of activity, combined with functional genomics including ChIP-seq to identify a consensus WalR DNA-binding motif and define the direct WalKR regulon. As previously shown, the direct regulon includes multiple autolysin genes. However, we also show WalR directly regulates at least five essential genes involved in (i) lipoteichoic acid synthesis ( ltaS ), (ii) translation (rplK ), (iii) DNA compaction ( hup ), (iv) initiation of DNA replication ( dnaA, hup ) and (v) purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus is a polyfunctional regulator with fundamental control over critical cell systems, linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings explain the essentiality of this locus and should reignite interest in WalKR as a bona fide target for novel anti-staphylococcal therapeutics. ### Competing Interest Statement The authors have declared no competing interest.