1. Introduction
1.5 PcsB, an essential putative cell wall hydrolase in S. pneumoniae
PcsB was first identified in Streptococcus agalactiae as a protein found to dominate in the supernatant. Homology searches show that PcsB is highly conserved among streptococci and
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lactococci, while a partial homologue is found in some enterococci. The name PcsB, short for
“protein required for cell separation in group B Streptococcus”, derives from the observation that pcsB deletion mutants of S. agalactiae grew in clusters comprising many cells instead of chains as in wild-type cells. New septa were not initiated at mid-cell but at random positions and angles in the pcsB mutant, and unseparated cells continued to form new septa. The pcsB gene could only be removed in the presence of an osmoprotectant, indicating that the cells became sensitive towards osmotic pressure [87]. Overexpression of pcsB did not lead to elevated levels of PcsB in the supernatant. Therefore, the amount of secreted PcsB must be regulated either at the level of translation or secretion [88].
The initial studies on pcsB in S. pneumoniae suggested that it was an essential gene in this bacterium. Since S. pneumoniae only contains one copy of pcsB, while S. agalactiae contains several pcsB paralogues, it is speculated that this redundancy might be the reason for the non-essential role of pcsB in S. agalactiae [89-92]. However, subsequent studies could report pcsB as non-essential in four different genetic backgrounds of S. pneumoniae, among them the virulent TIGR4 strain. Removal of pcsB in TIGR4 resulted in a similar phenotype as described for S.
agalactiae. In addition to the formation of large cell aggregates, it developed misplaced septa that were synthesized at abnormal angles relative to the old cell wall (Figure 4) [93, 94]. For the pneumococcal strains D39 (serotype 2) and R6 (descendant of serotype 2) pcsB is reported to be essential. The reason why TIGR4 can survive without pcsB, while the D39 and R6 strains cannot, is not known. A six days incubation at 37oC, however, allowed viable pcsB mutants of D39 to grow. It can therefore be argued that pcsB mutants in the virulent TIGR4 strain were obtained upon high selective pressure and accumulation of suppressor mutations [90].
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Figure 4. Morphology of a pcsB mutant of S. pneumoniae TIGR4. The left panel shows a pcsB mutant with unseparated cells growing in clusters with disrupted division angels. The panel to the right shows a pcsB mutant in which the pcsB expression has been restored by a plasmid containing the pcsB gene [94].
The figure is reprinted from Giefing-Kroll et al. [94] with permission from the Society for General Microbiology (SGM).
PcsB consists of four parts; (i) an N-terminal secretory signal peptide, (ii) a coiled-coil domain containing a leucine zipper motif, (iii) a linker and (iv) a C-terminal CHAP domain (Figure 5) [91, 94].
Figure 5. Predicted domain organization of pneumococcal PcsB. The N-terminal part consists of a secretory signal peptide. The coiled-coil domain is predicted to contain a leucine zipper motif. There is a linker between the coiled-coil domain and the predicted CHAP domain [91, 94].
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PcsB is shown to localize to the septum and to the equatorial lines where ongoing cell wall synthesis occurs in S. pneumoniae, where it interacts with the FtsEX complex [57]. PcsB is very abundant in S. pneumoniae, estimated to be present at approximately 5000 monomers in each cell.
However, the synthesis of PcsB must be even higher due to the high amount of PcsB that is found in the supernatant as well [90]. The reason why PcsB is needed in such high amounts is not clear, but normal cell growth is completely dependent on this high expression level. Only a small reduction in pcsB expression leads to severe defects in cell division [90]. Interestingly, PcsB is only detected in the supernatant and the membrane fractions of S. pneumoniae, but not in the cell wall fraction where it is predicted to act as a murein hydrolase [57, 95].
Transcriptional analyses of a TIGR4 pcsB mutant strain have shown altered expression of several genes. Of particular interest is the increased expression of two genes encoding so called LysM-domain proteins. Elevated expression of the corresponding genes, spr0096 and spr1875, was also identified in a pcsB depleted R6 strain. Since LysM domains are cell wall binding modules, it is thought that these two proteins participate in cell wall metabolism. An up-regulation of these LysM-containing proteins might partially compensate for the loss of PcsB [90, 94].
