Characterization of a dynamic S layer on Bacillus thuringiensis

Abstract

The surfaces of three BaciUus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and y = 73°. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pl of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential-and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize.Our increasing awareness of S layers, or surface arrays, as the outermost component of many archaebacteria and eubacteria (1,13,21,(35)(36)(37)40) exemplifies their possible importance for the vitality and integrity of free-living bacterial cells. Certainly, their importance as the single wall layer of Methanococcus, Thermoproteus, Sulfolobus, and Halobacterium spp. (1, 3, 38) cannot be denied, since this is all that stands between these bacteria and their external milieu.

A general strategy for identification of S-layer genes in the Bacillus cereus group: molecular characterization of such a gene in Bacillus thuringiensis subsp. galleriae NRRL 4045 The GenBank accession number for the slpA sequence is AJ249446.

“…Analysis of the deduced amino acid sequence showed that there was a 29 aa signal peptide followed by three SLH motifs. The mature protein thus starts, as predicted, with AGKTFPDV (Luckevich & Beveridge, 1989) and has a predicted molecular mass of 84n4 kDa, which is similar but not identical to the 91n4 kDa published (Luckevich & Beveridge, 1989). A difference between the predicted molecular mass and that deduced from electrophoretic migration has already been observed with Sap and EA1 (Etienne-Toumelin et al, 1995 ;Mesnage et al, 1997).…”

“…responding residues of Sap (Etienne-Toumelin et al, 1995 ;Luckevich & Beveridge, 1989). These residues are included in the SLH domain and in fact are, except for one conservative change, also identical to those of EA1 (Mesnage et al, 1997).…”

“…anthracis, MS confirmed the molecular mass deduced from the sequence for both Sap and EA1 proteins (O. Barzu# ,personal communication). A pI of 5n33 was calculated from the complete sequence and this is close to the value of 5n0 obtained by isoelectric focusing (Luckevich & Beveridge, 1989).…”

“…thuringiensis bacteria were grown overnight in BHI and pelleted by centrifugation. Pellets were washed and resuspended in 1\10 the initial volume of 2 M guanidinium hydrochloride (pH 2n5), as described by Luckevich & Beveridge (1989) to extract specifically the cellsurface-anchored proteins. The samples were then dialysed against Tris\HCl (pH 8n0), resuspended in Laemmli buffer (Laemmli, 1970), boiled and 20 µl was loaded onto 10 % polyacrylamide gel and subjected to electrophoresis.…”

“…thuringiensis subsp. galleriae NRRL 4045, for which biochemical characterization had been reported (Luckevich & Beveridge, 1989).…”