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Biotechnology |
Centre for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria1
Institute of Applied Microbiology, University of Agricultural Sciences, 1190 Vienna, Austria2
Author for correspondence: Margit Sára. Tel: +43 1 47 654 2208. Fax: +43 1 47 89 112. e-mail: sara{at}edv1.boku.ac.at
The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31–1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated high-molecular-mass exoamylase. To elucidate the structure–function relationship of distinct segments of SbsC, three N- and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC31–1099. With the various truncated forms it could be demonstrated that the N-terminal part (aa 31–257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of in vitro double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of Streptococcus gordonii. In contrast to the self-assembling N-terminal truncation rSbsC258–1099, two further N-terminal truncations (rSbsC343–1099, rSbsC447–1099) and three C-terminal truncations (rSbsC31–713, rSbsC31–844, rSbsC31–860) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC31–880, rSbsC31–900 and rSbsC31–920 revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.
Keywords: heterologous expression, self assembly, peptidoglycan, secondary cell wall polymer, exoamylase
Abbreviations: GHCl, guanidine hydrochloride; GPC, gel-permeation chromatograpy; HMMA, high-molecular-mass exoamylase; SLH, S-layer homologous
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