Genetic analysis of Bacillus anthracis Sap S-layer protein crystallization domain.
Bacillus anthracis, the aetiological agent of anthrax, synthesizes two surface-layer (S-layer) proteins. S-layers are two-dimensional crystalline arrays that completely cover bacteria. In rich medium, the B. anthracis S-layer consists of Sap during the exponential growth phase. Sap is a modular protein composed of an SLH (S-layer homology)-anchoring domain followed by a putative crystallization domain (Sap c). A projection map of the two-dimensional Sap array has been established on deflated bacteria. In this work, the authors used two approaches to investigate whether Sap c is the crystallization domain. The purified Sap c polypeptide (604 aa) was sufficient to form a crystalline structure, as illustrated by electron microscopy. Consistent with this result, the entire Sap c domain promoted auto-interaction in a bacterial two-hybrid screen developed for the present study. The screen was derived from a system that takes advantage of the Bordetella pertussis cyclase subdomain structure to enable one to identify peptides that interact. A screening strategy was then employed to study Sap c subdomains that mediate interaction. A random library, derived from the Sap c domain, was constructed and screened. The selected polypeptides interacting with the complete Sap c were all larger (155 aa and above) than the mean size of the randomly cloned peptides (approx. 60 residues). This result suggests that, in contrast with observations for other interactions studied with this two-hybrid system, large fragments were required to ensure efficient interaction. It was noteworthy that only one polypeptide, which spanned aa 148-358, was able to interact with less than the complete Sap c, in fact, with itself.
Candela
Thomas
T
Unité Toxines et Pathogénie Bactérienne (CNRS, URA 2172), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Mignot
Tâm
T
Unité Toxines et Pathogénie Bactérienne (CNRS, URA 2172), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Hagnerelle
Xavier
X
Unité de Biochimie Structurale (CNRS, URA 2185), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Haustant
Michel
M
Unité Toxines et Pathogénie Bactérienne (CNRS, URA 2172), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Fouet
Agnès
A
Unité Toxines et Pathogénie Bactérienne (CNRS, URA 2172), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
D001408:Bacillus anthracis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D015723:Gene Library; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; C055764:S-layer proteins