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Binding of the immunity protein inactivates colicin M
T Olschläger1, A Turba, V Braun
1Mikrobiologie II, Auf der Morgenstelle 28, Universität, Tübingen, Germany.
Abstract:
Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.
Insights
Colicin M immunity protein (Cmi) protects cells by binding Colicin M (Cma) at the cell membrane, preventing Cma from inhibiting essential cell wall synthesis. Cmi levels and Cma binding are key to this protective mechanism.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Colicin M (Cma) is a bacteriocin that inhibits peptidoglycan and lipopolysaccharide biosynthesis by disrupting bactoprenyl phosphate recycling.
- The Cma-producing cells are protected by an immunity protein, Cmi.
- Understanding the mechanism of Cmi-mediated immunity is crucial for comprehending Cma's action and developing countermeasures.
Purpose of the Study:
- To investigate the mechanism by which the immunity protein (Cmi) protects cells from Colicin M (Cma).
- To determine the role of Cmi concentration and cellular localization in Cma immunity.
Main Methods:
- In vitro assays measuring peptidoglycan synthesis inhibition by Cma.
- Genetic manipulation of Cma uptake systems and cmi gene copy number in bacterial cells.
- Proteolytic degradation studies of Cmi in sphaeroplasts with and without Cma.
Main Results:
- The amount of Cmi directly correlated with the inhibition of in vitro peptidoglycan synthesis by Cma.
- Overexpression of the Cma uptake system led to immunity breakdown, which was reversed by increasing cmi gene copy number.
- Cmi was degraded by trypsin in sphaeroplasts, but Cma addition prevented this degradation.
- The N-terminal hydrophobic region of Cmi suggests membrane anchoring.
Conclusions:
- Cmi likely functions by binding Cma at the periplasmic face of the cytoplasmic membrane, inactivating it without catalytic activity.
- Cmi does not interfere with Cma uptake or directly interact with Cma's cellular target.
- The primary mechanism of Cma immunity involves Cmi binding to Cma, with its localization facilitated by a hydrophobic N-terminus.