Collagen-like glycoprotein bclS is involved in the formation of filamentous structures of the Lysinibacillus sphaericus exosporium

Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.