Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis

Abstract

Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.

FIG. 1.

cbpD promoter activity in competent and noncompetent cells. The L1 mutant was collected at various cell densities (OD₅₅₀ of 0.1 to 0.5) and induced to competence by addition of 250 ng/ml of synthetic CSP-1. After 30 min at 30°C, cultures were lysed and assayed for β-galactosidase activity. Corresponding uninduced samples were run in parallel. Solid squares, cell lysates from cells induced with CSP-1; open squares, cell lysates from uninduced cells. The data presented are representative of results from three independent experiments. Strain L1 is comA egb cbpD::pEVP3.

FIG. 2.

Comparison of competence-induced release of cytoplasmic β-galactosidase from wild-type S. pneumoniae cells and a mutant lacking a functional cbpD gene. Bacterial cultures of strains EK4166 and EK4168 were collected at different cell densities (OD₅₅₀ of 0.1 to 0.5) and induced to competence by addition of 250 ng/ml of synthetic CSP-1. After 30 min at 30°C, culture supernatants were harvested by centrifugation followed by sterile filtration. The supernatants were subsequently assayed for β-galactosidase activity. Solid squares, supernatants from EK4166 cells; open squares, supernatants from EK4168 cells. The experiment has been repeated several times with similar results. Genotypes of strains: EK4166, comA egb hirL::pEVP3; and EK4168, comA egb hirL::pEVP3 cbpD.

FIG. 3.

Quantitative comparison of competence-induced DNA release in wild-type and ΔcbpD cells. When the bacterial cultures reached an OD₅₅₀ of 0.3, 15 pg/ml control DNA (pLNO2HM1) and 0.5 μg/ml salmon sperm DNA were added. The cultures were immediately split in two, and one half of the culture was induced to competence by addition of 250 ng/ml of synthetic CSP while the other was kept untreated. After 30 min of incubation at 30°C, supernatants were collected by centrifugation and sterile filtered. Subsequently, the collected supernatants were used in a real-time PCR assay with primers and probes directed against the pneumolysin gene or the hm1 gene. The amount of released DNA in untreated (noncompetent) cultures was set to 1. Data are plotted as the means of three independent experiments with error bars representing the standard deviations. Genotypes of strains: CP1415, comA; and L3, comA cbpD.

FIG. 4.

Ectopic expression of CbpD. When cultures of the L3 and L5 mutants reached an OD₅₅₀ of 0.3, 15 pg/ml control DNA (pLNO2HM1) and 0.5 μg/ml salmon sperm DNA were added. The cultures were immediately split in three and induced with 250 ng/ml CSP-1, 250 ng/ml BIP-1, or kept untreated. After 30 min incubation at 30°C, supernatants were collected by centrifugation followed by sterile filtration. Subsequently, the collected supernatants were used in a real-time PCR assay with primers and probes directed against the pneumolysin gene or the hm1 gene. The amount of released DNA in untreated (noncompetent) cultures was set to 1. Data are plotted as the means of three independent experiments with error bars representing the standard deviations. Genotypes of strains: L3, comA cbpD; and L5, comA cbpD qsrA::cbpD.

FIG. 5.

Competence-induced DNA release in ΔlytA and ΔlytC mutants. Cultures of the H3 and H5 mutants were incubated at 37°C until they reached an OD₅₅₀ of 0.3. Then 15 pg/ml control DNA (pLNO2HM1) and 0.5 μg/ml salmon sperm DNA were added. The cultures were immediately split in two, transferred to 30°C, and induced with 250 ng/ml CSP or kept untreated. After 30 min incubation at 30°C, supernatants were collected by centrifugation and sterile filtered. Subsequently, the collected supernatants were used in a real-time PCR assay with primers and probes directed against the pneumolysin gene or the hm1 gene. The amount of released DNA in untreated (noncompetent) cultures was set to 1. Data are plotted as the means of three independent experiments with error bars representing the standard deviations. Genotypes of strains: H3, comA lytA::pEVP3 Nov^r; and H5, comA egb lytC::pEVP3.