Summary
Bacillus anthracis, the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S‐layer. An array remained when sap, a gene described as encoding an S‐layer component, was deleted. The remaining S‐layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S‐layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram‐positive surface proteins, EA1 has three 'S‐layer‐homology’motifs immediately downstream from a signal peptide. Single‐ and double‐disrupted mutants were constructed. EA1 and Sap were co‐localized at the cell surface of the wild‐type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S‐layer, and is the major cell‐associated antigen.
SummaryMany surface proteins of Gram-positive bacteria contain motifs, about 50 amino acids long, called S-layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S-layer proteins, each with three SLH motifs towards the amino-terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitro, demonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surfaceexposed levansucrase retained its enzymatic and antigenic properties.
Bacillus anthracis synthesizes two S‐layer proteins, each containing three S‐layer homology (SLH) motifs towards their amino‐terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface‐exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.
Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracissurface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-γ-d-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.
Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S‐layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three ‘S‐layer homology’ motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co‐localized at the cell surface of both the non‐capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S‐layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a Gram‐positive bacterium.
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