The connection between peptidoglycan remodeling and cell division is poorly understood in ellipsoid-shaped ovococcus bacteria, such as the human respiratory pathogen Streptococcus pneumoniae. In S. pneumoniae, peptidoglycan homeostasis and stress are regulated by the WalRK (VicRK) two-component regulatory system, which positively regulates expression of the essential PcsB cysteine-and histidine-dependent aminohydrolases/peptidases (CHAP)-domain protein. CHAP-domain proteins usually act as peptidoglycan hydrolases, but purified PcsB lacks detectable enzymatic activity. To explore the functions of PcsB, its subcellular localization was determined. Fractionation experiments showed that cell-bound PcsB was located through hydrophobic interactions on the external membrane surface of pneumococcal cells. Immunofluorescent microscopy localized PcsB mainly to the septa and equators of dividing cells. Chemical cross-linking combined with immunoprecipitation showed that PcsB interacts with the cell division complex formed by membrane-bound FtsX Spn and cytoplasmic FtsE Spn ATPase, which structurally resemble an ABC transporter. Far Western blotting showed that this interaction was likely through the large extracellular loop of FtsX Spn and the amino terminal coiled-coil domain of PcsB. Unlike in Bacillus subtilis and Escherichia coli, we show that FtsX Spn and FtsE Spn are essential in S. pneumoniae. Consistent with an interaction between PcsB and FtsX Spn , cells depleted of PcsB or FtsX Spn had strikingly similar defects in cell division, and depletion of FtsX Spn caused mislocalization of PcsB but not the FtsZ Spn early-division protein. A model is presented in which the interaction of the FtsEX Spn complex with PcsB activates its peptidoglycan hydrolysis activity and couples peptidoglycan remodeling to pneumococcal cell division.WalRK Spn (VicRK) regulon | cell wall biosynthesis | cell surface protein | murein hydrolysis

PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB ؉ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass and that cell-associated PcsB was moderately abundant and present at Ϸ4,900 monomers per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also showed that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could be deleted. Unexpectedly, capsule influenced the cell shape and chain formation phenotypes of the wild-type D39 strain and mutants underexpressing PcsB or deleted for other genes involved in peptidoglycan biosynthesis, such as dacA. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-peptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB.Comparatively little is known about the exact mechanisms and interactions that occur during peptidoglycan (PG) biosynthesis and cell division of ovococcus bacteria, such as Streptococcus pneumoniae (pneumococcus) and other Streptococcus species (reviewed recently in reference 71). These ovoid, football-shaped bacteria divide along successive parallel planes perpendicular to the long axis of the cell. This mode of division contrasts with that of spherical coccus species such as Staphylococcus aureus, which divides in alternating perpendicular planes. Another major difference between these two bacterial types is that ovococcus species have two modes of PG biosynthesis (peripheral and septal), whereas spherical species have only septal PG biosynthesis (30,38,50,51,71). All models of PG biosynthesis require murein hydrolases, such as amidases or endopeptidases, to participate in PG remodeling and cell separation during division (15,48,66,67,71).One candidate murein hydrolase is the PcsB protein, which has been characterized to various degrees in S. pneumoniae (29,42,45,46), Streptococcus mutans (where it is called GbpB) (11,12,40,41), and Streptococcus agalactiae (55,56). PcsB orthologs contain four domains: a signal peptide (SP) that directs export, a predicted extended coiled-coil domain that contains leucine zipper motifs, an alanine-ric...

Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRK Spn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an L,D-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (D,D-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in D-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRK Spn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein.

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 .

Streptococcus pneumoniae is a serious human respiratory pathogen that has the capacity to evade capsule-based vaccines and to develop multidrug antibiotic resistance. This review summarizes recent advances in understanding the mechanisms and regulation of peptidoglycan (PG) biosynthesis that result in ellipsoid-shaped, ovococcus Streptococcus cells. New results support a two-state model for septal and peripheral PG synthesis at mid-cell, involvement of essential cell division proteins in PG remodeling, and mid-cell localization of proteins that organize PG biosynthesis and that form the protein translocation apparatus. PG biosynthesis proteins have already turned up as promising vaccine candidates and targets of antibiotics. Properties of several recently characterized proteins that mediate or regulate PG biosynthesis suggest a source of additional targets for therapies against pneumococcus.