Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Protoplast formation and regeneration of a thermophilic Clostridium sp.

Abstract Protoplasts of a thermophilic Clostridium sp. were prepared by lysozyme treatment using lactose as osmotic stabilizer. High frequency reversion (3–29.8%) to the bacillary form was obtained on hypertonic rich medium.     

Defined minimal medium for a thermophilic Bacillus sp. developed by a chemostat pulse and shift technique

Summary A new method for the design and optimization of a minimal medium is described. A chemostat pulse technique was used to identify growth limiting nutrients. By combining this pulse technique with medium shifts, the essential medium components were determined and the composition quantified. The described technique has useful applications for fastidious organisms and organisms which cannot be easily cultivated in shake flasks. A minimal medium is given for thermophilic Bacillus caldotenax, which exhibits methionine and biotin auxotrophy.     

High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA.

Summary A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycolinduced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4x10⁷ transformants per g of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

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.     

Physical map of alkaliphilic Bacillus firmus OF4 and detection of a large endogenous plasmid

Extremely alkaliphilic Bacillus firmus OF4 is among the best characterized of this group of alkaliphiles. Together with alkaliphilic Bacillus C-125 and numerous non-alkaliphilic Bacillus species whose chromosomes and gene organizations are currently being studied in detail, work on B. firmus OF4 offers the opportunity to discern whether there are features of chromosome and gene organization that are associated with alkaliphily. A physical map of the B. firmus OF4 is consistent with a circular chromosome of approximately 4 Mb, with an extrachromosomal element of 110 kb also detected. The previously identified cadmium-resistance locus and transposition functions in B. firmus OF4 were localized to the extrachromosomal element, whose genes exhibit a slightly different pattern of codon usage from chromosomal genes. No clustering of genes thus far identified with roles in alkaliphily has been found. Direct repeat sequences (DRS) were previously reported upstream of a gene encoding a Na⁺/H⁺ antiporter that has a role in pH homeostasis. In the current analyses, these sequences were found to be present in multiple copies on the chromosome, most of which are present in one 920-kb fragment. Such sequences might play a role in DNA rearrangements that allow amplification of important genes in this region. Received: March 3, 1998 / Accepted May 12, 1998     

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.     

Polyethylene glycol-facilitated transformation of Bacteroides fragilis with plasmid DNA.

A method for the transformation of Bacteroides fragilis with plasmid DNA was developed by using the clindamycin resistance plasmid pBFTM10 as the source of transforming DNA. The method was technically simple to perform and resulted in an average of 4.2 X 10(3) transformants per microgram of pBFTM10 added. A method for the preparation of frozen competent cells is also described.     

Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzymeIn partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.