1.5.1 Activation of cell wall hydrolases by the FtsEX complex during cell division
Many murein hydrolases involved in bacterial cell division are regulated by a protein complex consisting of FtsE and FtsX, as reported for E. coli and B. subtilis. FtsE is a cytoplasmic ATPase that interacts with FtsX which is embedded in the cytoplasmic membrane. In E. coli, the FtsEX complex contributes to constriction of the Z-ring during cell division [59-61]. In this bacterium, the proteins EnvC and NlpD activate three redundant amidases called AmiA, AmiB and AmiC that are responsible for septal cross-wall splitting during cell division. EnvC is shown to interact with a periplasmic loop of FtsX, and is only able to activate the amidases AmiA and AmiB when the FtsEX complex performes cytoplasmic hydrolysis. It is therefore believed that ATP-hydrolysis by FtsE results in a conformational change in FtsX that is transmitted to EnvC, leading to activation of the amidases AmiA and AmiB. This is an elegant way of controlling that these amidases are activated at the right place in the cell and not until assembly of the cytokinetic Z-ring is completed [96-99]. Similarly, a cell wall hydrolase called CwlO, needed for cell wall elongation
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in B. subtilis, is shown to interact with FtsX, and its activation depends on ATP-hydrolysis by FtsE as well [100, 101].
The regulatory role of the FtsEX complex in the control of murein hydrolase activity is also seen in S. pneumoniae. As previously mentioned, the putative murein hydrolase PcsB is shown to interact with FtsX [57, 62]. The coiled-coil domain of PcsB interacts with the two extracellular loops ECL1 and ECL2 of FtsX (Figure 6). Because of the fact that FtsE, FtsX and PcsB all are essential in pneumococcal cell division, and that depletion of ftsX or ftsE gives rise to a ΔpcsB phenotype, it is believed that activation of PcsB requires interaction with FtsX. The energy derived from ATP-hydrolysis by FtsE probably induces a conformational change in FtsX that is transmitted to PcsB, which then becomes active. However, it is not known whether PcsB functions as a murein hydrolase, a scaffolding protein or as a regulator of another cell wall hydrolase [57, 62].
Figure 6. Predicted model of PcsB activation in S. pneumoniae. Membrane embedded FtsX interacts with the coiled-coil domain of PcsB via its extracellular loops ECL1 and ECL2 (ECL2 is not shown in the figure). Hydrolysis of ATP by FtsE in the cytoplasm most probably induces a conformational change in FtsX that is transferred via the ECL1 and ECL2 loops to PcsB, which then becomes active [57, 62]. The figure is reprinted from Sham et al. [57] with permission from PNAS.
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1.5.2 Evidence for muralytic activity of PcsB are lacking
PcsB contains a CHAP domain, which is predicted to possess peptidoglycan hydrolytic activity.
Other examples of proteins carrying this domain are CbpD from S. pneumoniae (see section 1.4), the cell separation enzyme Cse from S. thermophilus and the competence induced murein hydrolase LytF from S. gordonii [102-104]. CHAP domains have been shown to possess either amidase activity or endopeptidase activity resulting in cleavage of stem peptides in the peptidoglycan [68]. All members of the CHAP superfamily are predicted to possess the same hydrolytic mechanism, but very few CHAP-containing enzymes have been characterized with respect to their site of cleavage within the stem peptide [68, 102]. The active site comprises a catalytic triad where a cysteine performs a nucleophilic attack, a histidine residue functions as a proton donor, and a polar residue, usually asparagine or aspartic acid, contributes by orienting the histidine residue in the correct position [102].
Hydrolytic activity of PcsB has never been detected. Several attempts to demonstrate its activity have been conducted, but not one has yet succeeded. Even when the pneumococcal PcsB was tested in the presence of a recombinant ECL1 extracellular loop of FtsX, no muralytic activity was seen [57, 87, 88]. Furthermore, addition of recombinant PcsB into the medium of a pcsB mutant of S. pneumoniae TIGR4 did not restore wild-type phenotype [94]. These observations have led to speculations that PcsB does not function as a cell wall hydrolase after all, but rather as a scaffolding protein or as an activator of another cell wall hydrolase [57, 93-95]. However, the point mutations C292A or H343A in the predicted active site of the CHAP domain are lethal, indicating that enzymatic activity of the CHAP domain is required for cell viability [91].