PARACRYSTALLINE SURFACE-LAYERS OF DAIRY PROPIONIBACTERIA

We examined 70 dairy propionibacteria and detected a crystalline surface layer (S-layer) in only 2 organisms (Propionibacterium freudenreichii CNRZ 722 and Propionibacterium jensenii CNRZ 87) by freeze-etching and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Both S-layers exhibited oblique (p2) symmetry (a = 9.9 nm; b = 5.4 nm; gamma = 80-degrees) and completely covered the cell surface. Treatment for 15 min at the ambient temperature with 5 M guanidine hydrochloride or acidic conditions (250 mM ammonium acetate, pH 2.7) efficiently extracted the S-layer protein from intact cells of strain CNRZ 722, whereas treatment with 5 M guanidine hydrochloride at 100-degrees-C for 15 min was necessary to isolate the S-layer protein of strain CNRZ 87. The precipitates obtained after dialysis of the extracting agents produced no regular patterns. The molecular masses of the two S-layer proteins, as estimated by SDS-PAGE, were 58.5 kDa for the strain CNRZ 722 and 67.3 kDa for the strain CNRZ 87. Mass spectrometry of the isolated S-layer protein of strain CNRZ 722 gave a molecular mass value close to the expected value (56,533 Da). The N-terminal sequences of the two purified S-layer proteins differed, as did their amino acid compositions, except that the same high hydrophobic amino acid content (52%) was observed.