Influence of the spxB gene on competence in Streptococcus pneumoniae

Abstract

In Streptococcus pneumoniae expression of pyruvate oxidase (SpxB) peaks during the early growth phase, coincident with the time of natural competence. This study investigated whether SpxB influences parameters of competence, such as spontaneous transformation frequency, expression of competence genes, and DNA release. Knockout of the spxB gene in strain D39 abolished spontaneous transformation (compared to a frequency of 6.3 x 10(-6) in the parent strain [P < 0.01]). It also reduced expression levels of comC and recA as well as DNA release from bacterial cells significantly during the early growth phase, coincident with the time of spontaneous competence in the parent strain. In the spxB mutant, supplementation with competence-stimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 x 10(-2)). This speaks against the role of SpxB as a necessary source of energy for competence. Neither supplementation with CSP-1 nor supplementation with the SpxB products H2O2 and acetate altered DNA release. Supplementation of the parent strain with catalase did not reduce DNA release significantly. In conclusion, the pneumococcal spxB gene influences competence; however, the mechanism remains elusive.

FIG. 1.

spxB gene expression in strain D39 during the lag and log phases. D39 was cultured in BHI broth with 5% FCS. Left y axis, the spxB transcription levels were determined by real-time RT-PCR and expressed as the copy number per 100 CFU. Mean values of triplicates from three independent experiments (±SE) are presented. Right y axis, CFU per ml at each OD at which the spxB expression was measured. Presented are mean values of triplicates from three independent experiments.

FIG. 2.

Transformation frequency of strain D39 Sm^r and its spxB mutant at an OD₆₀₀ of 0.15. Strain D39 Sm^r and its spxB mutant were grown to an OD₆₀₀ of 0.15 in BHI broth with 5% FCS. The transformation frequency was measured in TSB competence 8.0 medium with and without the addition of CSP-1, and the number of Rif^r transformants per CFU was calculated. Mean values of triplicates from three independent experiments (±SE) are presented.

FIG. 3.

comC and recA gene expression and spontaneous transformation frequencies of strain D39 Sm^r and its spxB mutant during the time of competence. Strain D39 Sm^r and its spxB mutant were grown to OD₆₀₀s of 0.05 to 0.45 in BHI broth with 5% FCS. (A and B) Left Y axes, the comC (A) and recA (B) gene transcription levels (gray and white bars) were determined by real-time RT-PCR and expressed as the copy number per 100 CFU in strain D39 Sm^r compared to its spxB-deficient mutant at each OD. Mean values of triplicates from three independent experiments (±SE) are presented. *, P < 0.05; the P values were calculated by comparing the comC or recA gene expression in D39 Sm^r with the comC or recA gene expression in D39 Sm^r ΔspxB at each OD. Right y axes, the spontaneous transformation frequencies were measured in TSB competence 8.0 medium, and the number of Rif^r transformants per CFU was calculated. Mean values of duplicates from two independent experiments (±SE) are presented. Gray squares and solid lines, D39 Sm^r; white squares and broken lines, D39 Sm^r spxB knockout mutant. (C) Growth curves in BHI broth supplemented with 5% FCS of strain D39 Sm^r and its spxB knockout mutant. (D) CFU per ml of each OD presented in panel C for strain D39 Sm^r and its spxB knockout mutant.

FIG. 4.

DNA release of strain D39 Sm^r and its spxB mutant during the lag and log phases. Bacteria were grown to different ODs in BHI broth with 5% FCS. DNA release into the supernatant was measured with and without the addition of CSP-1 by real-time PCR (A) or as the number of transformants as described by Moscoso and Claverys (21) (B). (A) The DNA quantity in the supernatant is expressed as the fold difference between DNA in the supernatant and that for D39 Sm^r without the addition of CSP-1 (±SE). (B) Values for DNA release represent streptomycin-resistant transformants per ml. Mean values of duplicates from three independent experiments (±SE) are presented. * and **, P < 0.05; the P values were calculated by comparing the DNA release of D39 Sm^r with the DNA release of D39 Sm^r ΔspxB at each OD without (*) or with (**) the addition of CSP-1.

FIG. 5.

DNA release of the spxB-deficient mutant grown in culture medium supplemented with acetate and/or H₂O₂. Bacteria were grown to different ODs in BHI broth plus 5% FCS with and without acetate and/or H₂O₂. No CSP-1 was added to the cultures. Values for DNA release represent streptomycin-resistant transformants per ml. Mean values of triplicates from three independent experiments (±SE) are presented.