Rhapidosomes in Clostridium Botulinum

Spirosomes, very fine spiral particles, were isolated from a protoplastlysate of Lactobacillus brevis ATCC 8287 by differential centrifugation and purified further by potassium tartrate density gradient centrifugation. The purified spirosome preparation showed a maximum peak around 275 nm on the ultraviolet absorption spectrum and it consisted of about 94.5% protein. The buoyant density in CsCI of the spirosomes was 1.320 gjcm 3 • The spirosomes were composed mainly of a single protein (spirosin) with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the spirosomes was found to be composed predominantly of neutral amino acids accompanied by approximately equal amounts of acidic and basic amino acids. The spirosomes showed one antigenic determinant in the immunodiffusion test. The spirosomes were readily degraded by the action of proteolytic enzymes and lost their antigenicity, but they were not affected by treatmentwith either deoxyribonuclease or ribonuclease. The spiral structure of the spirosome was also found to be disintegrated by treatment with 1 M guanidine hydrochloride, 4 M urea or 0.1 % SDS, but not by the action of deoxycholate, nonionic detergents or mercaptoethanol, as observed in the electron microscope.We observed earlier a large number of very fine spiral particles in membrane preparations of Lactobacillus fermentum and Lactobacillus casei, and called them spirosomes (18). Spirosomes were also isolated from Laclobacillus brevis and partially purified and their ultrastructure was examined by electron microscopy after negative staining (16). The spirosomes consist of a left-handed helical structure about 6.5 nm in width, 12.5 nm in height and II nm in pitch, and their length ranges from 30 to 900 nm with a high frequency of 100 to 200 nm. Furthermore, we found similar spiral particles in Escherichia coli (17). Similar minute spirals were detected in Iysates of different species of Clostridium after induction with mitomycin C (31, 37). In addition, similar helical structures have been found in three species of Acholeplasma (21). From these observations spirosomes or similar spiral particles appear to exist widely as minute inclusions in both gram-positive and gram-negative bacteria.The wide distribution in bacteria and the characteristic appearance of spirosomes or similar spiral inclusions has led to speculation on their biological function. 199

supporting

confidence: 80%

Purification and Characterization of Spirosomes in Lactobacillus brevis

Ueki

Masuda

Kawata

1982

“…The spirosomes consist of a left-handed helical structure about 6.5 nm in width, 12.5 nm in height and 11 nm in pitch, and its length ranges from 30 to 900 nm with a high frequency of 100 to 200 nm. Similar minute spirals have been observed in different species of Clostridium (15,17), Escherichia coli (6), and three species of Acholeplasma (8). Thus, spirosomes or similar spiral inclusions have been widely found in gram-positive and -negative bacteria.…”

Section: Superprecipitation-like Phenomenon and Destruction Induced Bsupporting

confidence: 76%

Superprecipitation‐Like Phenomenon and Destruction Induced by Adenosine 5′‐Triphosphate in Spirosomes Isolated from Lactobacillus brevis

