Pneumococci that have developed the competent state kill and lyse non-competent sister cells and members of closely related species during co-cultivation in vitro. The key component in this process, called fratricide, is the product of the late competence gene cbpD. In addition, the peptidoglycan hydrolases LytA and LytC are required for efficient lysis of target cells. Here, we have investigated the relative contribution and possible role of each of the proteins mentioned above. Previous studies have shown that CbpD is produced exclusively by competent cells, whereas LytA and LytC can be provided by the competent attackers as well as the non-competent target cells. By using an improved assay to compare the effect of cis-versus trans-acting LytA and LytC, we were able to show that target cells are lysed much more efficiently when LytA and LytC are provided in cis, i.e. by the target cells themselves. Western analysis demonstrated that considerable amounts of LytC are present in the growth medium. In contrast, we were not able to detect any extracellular LytA. This finding indicates that LytA-and LytC-mediated fratricide represent different processes. In the absence of LytA and LytC, only a tiny fraction of the target cells were lysed, demonstrating that CbpD does not function efficiently on its own. However, in the presence of 1 mM EDTA, the fraction of target cells lysed directly by CbpD increased dramatically, indicating that divalent cations are involved in the regulation of fratricide under natural conditions.

SummaryPneumococci that are competent for natural genetic transformation express a number of proteins involved in binding, uptake, translocation and recombination of DNA. In addition, they attack and lyse noncompetent sister cells present in the same environment. This phenomenon has been termed fratricide. The key effector of pneumococcal fratricide is CbpD, a secreted protein encompassing an N-terminal CHAP domain, two SH3b domains and a C-terminal choline-binding domain (CBD). CbpD is believed to degrade the cell wall of target cells, but experimental evidence supporting this hypothesis has been lacking. Here, we show that CbpD indeed has muralytic activity, and that this activity requires functional CBD and SH3b domains. To better understand the critical role played by the non-catalytic C-terminal region of CbpD, various translational fusions were constructed between the CBD and SH3b domains and green fluorescent protein (GFP). The results showed that the SH3b domains specifically recognize and bind peptidoglycan, while the CBD domain functions as a localization signal that directs CbpD to the septal region of the pneumococcal cell. Intriguingly, transmission electron microscopy analysis revealed that target cells attacked by CbpD ruptures at the septal region, in accordance with the binding specificity displayed by the CBD domain.

Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.

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