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Identification and molecular characterization of an N-Acetylmuraminidase, Aml, involved in Streptococcus mutans cell
Goh Yoshimura1, Hitoshi Komatsuzawa, Ikue Hayashi
1Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan.
Abstract:
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 Ca(2+) 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.
Insights
Streptococcus mutans automutanolysin (Aml) is a single gene product that selectively lyses cariogenic bacteria. Aml acts as an N-acetylmuraminidase and is crucial for S. mutans cell separation.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Streptococcus mutans produces two bacteriolytic enzymes.
- These enzymes were previously identified as 100 kDa and 80 kDa.
Purpose of the Study:
- Identify the protein sequence of the S. mutans bacteriolytic enzymes.
- Characterize the function and properties of the identified enzyme.
- Investigate the role of the enzyme in S. mutans cell division.
Main Methods:
- Protein sequencing and gene identification.
- Enzyme activity assays under varying conditions (pH, ions).
- Analysis of peptidoglycan fragments.
- Gene disruption and cell morphology analysis.
Main Results:
- The two enzymes originate from a single gene product, designated automutanolysin (Aml).
- Aml exhibits modular design with N-terminal repeats and a C-terminal active domain.
- Aml selectively lyses S. mutans and S. sobrinus, acting as an N-acetylmuraminidase.
- Enzyme activity is enhanced by Ca(2+) and inhibited by EGTA, EDTA, and iodoacetic acid, with optimal activity at pH 6-7.
- Disruption of the aml gene leads to longer bacterial cell chains, indicating Aml's role in cell separation.
Conclusions:
- Automutanolysin (Aml) is a novel, specifically acting bacteriolytic enzyme from S. mutans.
- Aml is an N-acetylmuraminidase essential for S. mutans cell separation.
- The findings provide insights into the mechanism of cell division in cariogenic streptococci.