Kawata

Masuda

Nomura

1982

We detected very fine spirals in membrane fractions of Lactobacillus fermentum and L. casei and called them spirosomes on the basis of their morphological characteristics (7). Later, we isolated spirosomes from protoplasts of Lactobacillus brevis and examined their ultrastructure under the electron microscope (5). The spirosomes consist of a left-handed helical structure about 6.5 nm in width, 12.5 nm in height and 11 nm in pitch, and its length ranges from 30 to 900 nm with a high frequency of 100 to 200 nm. Similar minute spirals have been observed in different species of Clostridium (15, 17), Escherichia coli (6), and three species of Acholeplasma (8). Thus, spirosomes or similar spiral inclusions have been widely found in gram-positive and -negative bacteria.In a recent publication (18), we reported further purification and characterization of spirosomes isolated from L. brevis, using potassium tartrate density gradient centrifugation. The spirosome sensitive to proteolytic digestion is composed of a single protein, termed spirosin, with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar cytoplasmic helical structure was isolated from Acholeplasma laidlawii and was found to be composed of a predominant polypeptide with a molecular weight of 100,000 (8); however, no evidence could be found for inferring the possibility of a cytoskeletal role for the helical structure. The function and origin of spirosomes or similar helical structures are uncertain, but it seems likely that they are nonfunctional. In the present study, we found that spirosomes purified from L. brevis are superprecipitated as in the case of actomyosin (2, 16) and morphologically destroyed immediately after addition of adenosine 5'-triphosphate (ATP).L. brevis ATCC 8287 was grown at 37 C in the liquid medium of Efthymiou and Hansen (3) and cells were harvested at the logarithmic growth phase to form protoplasts as described previously (5). Spirosomes were isolated from the protoplast-lysate of the cells and purified further by using potassium tartrate density gradient centrifugation as described previously (18). The purified spirosome preparation was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.2) to give a concentration of 0.75 mg of protein per ml. Protein concentration was determined by the method of Lowry et al (10). All the nucleo-979

“…Spirosomes were not found in the preparations from L. casei, E. coli, and Clostridium species by our weak sonication method but they were found in the preparations of cell lysates from L. casei and E. coli C as reported by some workers (5,6,9,11). Hence, the possibility that spirosomes exist in other "spirosome-absent" strains listed in Table 1 cannot be denied.…”

Section: Discussionsupporting

confidence: 57%

“…Holt and Canale-Parola (3) observed a helical array structure on the protoplasmic cylinder of Spirochaeta stenostrepta, and separated the individual helices or segments of helices from the cell by ballistic disintegration. Ueda and Takagi (11) found similar spiral structures in mitomycin-induced lysates of Clostridium botulinu m type B. Kawata et al (6) found similar particles in the mesosomal fraction obtained from protoplasts of Lactobacillus fermenti and Lactobacillus casei by osmotic disruption and named the particle "spirosome." Since then, spirosomes or similar particles have been observed in both gram-positive and gram-negative bacteria (4,5,7,9).…”

mentioning

confidence: 97%

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

Matayoshi

Oda

1985

Fine spiral structures (spirosomes) were observed in cell suspensions of five species of bacteria just after weak sonication. The structure is morphologically indistinguishable from the spirosome reported for Lactobacillus species. The molecular weight of the protein of the spirosomes from nine strains was about 94, 000 to 95, 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The difference in the molecular weight among these spirosomes was not very great, but there were slight differences among the strains from which the spirosomes were derived.Holt and Canale-Parola (3) observed a helical array structure on the protoplasmic cylinder of Spirochaeta stenostrepta, and separated the individual helices or segments of helices from the cell by ballistic disintegration. Ueda and Takagi (11) found similar spiral structures in mitomycin-induced lysates of Clostridium botulinu m type B. Kawata et al (6) found similar particles in the mesosomal fraction obtained from protoplasts of Lactobacillus fermenti and Lactobacillus casei by osmotic disruption and named the particle "spirosome." Since then, spirosomes or similar particles have been observed in both gram-positive and gram-negative bacteria (4, 5, 7, 9). However their true origin and function are still obscure.During our investigation of the surface ultrastructure of some anaerobic bacteria, we observed many spiral structures in the bacterial cell suspension just after weak ultrasonic treatment. The spiral structures are morphologically indistinguishable from the spirosomes reported for Lactobacillus species (6). In this paper, we describe the spirosomes revealed by weak sonication in various species of bacteria. MATERIALS AND METHODSOrganisms and cultivation. The 47 strains of bacteria used in this study are listed in Table 1. L. fermentum NCTC7230 and L. casei var. casei 1001 were obtained from the Yakult Central Institute (Tokyo). Strain HD-17 is an unusually coiled anaerobic, gram-positive, nonsporeforming rod isolated from human feces (2). The five strains of Peptostreptococcus productus, the two strains of Eubacterium lentum and the 1 3