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.     

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.     

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.     

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.     

Improvements in the transformation of Bacillus subtilis protoplasts with plasmid DNA

Abstract The transformation system currently used for Bacillus subtilis protoplasts has been improved. Special emphasis was made on three parameters of practical importance:
(a) conditions for direct selection of transformants, (b) optimization of the transformation system for Rec⁻ mutants, and (c) conservation of protoplast suspensions for further use.
Selective regeneration was efficiently achieved for kanamycin or neomycin. Chloramphenicol, tetracycline and erythromycin were only expressed when low concentrations of the antibiotics were used to select transformants during regeneration.     

The Na+/H+ antiporter of alkaliphilic Bacillus sp.

The Na⁺/H⁺ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily. The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electro-genic Na⁺/H⁺ antiport activity driven by ΔΨ (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions. On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na⁺/H⁺ antiport activity driven by ΔΨ. Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4). Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product. It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus. These homologues are present as a cluster of seven ORFs in each case. The shaA gene product of B. subtilis shows significant similarity to the ORF-1 product of strain C-125. Disruption of the shaA gene resulted in a decrease in Na⁺/H⁺ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na⁺ concentration was increased. We conclude that the shaA gene encodes a Na⁺/H⁺ antiporter, which plays an important role in extrusion of cytotoxic Na⁺. Received: May 29, 2000 / Accepted: July 18, 2000     

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.     

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.     

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     

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     

嗜碱芽孢杆菌Bacillus sp. N16-5表达载体的构建

【目的】构建可以在嗜碱芽孢杆菌Bacillus sp.N16-5中表达外源基因的表达载体。【方法】以枯草芽孢杆菌表达质粒pHCMC04为基本骨架,删除木糖诱导的启动子和木糖阻遏蛋白基因,分别插入枯草芽孢杆菌组成型强启动子P43和嗜碱芽孢杆菌N16-5的组成型启动子P EF,构建表达载体pABN165P43和pABN165P EF;将绿色荧光蛋白基因gfp作为报告基因连接至表达载体得到pABN165P43-gfp和pABN165P EF-gfp,利用原生质体转化法将其转化至Bacillus sp.N16-5,通过荧光显微镜和多功能荧光读板仪检测报告基因的表达情况。【结果】所构建的表达载体pABN165P43-gfp和pABN165P EF-gfp可以在Bacillus sp.N16-5中表达报告基因gfp,荧光定量数据显示,在7.5 h左右开始检测到绿色荧光蛋白的表达,从7.5 h到12 h荧光强度随时间增长迅速并在12 h左右达到最大,最大荧光值在7000左右。【结论】成功构建了2个嗜碱芽孢杆菌Bacillus sp.N16-5表达载体,实现了外源基因在嗜碱芽孢杆菌中的表达。     

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.