In a previous study, electron microscopic examinations of thin sections of ~?obacilIus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identilied as a layer of a nonglycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (Slayer) lattice (a = 4.5 nm, 6 = 9.6 nm, y = 77 ") which completely covers the cell surface. Treatment with 5M-LiCl extracted the

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... ayer protein from intact cells efficiently and selectively. Viability did not decrease signi6cantly. Moreover, the Slayer reappeared when treated cells were allowed to grow again. I n vitm self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in viva The purified Slayer protein had a high content (44 %) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid. Author for correspondence. Tel. 9928 5334; fax 9928 5350. Abbreviations: CE, crude extract; GHCI, guanidinium hydrochloride; PCF, polycationic ferritin; S-layer, surface layer. helveticus ATCC 12046, especially since an S-layer has been detected on other strains of L. helveticus by Masuda & Kawata (1983). In this paper the presence of an S-layer on L. helveticus ATCC 12046 was confirmed by analysis of the cell surface topography using freeze-etc hing techniques. Isolation procedures and reassembly experiments in vitro are described as well as re-formation of the S-layer on intact cells after extraction. The amino acid composition and the N-terminal sequence of the purified S-layer protein were determined. Methods Bacterial strain and growth conditions. Lactobacillus helveticus ATCC 12046 was obtained from the Pasteur Institute, Paris, France, and stored at -30 "C in MRS broth (De Man et al., 1960) containing 15% (v/v) glycerol. The strain was grown unstirred in MRS medium at 43 "C after inoculation with 1 % (v/v) overnight culture. Determination of the effect of LiCl and guanidium hydrochloride (GHCI) treatment on cell viability. Viability was determined as c.f.u. on MRS agar plates after incubation for 48 h at 43 "C under anaerobic conditions. Exponential-phase cells (1.6 mg dry mass ml-' ; 6.5 x lo9 c.f.u. mt1) were incubated for 15 min at several temperatures with LiCl or guanidinium hydrochloride (GHCl) to extract the S-layer OOOI-7105 O 1992 SGM Part of this work was done during a visit by S. L. to the Zentrum fiir Ultrastrukturforschung, Vienna, Austria, which was supported by grants from INRA and from the Fonds zur Fiirderung der Wissenschaftlichen Forschung and Bundesministerium fiir Wissenschaft und Forschung in Osterreich. The kindness and excellent technical assistance of Ms Andrea Sheberl and Mrs Jutta Frank were particularly appreciated by S. L., as well as the numerous interesting discussions about cheese, bacteria and humans with Seta Kii#. 149, 527-533.