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

Abstract

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 (WalR Spn ) 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 walRK Spn operon. To better understand the functions of the WalRKJ Spn proteins in S. pneumoniae, we performed experiments to determine their cellular localization and amounts. In contrast to WalK from Bacillus subtilis (WalK Bsu ), which is localized at division septa, immunofluorescence microscopy showed that WalK Spn is distributed throughout the cell periphery. WalJ Spn is also localized to the cell surface periphery, whereas WalR Spn was found to be localized in the cytoplasm around the nucleoid. In fractionation experiments, WalR Spn was recovered from the cytoplasmic fraction, while WalK Spn and the majority of WalJ Spn were recovered from the cell membrane fraction. This fractionation is consistent with the localization patterns observed. Lastly, we determined the cellular amounts of WalRKJ Spn by quantitative Western blotting. The WalR Spn response regulator is relatively abundant and present at levels of Ϸ6,200 monomers per cell, which are Ϸ14-fold greater than the amount of the WalK Spn histidine kinase, which is present at Ϸ460 dimers (920 monomers) per cell. We detected Ϸ1,200 monomers per cell of WalJ Spn ancillary protein, similar to the amount of WalK Spn .

Two-component signal transduction systems in oral bacteria

“…Staphylococcus, Enterococcus , Listeria , and Lactobacillus , except for Streptococcus spp. and Lactococcus lactis , in which the operons include only the yycJ orthologue, also known as vicX or walJ [100,102]. Proteins encoded by vicX are metal-dependent beta-lactamases of as-yet unknown function, although there is evidence suggesting that VicX modulates VicK activity in S. mutans , because vicX -defective strains showed altered phenotypes in VicRK-regulated functions [103].…”

“…Proteins encoded by vicX are metal-dependent beta-lactamases of as-yet unknown function, although there is evidence suggesting that VicX modulates VicK activity in S. mutans , because vicX -defective strains showed altered phenotypes in VicRK-regulated functions [103]. VicX of S. pneumoniae is localized in the membrane, implying an interaction with the VicK sensor protein [102]. Figure 2 illustrates differences between B. subtilis and streptococcal species in the domain architecture of YycG (VicK) and ancillary proteins of the VicRK TCS (YycFG).…”

“…Figure 2 illustrates differences between B. subtilis and streptococcal species in the domain architecture of YycG (VicK) and ancillary proteins of the VicRK TCS (YycFG). In B. subtilis and other non-streptococcal species, the N-terminus of the HK includes two TM domains flanking an extracellular sensor loop, while the streptococcal VicK proteins have a single TM linked to a short extracellular peptide [102]. In a model for function of YycFG and associated ancillary proteins, an extracellular signal activates YycG by affecting inhibitory interactions of YycI and YycH.…”

“…In B. subtilis , YycG (VicK) co-localized with FtsZ of the divisome, and it was proposed that VicK Bs senses cell division directly [133]. S. pneumoniae , VicK Spn does not co-localize with the FtsZ ring at division septa, but is peripherally distributed during exponential growth [102]. However, VicRK Spn target protein, PcsB, localizes at the cell-division septa by interacting with FtsX, a component of the divisome [102,121].…”

“…S. pneumoniae , VicK Spn does not co-localize with the FtsZ ring at division septa, but is peripherally distributed during exponential growth [102]. However, VicRK Spn target protein, PcsB, localizes at the cell-division septa by interacting with FtsX, a component of the divisome [102,121]. It is possible that signals sensed by human streptococci are different from those that activate YycG of B. subtilis , present in soil and water habitats, microbial communities of plants, and gastrointestinal tracts of animals [134].…”