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Visualization of penicillin-binding proteins during sporulation of Streptomyces griseus
1Department of Microbiology, Ohio State University, Columbus 43210, USA.
Abstract:
We used fluorescein-tagged beta-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.
Insights
Researchers visualized penicillin-binding proteins (PBPs) in Streptomyces griseus. An 85-kDa PBP was crucial for septum formation during bacterial sporulation, as shown by fluorescein-tagged antibiotics.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Penicillin-binding proteins (PBPs) are essential enzymes involved in bacterial cell wall synthesis.
- Sporulation in Streptomyces griseus is a complex developmental process involving significant morphological changes, including septum formation.
Purpose of the Study:
- To identify and characterize PBPs involved in septum formation during Streptomyces griseus sporulation.
- To investigate the role of a specific PBP in the process of sporulation.
Main Methods:
- Utilized fluorescein-tagged beta-lactam antibiotics to visualize PBPs in Streptomyces griseus.
- Performed membrane protein isolation and binding assays.
- Employed fluorescence microscopy to observe PBP localization and antibiotic interaction.
- Investigated the effect of cefoxitin on PBP binding and septum formation.
Main Results:
- Identified six PBPs in growing and sporulating Streptomyces griseus cultures.
- Observed a fourfold increase in the binding activity of an 85-kDa PBP during sporulation, coinciding with septum formation.
- Demonstrated that cefoxitin inhibits the 85-kDa PBP and prevents sporulation septum formation.
- Fluorescence microscopy confirmed specific labeling of sporulation septa by a fluorescein-tagged antibiotic (Flu-APA), which was blocked by cefoxitin.
Conclusions:
- The 85-kDa PBP is preferentially localized to sporulation septa in Streptomyces griseus.
- The 85-kDa PBP plays a critical role in septum formation during Streptomyces griseus sporulation.
- This PBP is a potential target for understanding and manipulating bacterial differentiation.