Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology

Abstract

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.

Fig. 1.

Identification of a novel spore protein in Bacillus anthracis. Bacillus anthracis coat proteins from the wild-type Sterne strain were extracted from spores, subjected to 16% SDS-PAGE and stained with Coomassie Blue G-250. The band indicated by an arrowhead was cut out and analyzed using matrix-assisted laser desorption/ionization time-of-flight MS. The peptide sequence is aligned with the best-fit ORF (BAS1956) from the annotated genome of the Sterne strain (inset). Molecular weight markers are indicated in kDa.

Fig. 2.

Effect of cotβ on spore coat-surface topography. Wild-type Sterne (a and a₁) and cotβ mutant (strain MGM68) spores (b, b₁, and c) were analyzed by AFM in the tapping mode. Spore outlines and spore coat ridges imaged in (a) and (b) are indicated by white-dotted lines and arrows in (a₁) and (b₁), respectively. Scale bars represent 0.5 μm in (a), (b) and (c).

Fig. 3.

Fluorescence microscopic and Western blot analysis of Cotβ-GFP in Ames and Sterne strain spores and sporulating cells. (a) Fluorescence microscopic localization of Cotβ-GFP in strain RG134. Cells were grown at room temperature and analyzed 24 h (corresponding to stage III), 28 h (corresponding to stage IV), and 48 h (after spore release) after inoculation. Membrane staining (MEM) is shown in the upper panels, GFP fluorescence (GFP) is shown in the middle panels, and the merged (MERGE) image is shown in the lower panels. Forespore membranes are not stained due to the exclusion of the dye by the coat. The sporangia pictured in the merged images are cartooned below for clarity; spores and forespores are indicated by dark grey ovals, and mother cells are indicated by white rounded rectangles. Released spores in (a) were prepared by water washing. Fluorescence of Hypaque-purified released spores was indistinguishable from that of water-washed spores (data not shown). (b) Deconvolved micrograph from a spore from strain MGM37. (c) Spore coat extracts of cotβ-gfp fusion bearing spores, from strains Ames-JAB-10 and RG134 (lanes 1 and 2, respectively), or from the wild-type Ames strain spores (lane 3) were fractionated using SDS-PAGE and transferred to a PVDF membrane, and then probed with anti-GFP antibodies. The asterisk (*) indicates a cross-reacting species, and arrowheads indicate the Cotβ-GFP-specific doublet that is present in both fusion-bearing strains. (d) After sporulation for 2 days at room temperature, spores bearing cotβ-gfp (from strain RG134, panels 1 and 2), or bearing cotβ-gfp and a cotE mutation (from strain MGM37, panels 3 and 4) were collected and imaged by fluorescence (β-GFP) or IFM (α-GFP).