The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface

Abstract

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.

FIG. 1.

The exosporium of exsA mutants is not tightly attached. Whole spores of AM1605 (exsA) (image on left) are compared to those of the wild-type parent (image on right). Bars represent 0.2 μm.

FIG. 2.

Coat and exosporium assembly are disrupted in an exsA mutant. Sections through AM1605 (exsA) (image on left) and wild-type (image on right) spores are shown. Bars represent 0.2 μm.

FIG. 3.

Predicted sequence of ExsA from B. cereus ATCC 10876. This is laid out to emphasize the repeated regions. The residues immediately preceding interruption of the sequence in mutants AM1606 and AM1607 are underlined.

FIG. 4.

Exosporium proteins of the wild type and exsA mutants. Proteins were separated by SDS-PAGE and visualized with Sypro Ruby. Lane 1, wild type (B. cereus ATCC 10876); lane 2, AM1605; lane 3, AM1606; lane 4, AM1607; lanes 5 and 6, low- and high-range molecular mass markers (Sigma), respectively.

FIG. 5.

Western blots of SDS-PAGE-separated exosporium before and after deglycosylation. Blots were probed with an anti-BclA antibody provided by J. F. Kearney (28). Lane 1, wild type; lane 2, AM1605; lane 3, wild type, deglycosylated; lane 4, AM1605, deglycosylated. Lanes 1 and 2 contained 30 and 18 μg protein, respectively.

FIG. 6.

Glycoprotein-stained exosporium proteins. After separation by SDS-PAGE, glycoproteins were visualized on polyvinylidene difluoride blots by using the ECL glycoprotein detection kit (Amersham). Lane 1, AM1605; lane 2, wild type; lane 3, AM1605, deglycosylated; lane 4, wild type, deglycosylated.

FIG. 7.

Expression of exsA during synchronous sporulation. This was estimated using the β-galactosidase reporter activity of AM1606, which carries an exsA-lacZ transcriptional fusion. Time zero corresponds to the synchronous transfer of the cells to sporulation medium. Circles represent LacZ specific activity (picomoles of methylumbelliferone produced/minute/OD unit); open circles are for reporter strain AM1606, and closed circles are for the wild-type UM20.1 parent. Aliquots of the sporulating cells were visualized by light microscopy to estimate the percentage containing phase-grey or phase-bright forespores (represented as closed squares).