title: Are the surface layer homology domains essential for cell surface display and glycosylation of the S-layer protein from Paenibacillus alvei CCM 2051T?
abstract: Paenibacillus alvei CCM 2051(T) cells are decorated with a two-dimensional (2D) crystalline array comprised of the glycosylated S-layer protein SpaA. At its N terminus, SpaA possesses three consecutive surface layer (S-layer) homology (SLH) domains containing the amino acid motif TRAE, known to play a key role in cell wall binding, as well as the TVEE and TRAQ variations thereof. SpaA is predicted to be anchored to the cell wall by interaction of the SLH domains with a peptidoglycan (PG)-associated, nonclassical, pyruvylated secondary cell wall polymer (SCWP). In this study, we have analyzed the role of the three predicted binding motifs within the SLH domains by mutating them into TAAA motifs, either individually, pairwise, or all of them. Effects were visualized in vivo by homologous expression of chimeras made of the mutated S-layer proteins and enhanced green fluorescent protein and in an in vitro binding assay using His-tagged SpaA variants and native PG-containing cell wall sacculi that either contained SCWP or were deprived of it. Experimental data indicated that (i) the TRAE, TVEE, and TRAQ motifs are critical for the binding function of SLH domains, (ii) two functional motifs are sufficient for cell wall binding, regardless of the domain location, (iii) SLH domains have a dual-recognition function for the SCWP and the PG, and (iv) cell wall anchoring is not necessary for SpaA glycosylation. Additionally, we showed that the SLH domains of SpaA are sufficient for in vivo cell surface display of foreign proteins at the cell surface of P. alvei.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Vienna, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Janesch
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002473:Cell Wall / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D056507:Paenibacillus / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins