Fratricide is essential for efficient gene transfer between pneumococci in biofilms

Abstract

Streptococcus pneumoniae and a number of commensal streptococcal species are competent for natural genetic transformation. The natural habitat of these bacteria is multispecies biofilms in the human oral cavity and nasopharynx. Studies investigating lateral transfer of virulence and antibiotic resistance determinants among streptococci have shown that interspecies as well as intraspecies gene exchange takes place in these environments. We have previously shown that the action of a competence-specific murein hydrolase termed CbpD strongly increases the rate of gene transfer between pneumococci grown in liquid cultures. CbpD is the key component of a bacteriolytic mechanism termed the fratricide mechanism. It is secreted by competent pneumococci and mediates the release of donor DNA from sensitive streptococci present in the same environment. However, in nature, gene exchange between streptococci takes place in biofilms and not in liquid cultures. In the present study, we therefore investigated whether CbpD affects the rate of gene transfer in laboratory-grown biofilms. Our results show that the fratricide mechanism has a strong positive impact on intrabiofilm gene exchange, indicating that it is important for active acquisition of homologous donor DNA under natural conditions. Furthermore, we found that competent biofilm cells of S. pneumoniae acquire a Nov(r) marker much more efficiently from neighboring cells than from the growth medium. Efficient lysis of target cells requires that CbpD act in conjunction with the murein hydrolase LytC. In contrast, the major autolysin LytA does not seem to be important for fratricide-mediated gene exchange in a biofilm environment.

Fig 1

S. pneumoniae biofilm development followed over a period of 24 h. DIC micrographs were taken 2 h (A), 4 h (B), 6 h (C), 9 h (D), 12 h (E), and 24 h (F) after inoculation.

Fig 2

Confocal microscopy of strain RH14 (ΔlytA) biofilms grown for 12 h (A) and 24 h (B) at 37°C. The biofilms were stained with calcein red-orange to visualize the entire biofilm structure.

Fig 3

Transformation in S. pneumoniae biofilms cultivated for different periods of time. (A) Transformation efficiencies obtained in RH1 and RH14 biofilms subjected to CSP (100 ng ml⁻¹) and purified Nov^r DNA (1 μg ml⁻¹) 4, 6, 8, and 12 h postinoculation. CSP and transforming DNA were added to the biofilm medium concomitantly. (B) Intrabiofilm transfer of a Nov^r marker in mixed biofilms consisting of RH1 (Spc^r) and RH401 (ΔcomE Nov^r) cells seeded at a 1:1 ratio. To induce the competent state, CSP (100 ng ml⁻¹) was added 4, 6, 8, and 12 h postinoculation. Results are means ± standard errors from three independent experiments.

Fig 4

Comparison of the biofilm-forming capacities of different S. pneumoniae mutant strains used in the present study. The strains were grown in glass-bottom petri dishes for 4 h at 37°C and then transferred to 30°C for 2 h in the presence of CSP (100 ng ml⁻¹). An identical procedure was followed in the intrabiofilm transformation experiments whose results are presented in Table 3. Results are the means ± standard errors from at least three independent experiments.

Fig 5

Fratricide in biofilm visualized by confocal laser scanning microscopy and BacLight Live/Dead staining. (A) Mixed biofilm consisting of CbpD-proficient RH14 attacker cells and noncompetent SPH30 (ΔcomE) target cells seeded in a 1:1 ratio. Competence was induced 4 h postinoculation by addition of CSP (100 ng ml⁻¹) to the biofilm medium. After addition of CSP, the biofilm was transferred from 37°C to 30°C for 20 min. Before being examined by confocal microscopy the biofilm was stained using the BacLight Live/Dead kit, which stains live bacteria green and dead bacteria red. (B) Mixed biofilm consisting of CbpD-deficient SPH106 attacker cells and noncompetent SPH30 target cells. The biofilms in both panels were grown and treated identically. The only difference between them is that the SPH106 attacker strain in panel B is not able to produce CbpD when induced to competence by addition of CSP.