A novel spore protein, ExsM, regulates formation of the exosporium in Bacillus cereus and Bacillus anthracis and affects spore size and shape

Abstract

Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a beta-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 +/- 0.56 microm, versus that for the exsM mutant, 1.66 +/- 0.38 microm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis DeltaSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.

FIG. 1.

Fluorescence microscopic localization of ExsM-GFP during sporulation. (A) Fluorescence micrographs (magnification, ×1,000) of representative cells producing ExsM-GFP that were collected at 3 h (T3), 4 h (T4), 6 h (T6), and 8 h (T8) after sporulation. The cell membranes were stained with FM4-64 (red fluorescence). The figure also shows the merged images of the ExsM-GFP fusion protein and FM4-64 and the same field view under phase contrast. The brackets indicate individual sporangia. (B) Fluorescence and phase-contrast micrographs (magnification, ×2,000) of ExsM-GFP carrying mature spores. The white arrows point to the spore exosporium.

FIG. 2.

Ultrastructural analysis of mutant spores. (A and B) Scanning electron micrographs of B. cereus ATCC 4342 wild-type spores showing the typical morphology (A) or exsM mutant spores (B). In panel A, the white arrow points to the extended exosporium. Scale bar, 1 μm. (C, D, and E) Transmission electron micrographs of wild-type (C) or exsM mutant (D) spores. The black arrow(s) points to a single-layer exosporium in panel C or a double-layer exosporium in panel D. Panel E shows a representative exsM mutant spore stained with Alcian blue to increase contrast. The brackets denote the presence of the hair-like nap on both exosporium layers. Scale bar, 250 nm.

FIG. 3.

SDS-PAGE analysis of exosporium and exosporium plus coat proteins in wild-type and exsM mutant spores. Proteins were separated in a 4 to 20% polyacrylamide gel and stained with colloidal Coomassie blue. Panel A shows the exosporium protein profile from the B. cereus ATCC 4342 wild-type (lane 2) or exsM mutant (lane 3) spores obtained after treatment with 2% β-mercaptoethanol for 2 h at 37°C. In panel B, exosporium proteins from wild-type (lane 5) and exsM mutant (lane 6) spores were extracted by sonication and boiled in Laemmli sample buffer for 10 min. Panel C shows the exosporium and coat protein profile extracted with 8 M urea and 4% SDS from wild-type (lane 7) and exsM mutant (lane 8) spores. Lanes 1 and 4 correspond to the protein standard, and each standard molecular mass is indicated in kDa on the left. The black arrows indicate the positions of bands whose intensities changed in the exsM mutant.

FIG. 4.

Lysozyme resistance assay. Wild-type or exsM mutant spores were incubated with 250 μg/ml of lysozyme, and aliquots were taken at 0, 5, 10, 20, and 40 min. Serial dilutions of each aliquot were plated on BHI, and after overnight incubation, the survivors were enumerated. The graphic shows the mean and standard deviation (SD) of results from three independent experiments done with different preparations of wild-type and mutant spores. *, P < 0.05; **, P < 0.005 (wild-type versus exsM mutant; Student's t test).

FIG. 5.

Spore germination in the presence of l-alanine. Heat activated wild-type or exsM mutant spores were incubated in the presence of 50 mM l-alanine, and germination was monitored by loss of heat resistance at selected time points. Means and SD of results from one representative experiment performed in duplicate are shown.

FIG. 6.

Effect of DCS on spore germination. Loss of OD₅₈₀ was measured as an indicator of spore germination. Wild-type and exsM mutant spores were germinated in the presence of 50 mM l-alanine (A) or were pretreated with 1 mM DCS and then germinated with 50 mM l-alanine (B). Also, spores were germinated with 1 mM l-alanine and 1 mM inosine (C) or were pretreated with 1 mM DCS and then germinated with 1 mM l-alanine and 1 mM inosine (D). Means and SD of triplicate measurements of representative experiments are shown.

FIG. 7.

Spore outgrowth in complete medium. (A) A photograph of a representative tetrazolium overlay assay of exsM mutant and wild-type strains is shown. (B) Changes in the OD₅₈₀ were used to assess wild-type and exsM mutant spore germination and outgrowth in BHI medium. Means and SD of results from one representative experiment performed in triplicate are shown.

FIG. 8.

Ultrastructural analysis of B. anthracis exsM mutant spores. (A to C) Scanning electron micrographs of B. anthracis ΔSterne wild-type (A) or exsM mutant (B and C) spores. In panel C, the white arrows indicate spores that have partially lost their exosporium. Scale bar, 500 nm. (D and E) Transmission electron micrographs of B. anthracis ΔSterne wild-type (D) or exsM mutant (E) spores. The black arrows points to a single-layer exosporium in panel D and a double-layer exosporium in panel E. Panel F shows a representative exsM mutant spore stained with Alcian blue to increase the contrast. The brackets denote the presence of the hair-like nap on top of the exosporium in both layers. Scale bar, 250 nm.