Identification and molecular characterization of an N-acetylmuraminidase, Aml, involved in Streptococcus mutans cell separation

We previously demonstrated Streptococcus mutans produces two bacteriolytic enzymes of 100 kDa and 80 kDa (G. Yoshimura et al. Microbiol. Immunol. 48,465-469,2004). Here, we identified the protein sequence of these enzymes and found they come from a single gene product designated as automutanolysin (Aml). Aml has a modular design where the N-terminus contains five 13-amino-acid repeats and a C-terminal enzyme active domain. Aml selectively lyses S. mutans and S. sobrinus but no other oral streptococci. This suggests Aml possesses strong substrate specificity towards cariogenic bacteria present in the human oral cavity. Analysis of S. mutans peptidoglycan fragments released by Aml shows the enzyme is an N-acetylmuraminidase. We found Ca2+ enhances the activity; and EGTA, EDTA and iodoacetic acid inhibit the activity. The optimum pH range for lytic activity was 6 to 7. Disruption of the aml gene in S. mutans results in the formation of a longer bacterial cell chain length that was dispersed by the addition of a low concentration of Aml. This suggests Aml is involved in S. mutans cell separation.