Investigating a Competence-Controlled Murein Hydrolase in Streptococcus sanguinis

Objectives: The competence pathway is a stress response pathway. In the oral commensal Streptococcus sanguinis, competence plays a major role in biofilm formation via the uptake and release of DNA. This DNA contributes genetically and structurally to the oral biofilm. It is hypothesized that LytF, a competence-associated, putative murein hydrolase in S. sanguinis, plays a role in competence and biofilm development; thus, the objective of this study was to investigate the regulation and activity of LytF in S. sanguinis.
Methods: Expression of lytF and competence-associated genes was examined at mid-log phase using qRT-PCR after induction with 0.5µg/ml CSP. lytF mutant and complemented strains were created using overlap-extension PCR. Chain length and cell morphology was examined at mid-log phase. Zymograms were performed with mid-log phase whole cell extracts using S. sanguinis, S. gordonii, S. mutans, and S. oralis as substrate cells. Proteins were refolded at 37°C overnight in buffer containing Triton X-100. Proteins were precipitated from S. sanguinis supernatant using ammonium sulfate.
Results: lytF expression could be induced with the addition of CSP. Expression of the remaining competence pathway was not altered in the lytF mutant. Bacterial chains were elongated and cells more circular in the lytF mutant and complemented strain, respectively. Zymograms showed LytF of S. sanguinis to have muralytic activity against a variety of streptococcal species. A protein at the expected size of LytF could be precipitated from culture supernatants.
Conclusions: lytF was shown to be competence-associated and its gene product a murein hydrolase that appears to affect cell physiology. After secretion, LytF remained active against multiple streptococcal species, as shown through zymograms. This warrants further investigation into the release of cellular components via LytF activity. The competence-controlled LytF may, thus, play a major role in biofilm formation in the oral cavity.