Localization and Cellular Amounts of the WalRKJ (VicRKX) Two-Component Regulatory System Proteins in Serotype 2 Streptococcus pneumoniae

The WalRK two-component regulatory system coordinates gene expression that maintains cell wall homeostasis and responds to antibiotic stress in low-GC Gram-positive bacteria. Phosphorylated WalR (VicR) of the major human respiratory pathogen Streptococcus pneumoniae (WalRSpn) positively regulates transcription of several surface virulence genes and, most critically, pcsB, which encodes an essential cell division protein. Despite numerous studies of several species, little is known about the signals sensed by the WalK histidine kinase or the function of the WalJ ancillary protein encoded in the walRKSpn operon. To better understand the functions of the WalRKJSpn proteins in S. pneumoniae, we performed experiments to determine their cellular localization and amounts. In contrast to WalK from Bacillus subtilis (WalKBsu), which is localized at division septa, immunofluorescence microscopy showed that WalKSpn is distributed throughout the cell periphery. WalJSpn is also localized to the cell surface periphery, whereas WalRSpn was found to be localized in the cytoplasm around the nucleoid. In fractionation experiments, WalRSpn was recovered from the cytoplasmic fraction, while WalKSpn and the majority of WalJSpn were recovered from the cell membrane fraction. This fractionation is consistent with the localization patterns observed. Lastly, we determined the cellular amounts of WalRKJSpn by quantitative Western blotting. The WalRSpn response regulator is relatively abundant and present at levels of ≈6,200 monomers per cell, which are ≈14-fold greater than the amount of the WalKSpn histidine kinase, which is present at ≈460 dimers (920 monomers) per cell. We detected ≈1,200 monomers per cell of WalJSpn ancillary protein, similar to the amount of WalKSpn.