Protoplast formation and cell wall regeneration in Clostridium perfringens

A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts. Cell wall regeneration frequencies of up to 5% were obtained.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

Electroporation-induced transformation of intact cells of Clostridium perfringens.

Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMB1 and pHR106 resulted in 3.8 X 10(-5) and 4.2 X 10(-4) transformants per viable cell, respectively. With respect to shuttle plasmid pHR106, these values represent a greater than 100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.     

Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E).     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444.

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.