Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene

Abstract

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp␣ protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.Bacillus anthracis, the causative agent of anthrax, is a grampositive, spore-forming bacterium. Fully virulent bacilli are both capsulated and toxinogenic. The two toxins (lethal and edema toxins) are encoded by the virulence plasmid pXO1, and capsule synthesis is dependent on the presence of a second virulence plasmid, pXO2. When the capsule is absent, the cell wall of B. anthracis appears layered and is composed of small fragments displaying a highly patterned ultrastructure (8). Holt and Leadbetter (9) previously described a hexagonal lattice on the surface of vegetative cells of B. anthracis. This cell surface structure most likely represents what is called an S-layer, with p6 symmetry and a center-to-center spacing of the particles of 7 to 10 nm. S-layers, or surface arrays, have been found to be the outermost component of many archaebacteria and eubacteria. In most cases, single proteins compose these structures. When present, they represent 5 to 10% of the total cell protein, implying that their synthesis is energy-consuming for the bacterium. The fact that S-layers are found ubiquitously suggests that they play vital roles in the interaction between the cell and its environment. It has been suggested that the S-layer is an important virulence factor for bacteria such as Aeromonas salmonicida, Campylobacter fetus, and Rickettsia spp., protecting against complement killing, facilitating binding of the bacterium to host molecules, or enhancing its ability to associate with macrophages (see reference 21).Unlike most S-layers, that of B. anthracis is not the outermost component of the virulent bacilli since they are encapsulated; Azotobacter spp. are another of the rare examples of bacteria possessing both a capsule and an S-layer. The S-layer may have an important function in linking the capsule to the peptidoglycan wall or controlling the exchange of molecules with the environment. Bacillus thuringiensis, a closely related entomopathogen bacillus, possesses an S-layer (12). The B. thuringiensis S-layer is composed of linear arrays of sm...

A general strategy for identification of S-layer genes in the Bacillus cereus group: molecular characterization of such a gene in Bacillus thuringiensis subsp. galleriae NRRL 4045 The GenBank accession number for the slpA sequence is AJ249446.

“…The mature protein thus starts, as predicted, with AGKTFPDV (Luckevich & Beveridge, 1989) and has a predicted molecular mass of 84n4 kDa, which is similar but not identical to the 91n4 kDa published (Luckevich & Beveridge, 1989). A difference between the predicted molecular mass and that deduced from electrophoretic migration has already been observed with Sap and EA1 (Etienne-Toumelin et al, 1995 ;Mesnage et al, 1997). Furthermore, in Bac.…”

“…responding residues of Sap (Etienne-Toumelin et al, 1995 ;Luckevich & Beveridge, 1989). These residues are included in the SLH domain and in fact are, except for one conservative change, also identical to those of EA1 (Mesnage et al, 1997).…”

“…cereus group, except in Bac. anthracis (Etienne-Toumelin et al, 1995 ;Mesnage et al, 1997). In that organism, there are two S-layer proteins, Sap and EA1, and their genes were disrupted independently and simultaneously (Etienne-Toumelin et al, 1995 ;Mesnage et al, 1997).…”

Analysis of the structure–function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms

“…In contrast to Sap of B. anthracis (Etienne-Toumelin et al, 1995) and CTC of Bacillus thuringiensis (M. Sun & Z. Yu, GenBank accession no. AJ012290), SbsC possesses a longer N-terminal part and no SLH motifs, as well as a longer C-terminal part.…”

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

“…S-layer proteins are initially synthesized in the bacterial cytoplasm as precursors with Nterminal signal peptides, which are removed during translocation across the plasma membrane (2). Six consecutive immunoglobulin-like domains of Geobacillus stearothermophilus S-layer protein SbsB, organized into -shaped, disk-like monomeric crystallization units, are stabilized by interdomain calcium ion coordination, which functions as a switch for the formation of a condensed quaternary structure in the mature S-layer (3).…”