Detection of Fine Spiral Structures (Spirosomes) by Weak Sonication in Some Bacterial Strains

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.We previously found very fine spiral structures in membrane fractions from Lactobacillus fermentum and L. casei by electron microscopy, and named them spirosomes on the basis of their morphological characteristics (12). Later, we also observed spirosomes in Escherichia coli (14) and the other members of the family Enterobacteriaceae (11). Similar spiral structures had already been detected in Spirochaeta stenostrepta (6) and Clostridium botulinum type B (28). Using potassium tartrate density gradient centrifugation, we purified and characterized spirosomes from Lactobacillus brevis, indicating that the spirosomes are sensitive to proteolytic addresses: 1

mentioning

confidence: 57%

Section: Antigenicity Of Spirosomesmentioning

confidence: 99%

Comparative Characterization of Spirosomes Isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Nomura

Masuda

Kawata

1989

“…With respect to the localization of spirosome, Kawata et al suggested that it might exist in the cytoplasmic membrane of Lactobacillus (8). On the other hand, Matayoshi and Oda detected the spirosomes by electron microscopy in extracellular fluids of some bacteria which were sonicated weakly but not disrupted at all, suggesting their presence both in the cytoplasm and cell surfaces of these bacteria (15). In our study with Y. enterocolitica by use of MAb, spirosome has proved itself present mostly in the cytoplasm.…”

Section: Localization Of Spirosomementioning

confidence: 99%

“…Spirosome is a spiral inclusion found in various microorganisms, such as Lactobacillus (6), Clostridium (23), Enterobacteriaceae (8,10), Mycoplasma (11), Spirochaeta (3) and others (15). In Lactobacillus, it is composed of protein subunits with a molecular weight of 95 kDa called spirosin (24).…”

mentioning

confidence: 99%

Monoclonal Antibodies to Spirosin of Yersinia enterocolitica and Analysis of the Localization of Spirosome by Use of Them

Yamato

Takahashi

Tomotake

et al. 1994

Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0-Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies produced by them were designated MAbs-S5 and S27. They were IgG2a and IgGi, respectively, both with k light chains. MAbs-S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb-S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.

“…These results might indicate differences in the molecular structure of spirosin between Enterobacteriaceae and Lactobacillus, and even among Lactobacillus. Neither MAb reacted with the cell lysate of Bacillus and Pseudomonas, which was related to the findings that there was no spirosome found in these bacteria (6,8). As for the reactivity with V parahaemolyticus, the bacterium might well produce spirosin because it is a Gram-negative facultative anaerobic rod, as is Enterobacteriaceae.…”

Section: Resultsmentioning

confidence: 96%

Chromatographic Study of Spirosin by Use of Monoclonal Antibodies to Spirosin of Yersinia enterocolitica

Yamato

Tomotake

Shiomoto

et al. 1995

Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica . The antibodies were designated MAbs-S44 and S50. They were IgG2b and IgG2a,respectively, both with lc light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE-cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.