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Growth of Streptococcus mutans protoplasts is not inhibited by penicillin
Abstract:
A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.
Insights
Streptococcus mutans protoplasts, which lack cell walls, can grow even when exposed to benzylpenicillin. This suggests cell wall synthesis, not earlier steps, causes penicillin
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Streptococcus mutans is a key bacterium in dental caries.
- Benzylpenicillin inhibits growth of intact S. mutans by affecting cell wall synthesis.
- The precise mechanism of benzylpenicillin inhibition in streptococci requires further investigation.
Purpose of the Study:
- To investigate the effect of benzylpenicillin on Streptococcus mutans protoplasts.
- To determine if cell wall synthesis is essential for benzylpenicillin-induced growth inhibition.
- To elucidate the role of cell wall assembly in the mechanism of benzylpenicillin action.
Main Methods:
- Conversion of exponential-phase S. mutans BHT cells to protoplasts using mutanolysin.
- Incubation of protoplasts in osmotically stabilized medium with and without benzylpenicillin.
- Measurement of protoplast growth via turbidity, protein, RNA, and DNA synthesis.
Main Results:
- S. mutans protoplasts grew and synthesized macromolecules in the presence of benzylpenicillin.
- Growth of protoplasts was unaffected by benzylpenicillin, unlike intact cells.
- Pre-treatment of intact cells with benzylpenicillin followed by conversion to protoplasts also allowed growth.
Conclusions:
- Wall-free protoplasts of S. mutans can grow in the presence of benzylpenicillin.
- The inhibition of growth and macromolecular synthesis in intact S. mutans by benzylpenicillin is linked to cell wall assembly.
- This study suggests that factors related to intact cell wall formation, not early peptidoglycan synthesis, mediate benzylpenicillin's inhibitory effects.