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Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell
Abstract:
Furazlocillin (1 microgram/ml) and piperacillin (5 microgram/ml) bound specifically to penicillin-binding protein 3 (PBP-3) and not to the other major PBPs in intact Escherichia coli cells. The effect of this specific binding to PBP-3 on murein synthesis of elongating and synchronously septating cells was investigated in two thermosensitive division mutants, E. coli BUG6 and E. coli JE10730, the latter possessing a thermolabile PBP-3. Synchronous cell division was induced by shifting the cultures from the nonpermissive temperature (42 degrees C) to 30 degrees C. Both [14C]diaminopimelic acid incorporation into murein of intact cells and [14C]N-acetylglucosamine incorporation into murein of cells permeabilized with ether was inhibited by an average of 42% in septating cells. In filaments growing at the nonpermissive temperature, we detected no inhibition and, frequently, a 10 to 15% stimulation of murein synthesis. The two drugs, at concentrations used in the above experiments, bound exclusively to PBP-3 both in elongating and septating intact cells and in ether-treated cells. These results support the hypothesis that PBP-3 activity is exclusively required for septal murein synthesis.
Insights
Furazlocillin and piperacillin specifically target penicillin-binding protein 3 (PBP-3) in E. coli. This binding inhibits murein synthesis during cell division, suggesting PBP-3 is crucial for septal murein production.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Penicillin-binding proteins (PBPs) are essential enzymes involved in bacterial cell wall synthesis.
- Penicillin-binding protein 3 (PBP-3) in Escherichia coli is implicated in cell division and peptidoglycan synthesis.
- Understanding the specific roles of individual PBPs is crucial for developing targeted antibiotics.
Purpose of the Study:
- To investigate the specific binding of furazlocillin and piperacillin to PBP-3 in E. coli.
- To determine the effect of PBP-3 inhibition on murein synthesis during cell elongation and division.
- To elucidate the role of PBP-3 in septal murein synthesis.
Main Methods:
- Utilized thermosensitive E. coli division mutants (BUG6 and JE10730) with a thermolabile PBP-3.
- Synchronized cell division by temperature shift from 42°C to 30°C.
- Measured [14C]diaminopimelic acid and [14C]N-acetylglucosamine incorporation into murein of intact and permeabilized cells.
Main Results:
- Furazlocillin and piperacillin selectively bound to PBP-3 in both elongating and septating E. coli cells.
- Murein synthesis was inhibited by approximately 42% in synchronously septating cells.
- No inhibition, and even a slight stimulation (10-15%), of murein synthesis was observed in elongating filaments.
Conclusions:
- PBP-3 activity is specifically required for the synthesis of septal murein in E. coli.
- Targeting PBP-3 with antibiotics like furazlocillin and piperacillin effectively inhibits cell division.
- These findings support the hypothesis that PBP-3 plays a distinct role in bacterial cytokinesis.