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

Åvall-Jääskeläinen, Silja; Hynönen, Ulla; Ilk, Nicola; Pum, Dietmar; Sleytr, Uwe B.; Palva, Airi

doi:10.1186/1471-2180-8-165

Cited by 60 publications

(65 citation statements)

References 49 publications

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“…All of the residues that were almost or totally inaccessible in the monomeric form were found within the Cterminal half of the protein, suggesting that these residues are located inside a structurally compact region of the self-assembly domain. This finding is consistent with a previous trypsin cleavage experiment (3), which identified a trypsin-resistant region encompassing residues (190) 209 to 423 (see below). The ability of the recombinant SlpA protein to self-assemble in dialysis as a lattice on isolated cell wall fragments has been shown using cryo-EM and SAXS (Jääskeläinen et al, unpublished).…”

Section: Discussionsupporting

confidence: 82%

“…Isolated cell wall fragments of L. brevis ATCC 8287 and the cell wall-bound protein preparations were obtained as described previously (3). The cell wall-bound cysteine-containing proteins were modified with TMM(PEG) 12 using the calculated amount of 2 g of each mutant protein on the cell wall fragments and the molecular ratio of 12 ] in the total reaction mixture volume of 16 l. The reaction mixtures were incubated for 1, 3, or 5 min at room temperature and stopped by NEM as described above, and the modified proteins were analyzed by SDS-PAGE as described for the monomeric proteins.…”

Section: Bacterialmentioning

confidence: 99%

“…Suspensions of all 46 heterologously expressed, isolated, and dialyzed SlpA mutant proteins were examined by negative staining to visualize the self-assembly products by electron microscopy (EM) as described previously (3,20).…”

Section: Bacterialmentioning

confidence: 99%

“…This thermal stability could prove potentially useful in a variety of in vitro S-layer applications currently being planned or under development (27,30,31). Recently, SlpA was characterized to consist of an N-terminal cell wall binding domain (residues 1 to 178) and a C-terminal self-assembly domain (179 to 435) (3). For the development of applications that take advantage of these characteristics, further investigation of SlpA at the molecular level is essential.…”

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confidence: 99%

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Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

et al. 2009

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Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many Eubacteria and most Archaea species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium Lactobacillus brevis ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG) 12 (molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW ‫؍‬ 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of Lactobacillus S-layers.Bacterial surface layers (S-layers) are cell envelope structures ubiquitously found in gram-positive and gram-negative bacteria as well as in Archaea. S-layers are composed of identical (glyco)protein subunits with a molecular mass in the range of 40 to 200 kDa. The proteins self-assemble into two-dimensional crystalline structures with oblique (p1, p2), square (p4), or hexagonal (p3, p6) symmetry, covering the entire cell surface. The subunits are held together and attached to the underlying cell wall by noncovalent interactions and they have an intrinsic ability to spontaneously form regular layers in solution and on solid supports (24). S-layers have been shown to have roles in the determination and maintenance of cell shape as virulence factors, as mediators of cell adhesion, and as regulators of immature dendritic and T cells. Moreover, they can also function as a protective coat, molecular sieve, murein hydrolase, and ion trap (4,8,13,17,19,25,29).S-layer proteins have several properties that make them an attractive target for the development of nanobiotechnological applications both in vivo and in vitro. In particular, a high number of protein subunits are displayed at the bacterial cell surface. Moreover, the prot...

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Section: Discussionsupporting

confidence: 82%

Section: Bacterialmentioning

confidence: 99%

Section: Bacterialmentioning

confidence: 99%

mentioning

confidence: 99%

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Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

et al. 2009

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“…Typically, it consists of two structural regions in which two essential functions reside. One region is involved in the attachment of the S-layer subunit to the cell envelope (transmembrane region), while the other is involved in assembly (47).…”

Section: Discussionmentioning

confidence: 99%

Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

Liew

Jong

Najimudin

2015

Appl Environ Microbiol

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A proteomic analysis of a soil-dwelling, plant growth-promoting Azotobacter vinelandii strain showed the presence of a protein encoded by the hypothetical Avin_16040 gene when the bacterial cells were attached to the Oryza sativa root surface. An Avin_16040 deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type. Escherichia coli transformed with the wild-type Avin_16040 gene displayed on its cell surface organized motifs which looked like the S-layer monomers of A. vinelandii. The recombinant E. coli also demonstrated enhanced adhesion to the root surface. Azotobacter vinelandii is a Gram-negative free-living and obligate aerobic soil bacterium. It is well known to be a plant growth-promoting bacterium capable of fixing nitrogen and forming desiccation-resistant cysts under unfavorable growth condition (1, 2). The former activity requires it to house several oxygen-sensitive mechanisms while being an obligate aerobic bacterium (3). A. vinelandii also has characteristics such as production of plant growth hormones and antibiotics (4) as well as industrially important substances such as extracellular polysaccharide (EPS) alginate, poly-␤-hydroxybutyrate (PHB), and siderophore compounds (5).Many diverse genera of nitrogen-fixing bacteria are present in the plant rhizosphere. The effectiveness of their plant growthpromoting activity depends upon the establishment of their cells in the rhizosphere. This interaction depends upon many factors, one of them being plant exudates. As a diazotroph, A. vinelandii provides fixed nitrogen to the plant while acquiring sugars and other nutrients that leak from the roots (6).The complete A. vinelandii genome (GenBank accession number NC_012560) has explained many biochemical pathways and structures of the bacterium (7). It has also revealed hypothetical genes with unannotated functions. The advances in proteomic technology have led to new understanding of and insights into many important proteins and their related mechanisms.Studies have shown that plant-microbe communication is a two-way interaction involving various signal molecules that cause metabolic changes in both organisms (8-10). Nevertheless, there is limited information on the plant-bacterium interaction, especially with the roots and the rhizosphere. In this study, a proteomic approach was successfully used to study the interaction between a root-associated bacterium and rice plant in the rhizosphere (11-13).In this study, a differential proteomic analysis of A. vinelandii ATCC 12837 in response to different conditions and at different locations within the Oryza sativa MR 219 rhizosphere was performed. By two-dimensional gel electrophoresis (2DE) followed by tandem mass spectrometry (MS/MS) analyses, several known and hypothetical p...

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Molecular and biochemical properties of the S-layer protein from the wine bacterium Lactobacillus hilgardii B706

et al. 2011

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Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.g. due to the production of histamine. Many lactobacilli form an S-layer as the outermost cell wall component which has been found to facilitate the colonization of special ecological niches. A detailed understanding of the properties related to their S-layer proteins is necessary to improve the knowledge of the interactions between different bacterial cells and with the surrounding environments. The S-layer protein from the wine-related L. hilgardii strain B706 has been isolated and its gene sequence determined. The deduced amino acid sequence corresponds to a 41 kDa protein with an isoelectric point of 9.6 without additional posttranslational modifications after splitting off the leader peptide. The complete protein is organized in a 32 amino acids signal sequence for membrane translocation, a positively charged N-terminal domain that binds to the cell wall and a negatively charged C-terminal domain. When the S-layer was removed, the corresponding L. hilgardii B706 cells became more sensitive to bacteriolytic enzymes and some wine-related stress conditions. From a practical point of view, the S-layer may be considered as a target for the inhibition of food-spoiling lactobacilli.

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

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.

Cited by 60 publications

References 49 publications

Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

Molecular and biochemical properties of the S-layer protein from the wine bacterium Lactobacillus hilgardii B706

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