Transformation of Clostridium perfringens L forms with shuttle plasmid DNA

L-form (L-phase) cultures of Clostridium perfringens were tested for their transformability with plasmid DNA. Three L-form strains were transformable, but one, strain L-13, was superior to the others. This strain was easily and reproducibly transformed with previously described shuttle vectors which were derived from either C. perfringens or Escherichia coli. Strain L-13 was transformable by a variety of methods, and a new micromethod worked well under both aerobic and anaerobic conditions. The maximal number of transformants was attained after strain L-13 was exposed for 4 h to the transforming DNA and polyethylene glycol. Viable counts determined in tubes of semisolid brain heart infusion medium containing 10% sucrose, with or without 2 micrograms of tetracycline per ml, showed a transformation rate of 3.9 X 10(-5) (transformants per viable cells).     

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.     

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.     

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.     

Role of DNase in recovery of plasmid DNA from Clostridium perfringens.

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Role of DNase in recovery of plasmid DNA from Clostridium perfringens

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum.

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Transformation of bile acids by Clostridium perfringens.

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.     

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Transformation of bile acids by Clostridium perfringens

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.     

Transformation of Azotobacter vinelandii with plasmid DNA.

Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J. Bacteriol. 139:1058-1061, 1979) for chromosomal DNA-mediated transformation. The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively. With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2). With each plasmid, the frequency of transformation was independent of the phase of the growth cycle. When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA. This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained. Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen. However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells. Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure. The present protocol should facilitate the molecular cloning of genes in Azotobacter spp.     

Transformation of Thiobacillus versutus with plasmid DNA.

The representative of the facultatively chemolithotrophic thiobacilli, Thiobacillus versutus has been successfully transformed for the first time with plasmid DNA. The plasmid used for the transformation study was pKK2, a derivative of the broad host range pSa plasmid conferring Km resistance being effectively expressed in T. versutus. Different methods inducing an artificial state of competence were tested. Transformants were obtained at the efficiency of about 10(3) per micrograms of DNA. pKK2 appeared to be compatible with T. versutus indigenous plasmids, but for stable maintenance it required constant selective pressure.     

Transformation of Bacillus polymyxa with plasmid DNA.

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Response of Clostridium perfringens and its L form to bacteriocins of C. perfringens

Clostridium perfringens strain No. 28 and its penicillin-induced stable L form were treated with 10 different bacteriocins of C. perfringens. Viable count and labelled amino acid incorporation experiments revealed that the L form was sensitive to two and possibly three bacteriocins to which the bacillus was not, while both forms were commonly sensitive to two other bacteriocins and resistant to five others. Adsorption of bacteriocin, immunity factors, or perhaps uptake of bacteriocin might be proposed to explain the responses of these organisms to bacteriocins.     

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.     

Transformation of Bacillus polymyxa with plasmid DNA

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Transformation of Dictyostelium discoideum with plasmid DNA

DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.     

Transformation of Acetobacter xylinum with plasmid DNA by electroporation.

Genetic analysis of Acetobacter xylinum, a cellulose-synthesizing bacterium, has been limited by lack of a successful transformation method. Transformation of A. xylinum was attempted using two broad-host-range plasmids (pUCD2 and pRK248) and a variety of transformation methods. Methods using CaCl2, freeze/thaw treatments, and polyethylene glycol were unsuccessful. Transformation of a cellulose-negative strain of A. xylinum with plasmid DNA has been achieved with high-voltage electroporation. Electroporation conditions of 25 microF capacitance, 2.5 kV, 400 ohms resistance, and pulse lengths of 6-8 ms were applied to a cell/DNA mixture in a 0.2-cm cuvette. Plasmid pUCD2 transformed at an efficiency of 10(6)-10(7) transformants/micrograms DNA and pRK248 yielded 10(5) transformants/micrograms DNA. The frequency of transformation increased linearly with increasing DNA concentration, while transformation efficiency remained constant. pUCD2 was recovered from transformants following chloramphenicol amplification and observed by agarose gel electrophoresis. Both plasmids could be reisolated from Escherichia coli after back-transformation with alkaline lysis DNA preparations from Acetobacter transformants. Electro-transformation of A. xylinum with plasmid DNA suggests its potential use for analysis of the A. xylinum genome.