Influence of thespxBGene on Competence inStreptococcus 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 ؋ 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 competencestimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 ؋ 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 H 2 O 2 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.The human nasopharynx is the natural habitat of Streptococcus pneumoniae. Colonization is the prerequisite for invasion, transmission, and genetic evolution of pneumococci. An increasing number of adherence factors that support colonization are being discovered (13). In addition, production of hydrogen peroxide (H 2 O 2 ) is thought to inhibit competitive nasopharyngeal flora (23). In the pneumococcus, H 2 O 2 is produced under rich and aerobic conditions by the enzyme pyruvate oxidase (SpxB), encoded by the spxB gene. Besides the production of H 2 O 2 , SpxB decarboxylates pyruvate to acetyl phosphate plus CO 2 (24). Spellerberg et al. (29) showed that an spxB-deficient mutant exhibits reduced virulence for nasopharyngeal colonization, pneumonia, and sepsis. Expression of SpxB peaks during the early growth phase (18). This coincides with the time of pneumococcal competence.S. pneumoniae shares with at least 40 bacterial species the property of natural transformation (19). The importance of transformation for genetic evolution has been illustrated by the emergence of penicillin-resistant pneumococcal isolates and pneumococcal strains undergoing capsule switch (8,9,22). In S. pneumoniae, competence is induced by the competencestimulating peptide (CSP) (7). Induction of competence-specific (com) genes leads to DNA uptake and processing. Competence also triggers cell lysis and DNA release from a fraction of bacterial cells.This study investigated whether the spxB gene or the products of SpxB have any influence on competence in S. pneumoniae. It is shown that in strain D39, deletion of spxB abolished spontaneous transformation, reduced expression levels of an early competence gene (comC) and a late competence gene (recA), and reduced competence-associated DNA release. There was, however, no evidence for a role of the products ...

Cell Invasion and Pyruvate Oxidase-Derived H 2 O 2 Are Critical for Streptococcus pneumoniae-Mediated Cardiomyocyte Killing

“…In fact, the corresponding gene has been shown to be important for colonization (36), competence (37), and capsule production (29). Its deletion, therefore, can also attenuate the bacterium in a manner that is substantially greater than inhibition of H 2 O 2 production.…”

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

“…For experiments, pneumococci were at midlog phase (OD 600 of 0.3-0.35), and A549 cells were at 70-80% confluency. The spxB gene was cloned into pGEMT, and the kanR gene was introduced at the HindIII site of the spxB gene (70). To create type 4 ΔspxB, plasmid was transformed into bacteria in the presence of 200 ng CSP-1 peptide.…”

Human Lung Mast Cells Mediate Pneumococcal Cell Death in Response to Activation by Pneumolysin

“…pneumoniae serotype 2, strain D39 was obtained from the National Collection of Type Cultures, London, U.K. (NCTC 7466). The phenotypic characteristics of the D39 isogenic pneumolysin-deficient mutant (PLN-A) and the H 2 O 2 -deficient mutant (SpxB) were described in detail previously (27,29,36). The SpxB mutant was kindly provided by Prof. K. Muhlemann (University of Bern, Bern, Switzerland).…”

“…WT bacteria were identified as pneumococci prior to experiments by Gram stain, catalase test, a-hemolysis on blood agar plates, and optochin sensitivity. Isogenic mutants PLN-A and SpxB were identified by growth on erythromycin and chloramphenicol-supplemented blood agar plates, as previously described (27,29,36).…”