Summary auto-generated
This study characterized the surface layer (S-layer) protein of Lactobacillus helveticus ATCC 12046, a bacterium important in cheese production. The researchers used freeze-etching electron microscopy to confirm the presence of an oblique crystalline S-layer lattice completely covering the cell surface. The S-layer was composed of a single 52 kDa non-glycosylated protein. Treatment with 5M lithium chloride (LiCl) efficiently extracted the S-layer protein while maintaining cell viability (80% survival). Notably, when LiCl-extracted cells were grown in culture medium, the S-layer spontaneously reformed. The isolated S-layer protein contained 44% hydrophobic amino acids and had an N-terminal sequence rich in alanine, threonine, asparagine, and aspartic acid. In vitro reassembly experiments showed that the isolated protein subunits spontaneously self-assembled into sheets with the same oblique lattice symmetry observed on intact cells. Testing with lytic enzymes revealed that the S-layer does not protect peptidoglycan from lysozyme or mutanolysin, suggesting the pores in the lattice are larger than 3.5 nm.
Key findings
- L. helveticus ATCC 12046 possesses an oblique crystalline S-layer composed of a single 52 kDa non-glycosylated protein that completely covers the cell surface
- 5M LiCl effectively extracts the S-layer protein while preserving cell viability (~80%), and the S-layer spontaneously reforms when extracted cells resume growth
- Isolated S-layer protein subunits self-assemble in vitro into crystalline sheets with identical oblique lattice symmetry to those found on intact cells
- The S-layer protein is 44% hydrophobic amino acids but provides no protection against muramidase enzymes, indicating the lattice pores exceed 3.5 nm in diameter
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Abstract
Summary: In a previous study, electron microscopic examinations of thin sections of Lactobacillus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identified as a layer of a non-glycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (S-layer) lattice (a = 4·5 nm, b = 9·6 nm, γ = 77 °) which completely covers the cell surface. Treatment with 5 M-LiCl extracted the S-layer protein from intact cells efficiently and selectively. Viability did not decrease significantly. Moreover, the S-layer reappeared when treated cells were allowed to grow again. In vitro self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in vivo. The purified S-layer protein had a high content (44%) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid.