Influences of Capsule on Cell Shape and Chain Formation of Wild-Type andpcsBMutants of Serotype 2Streptococcus pneumoniae

Abstract

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...

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

“…To investigate whether full-length PcsB has muralytic activity, we performed zymography with purified recombinant PcsB and pneumococcal cells mixed into the separating SDS-PAGE gel as substrate. Consistent with the results reported by others 8,11,12 , no muralytic activity was detected. Next, we expressed only the C-terminal CHAP domain of PcsB.…”

“…Due to the net-like polymeric structure of PG, the mobility of PcsB dimers is probably restricted. Thus, it is likely that PcsB, which is a moderately abundant protein in the pneumococcal cell (about B5,000 monomers per cell 12 ), only acts locally and is then discarded. This is supported by the fact that relatively large amounts of PcsB are released to the medium during growth 12 .…”

“…Thus, it is likely that PcsB, which is a moderately abundant protein in the pneumococcal cell (about B5,000 monomers per cell 12 ), only acts locally and is then discarded. This is supported by the fact that relatively large amounts of PcsB are released to the medium during growth 12 . In sum, our results indicate that the unique modular structure and dimeric organization of PcsB have evolved to allow precise splitting of the septum during cell division, while avoiding damage to other parts of the PG sacculus.…”

“…Despite the presence of this putative catalytic domain in the PcsB sequence, attempts to demonstrate hydrolytic activity of full-length PcsB have been unsuccessful 8,11,12 . Recently, it was discovered that PcsB interacts with the membrane-embedded protein FtsX through its N-terminal CC domain.…”

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

“…S12). These morphologies were observed to a lesser extent in the D39 strain, perhaps because the capsule can change and/or mask certain division phenotypes (45).…”