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Reassembly in vitro of hexagonal surface arrays in a protein-producing bacterium, Bacillus brevis 47
Abstract:
Bacillus brevis 47 had two protein layers (the outer and middle walls) and a peptidoglycan layer (the inner wall) and contained two major proteins with approximate molecular weights of 130,000 and 150,000 in the cell wall. Both the total and Triton-insoluble envelopes revealed a hexagonal lattice array with a lattice constant of 14.5 nm. The proteins of 130,000 and 150,000 molecular weight isolated from the Triton-insoluble envelopes were serologically different from each other and assembled in vitro on the peptidoglycan layer. A mixture of 130,000- and 150,000-molecular-weight proteins led to the formation of a five-layered cell wall structure, two layers on each side of the peptidoglycan layer, which resembled closely the Triton-insoluble envelopes. A three-layered cell wall structure, one layer on each side of the peptidoglycan layer, was reconstituted when only the 150,000-molecular-weight protein was used. Both five- and three-layered cell walls reconstituted in vitro also contained hexagonally arranged arrays with the same lattice constant as that of the total and Triton-insoluble envelopes. A mutant, strain 47-57, which was isolated as a phage-resistant colony, had a two-layered cell wall consisting of the middle and inner wall layers and contained only 150,000-molecular-weight protein as the major cell wall protein. The cell envelopes of the mutant revealed the hexagonal arrays with the same lattice constant as that of the wild-type cell envelopes. We conclude that the outer and middle wall layers consist of proteins with approximate molecular weights of 130,000 and 150,000, respectively. Furthermore, the 150,000-molecular-weight protein formed the hexagonal arrays in the middle wall layer.
Insights
Bacillus brevis cell walls feature two major proteins (130,000 and 150,000 MW) forming distinct layers. The 150,000 MW protein is crucial for the hexagonal array structure in the middle wall.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Bacillus brevis possesses a complex cell wall structure.
- Understanding the protein composition and arrangement is key to bacterial cell envelope research.
Purpose of the Study:
- To elucidate the protein composition and structural organization of the Bacillus brevis 47 cell wall.
- To investigate the self-assembly properties of major cell wall proteins in vitro.
Main Methods:
- Isolation and characterization of major cell wall proteins from Bacillus brevis 47.
- In vitro reconstitution experiments of cell wall layers.
- Analysis of cell envelope structure using electron microscopy (hexagonal lattice array).
- Characterization of a mutant strain (47-57) with altered cell wall composition.
Main Results:
- Bacillus brevis 47 cell wall contains two major proteins (130,000 and 150,000 MW) in outer and middle layers.
- These proteins self-assemble on the peptidoglycan layer, forming multi-layered structures in vitro.
- A hexagonal lattice array (14.5 nm lattice constant) was observed in both wild-type and reconstituted cell envelopes.
- A mutant lacking the 130,000 MW protein retained the hexagonal array, indicating the 150,000 MW protein's role.
Conclusions:
- The 130,000 MW protein constitutes the outer wall layer, and the 150,000 MW protein forms the middle wall layer.
- The 150,000 MW protein is responsible for forming the hexagonal arrays in the middle cell wall layer.
- The structural organization of Bacillus brevis cell wall proteins is hierarchical and involves self-assembly.