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Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287.

Avall-Jääskeläinen S, Hynönen U, Ilk N, Pum D, Sleytr UB, Palva A - BMC Microbiol. (2008)

Bottom Line: The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far.Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Finland. silja.avall@helsinki.fi

ABSTRACT

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group.

Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179-435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.

Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

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Self-assembly of SlpA, rSlpA and rSlpA C-terminal domain. (a-c) Transmission electron micrographs showing self-assembly products of (a) nontruncated rSlpA, (b) rSlpA179–435, and (c) wild type SlpA isolated from L. brevis ATCC 8287 cells. (d) Transmission electron micrograph showing a freeze-etched preparation of L. brevis ATCC 8287 cells completely covered with the oblique S-layer lattice formed by SlpA. Arrows indicate the base vectors of the oblique lattice.
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Figure 4: Self-assembly of SlpA, rSlpA and rSlpA C-terminal domain. (a-c) Transmission electron micrographs showing self-assembly products of (a) nontruncated rSlpA, (b) rSlpA179–435, and (c) wild type SlpA isolated from L. brevis ATCC 8287 cells. (d) Transmission electron micrograph showing a freeze-etched preparation of L. brevis ATCC 8287 cells completely covered with the oblique S-layer lattice formed by SlpA. Arrows indicate the base vectors of the oblique lattice.

Mentions: Precipitate-forming N-terminally truncated SlpA proteins were chosen for transmission electron microscopical analysis of lattice formation. In these studies, rSlpA formed an oblique lattice that was identical with that formed by SlpA isolated from wild type L. brevis ATCC 8287 cells (compare Figures 4a and 4c), as well as with the lattice seen on L. brevis ATCC 8287 cells in the freeze-etched preparation (Fig. 4d), proving the native conformation of recombinant SlpA. In accordance, lattice constants for the self-assembly products of rSlpA (a = 10.38, b = 6.36 and 72.7°) and for the self-assembly products of SlpA isolated from L. brevis ATCC 8287 cells (a = 9.39, b = 6.10 and 79.8°) were practically identical. The recombinant protein SlpA179–435 (Fig. 4b) was found to form a regular, oblique lattice indistinguishable from that formed by full length rSlpA, but the removal of eleven residues more from the N-terminus resulting in rSlpA190–435 prevented lattice formation (Fig. 2). Surprisingly, the two larger N-terminally truncated proteins, rSlpA167–435 and rSlpA149–435, were unable to form regular lattice structures. Thus, residues 179–435 in mature SlpA define the region responsible for the crystallization of SlpA monomers.


Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287.

Avall-Jääskeläinen S, Hynönen U, Ilk N, Pum D, Sleytr UB, Palva A - BMC Microbiol. (2008)

Self-assembly of SlpA, rSlpA and rSlpA C-terminal domain. (a-c) Transmission electron micrographs showing self-assembly products of (a) nontruncated rSlpA, (b) rSlpA179–435, and (c) wild type SlpA isolated from L. brevis ATCC 8287 cells. (d) Transmission electron micrograph showing a freeze-etched preparation of L. brevis ATCC 8287 cells completely covered with the oblique S-layer lattice formed by SlpA. Arrows indicate the base vectors of the oblique lattice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571106&req=5

Figure 4: Self-assembly of SlpA, rSlpA and rSlpA C-terminal domain. (a-c) Transmission electron micrographs showing self-assembly products of (a) nontruncated rSlpA, (b) rSlpA179–435, and (c) wild type SlpA isolated from L. brevis ATCC 8287 cells. (d) Transmission electron micrograph showing a freeze-etched preparation of L. brevis ATCC 8287 cells completely covered with the oblique S-layer lattice formed by SlpA. Arrows indicate the base vectors of the oblique lattice.
Mentions: Precipitate-forming N-terminally truncated SlpA proteins were chosen for transmission electron microscopical analysis of lattice formation. In these studies, rSlpA formed an oblique lattice that was identical with that formed by SlpA isolated from wild type L. brevis ATCC 8287 cells (compare Figures 4a and 4c), as well as with the lattice seen on L. brevis ATCC 8287 cells in the freeze-etched preparation (Fig. 4d), proving the native conformation of recombinant SlpA. In accordance, lattice constants for the self-assembly products of rSlpA (a = 10.38, b = 6.36 and 72.7°) and for the self-assembly products of SlpA isolated from L. brevis ATCC 8287 cells (a = 9.39, b = 6.10 and 79.8°) were practically identical. The recombinant protein SlpA179–435 (Fig. 4b) was found to form a regular, oblique lattice indistinguishable from that formed by full length rSlpA, but the removal of eleven residues more from the N-terminus resulting in rSlpA190–435 prevented lattice formation (Fig. 2). Surprisingly, the two larger N-terminally truncated proteins, rSlpA167–435 and rSlpA149–435, were unable to form regular lattice structures. Thus, residues 179–435 in mature SlpA define the region responsible for the crystallization of SlpA monomers.

Bottom Line: The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far.Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Finland. silja.avall@helsinki.fi

ABSTRACT

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group.

Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179-435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.

Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

Show MeSH
Related in: MedlinePlus