Extracts Of Sporulating Cells Research Articles

Bacillus subtilis spoVE gene is transcribed by sigma E-associated RNA polymerase.

Expression of the Bacillus subtilis sporulation gene spoVE was examined by runoff transcription assay with an RNA polymerase preparation obtained from vegetative and sporulating cells. Transcripts from tandem promoters (P1 and P2 promoters) located just upstream of the spoVE structure gene were detected. The transcription of spoVE initiated within an hour after the onset of sporulation and coincided with the presence of RNA polymerase associated with a 33-kDa protein. Amino acid sequence analysis of the 33-kDa protein revealed that it is a sigma factor, sigma E. Reconstitution analysis of sigma E purified from the sporulating cell extracts and vegetative core RNA polymerase showed that sigma E recognizes the P2 promoter. SpoVE protein could not be synthesized in the transcription-translation coupled system prepared from vegetative cells (M. Okamoto, S. Fukui, and Y. Kobayashi, Agric. Biol. Chem. 49:1077-1082, 1985). However, addition of sigma E-associated RNA polymerase to the coupled system restored SpoVE protein synthesis. These results indicate that spoVE expression in sporulating cells is controlled essentially by sigma E-associated RNA polymerase.

A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells

Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum. A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types. The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Purification and characterization of intracellular proteases of Clostridium perfringens type A

Five intracellular proteases from sporulating cells of Clostridium perfringens type A were identified and three could be separated by DEAE-Sephacel. Two, I-A and I-B, had caseinolytic activity and one, I-C, was only active on N-benzoyl-DL-arginine-p-nitroanilide. I-A and I-B could each be further separated by Sephacryl S-300 into I-A-1 and I-A-2 and I-B-1 and I-B-2, respectively. I-A-1, a chymotrypsin-like enzyme, was the major intracellular protease, constituting 74% of the intracellular caseinolytic activity. In addition to cytoplasmic proteases both trypsin and chymotrypsin-like enzyme activity was associated with the membrane fraction. I-A-1 had a molecular weight of 330,000, with subunits of 120,000 and 138,000. I-A-1 cleaved a 1200 molecular weight peptide from C. perfringens enterotoxin. Early sporulating cell extracts of C. perfringens contained three presumptive enterotoxin precursors, which disappeared following treatment with I-A. Such cells also contained at least 10 spore coat related proteins, only one (51,500 molecular weight) of which was sensitive to I-A-1. The results indicate a possible role for the major intracellular protease in the processing of C. perfringens enterotoxin and a less important role, if any, in spore coat formation.

ISOLATION AND PURIFICATION OF Clostridium perfringens ENTEROTOXIN BY AFFINITY CHROMATOGRAPHY

ABSTRACTSporulating cells of certain strains of Clostridium perfringens produce an intracellular enterotoxin believed to be the causative agent of C. perfringens food poisoning. Rabbit antibody preparations to both vegetative and sporulating cell extracts were used to purify toxin by differential immuno‐affinity chromatography. Solid phase immune adsorbents were prepared by attaching antibody via α‐amino groups to succinylaminoethyl Sepharose‐4B to give two resins: one binding anti‐vegetative immunoglobulin G (IgG), (V‐resin), the other, antisporulating IgG, (S‐resin). Sporulating cell extract was passed through resin with bound IgG to vegetative cell extract. A large protein fraction representing antigens common to both C. perfringens forms was retained, but toxin was not measurably absorbed to the resin as determined by erythemal activity in rabbits. The toxic fraction was then passed through resin with bound IgG to sporulating cell extract. The majority of the protein did not adhere to this resin; however, that which did, showed erythemal activity in rabbits. Disc‐gel electrophoresis of the protein fraction eluted from resin with bound IgG to sporulating cell extract demonstrated the presence of five components. One elicited erythemal activity in rabbits. The maximum capacity of the V‐resin column was 102 μg of bound protein/ml of resin, and of the S‐resin column, 32 μg of bound protein/ml of resin. A 150‐fold purification was achieved by the procedure. Resins could be used repeatedly.

Antibody directed against Bacillus subtilis rho factor purified by sodium dodecyl sulfate slab gel electrophoresis. Effect on transcription by RNA polymerase in crude extracts of vegetative and sporulating cells.

Antibody directed against rho factor from vegetative Bacillus subtilis was prepared by immunizing a rabbit with denaturated rho polypeptide isolated by electrophoresis of partially purified DNA-dependent RNA polymerase on a sodium dodecyl sulfate-polyacrylamide slab gel. Antiserum to rho reacted specifically with native rho polypeptide but not with core RNA polymerase as judged by complement fixation and by an immunodiffusion assay. Anti-rho antibody also inhibited the ability of rho to stimulate transcription of phage phie DNA but failed to inhibit transcription of poly(dA-dT) by core enzyme. Specific antibody was also raised against a mixture of the beta and beta' subunits of RNA polymerase purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The effect of the anti-rho gamma-globulin on the transcription of phage phie DNA by RNA polymerase in crude extracts of vegetative and sporulating cells was examined. Anti-rho antibody markedly inhibited the transcription of phage DNA by RNA polymerase partially purified from vegetative bacteria by ammonium sulfate fractionation but had little effect on transcription of the phage DNA template by enzyme from sporulating cells. Addition of purified rho to a vegetative extract that had been depleted of rho by treatment with the anti-rho antibody restored active transcription of phage DNA. However, addition of purified rho to an antibody-treated extract of sporulating cells had little effect on phie RNA synthesis. These findings suggest that sporulating cells contain a component that interferes with the activity of the rho subunit of RNA polymerase.

RADIOSENSITIVITY OF SEVERAL DEHYDROGENASES AND TRANSAMINASES DURING SPOROGENESIS OF BACILLUS SUBTILIS.

Rowley, D. B. (Syracuse University, Syracuse, N.Y.), and H. R. Newcomb. Radiosensitivity of several dehydrogenases and transaminases during sporogenesis of Bacillus subtilis. J. Bacteriol. 87:701-709. 1964.-Cells of Bacillus subtilis grown in a chemically defined medium for 17 hr, subsequently washed, and transferred to a phosphate solution containing CaCl(2) sporulated in a reproducible pattern. The synthesis of dipicolinic acid essentially paralleled the appearance of thermoresistant cells. X-ray resistant cells appeared 2 to 3 hr before the appearance of thermoresistant cells and the formation of dipicolinic acid. Changes in enzyme activity of cells undergoing sporogenesis varied among the enzymes studied. The transaminases exhibited a marked decrease in activity during early sporogenesis, whereas the dehydrogenases exhibited maximal activity. The determination of enzyme radioresistance in extracts of sporulating cells indicated that the resistance of transaminases remained essentially unchanged, whereas the dehydrogenases exhibited a decreased resistance early in sporogenesis. Although both glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase (GOT) were demonstrated in vegetative cells, only the latter was detected in mature spores. The radioresistance of GOT in vegetative-cell extracts was two to three times greater than in spore extracts.