The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.

Across Genus Plasmid Transformation Between Bacillus subtilis and Escherichia coli and the Effect of Escherichia coli on the Transforming Ability of Free Plasmid DNA

Horizontal gene transfer (HGT) is a driving force for bacterial evolution that occurs via conjugation, transduction, and transformation. Whereas conjugation and transduction depend on nonbacterial vehicles, transformation is considered a naturally occurring process in which naked DNA molecules are taken up by a competent recipient cell. Here, we report that HGT occurred between two Bacillus subtilis strains cocultured on a minimum medium agar plate for 10 h. This process was almost completely resistant to DNase treatment and appeared to require close proximity between cells. The deletion of comK in the recipient completely abolished gene transfer, indicating that the process involved transformation. This process was also highly efficient, reaching 1.75 × 106 transformants/μg DNA compared to 5.3 × 103 and 1.86 × 105 transformants/μg DNA for DNA-to-cell transformation by the same agar method and the standard two-step procedure, respectively. Interestingly, when three distantly localized chromosomal markers were selected simultaneously, the efficiency of cell-to-cell transformation still reached 6.26 × 104 transformants/μg DNA, whereas no transformants were obtained when free DNA was used as the donor. Stresses, such as starvation and exposure to antibiotics, further enhanced transformation efficiency by affecting the donor cells, suggesting that stress served as an important signal for promoting this type of HGT. Taken together, our results defined a bona fide process of cell-to-cell natural transformation (CTCNT) in B. subtilis and related species. This finding reveals the previously unrecognized role of donor cells in bacterial natural transformation and improves our understanding of how HGT drives bacterial evolution at a mechanistic level.IMPORTANCE Because DNA is easily prepared, studies of bacterial natural genetic transformation traditionally focus on recipient cells. However, such laboratory artifacts cannot explain how this process occurs in nature. In most cases, competence is only transient and involves approximately 20 to 50 genes, and it is unreasonable for bacteria to spend so many genetic resources on unpredictable and uncertain environmental DNA. Here, we characterized a donor cell-dependent CTCNT process in B. subtilis and related species that was almost completely resistant to DNase treatment and was more efficient than classical natural transformation using naked DNA as a donor, i.e., DNA-to-cell transformation, suggesting that DNA donor cells were also important in the transformation process in natural environments.

Stress-Induced, Highly Efficient, Donor Cell-Dependent Cell-to-Cell Natural Transformation in Bacillus subtilis.

By mutagenizing with Co~(60)-γ-rays and subsequent enzyme production condition optimization, a mutant strain Bacillus licheniformis L106 was obtained from B. licheniformis C6. Around 19 000 U/mL of the enzyme productivity could usually be achieved by the strain, which was 1.9 fold higher than that achieved by the strain C6. Compared to the start strain C6, L106 has higher substantial application potential since it shortened the enzyme production period while still kept the merit of forming less spores during enzyme fermentation, and the optimal reaction pH and temperature of the produced enzyme were also unchanged.

The Breeding of Alkaline Protease High-Yielding Strain

By employing the plate transformation method, the study on Bacillus subtilis cell-to-cell natural genetic transformation was carried out The results showed that the bacteria cultured in the both LB and MM media could actively excrete transforming DNA at exponential growth stage. The cell to cell contact could facilitate the transformation. It was found that the B. subtilis strain, cultured not only in MM medium, but also in LB medium, could be transformed by cell released or chemically extracted DNA by the method of agar plate transformation, although the LB medium was previously believed not to support this process in traditional transformation protocol. Both of the transformation frequency of B. subtilis MM and LB culture peaks appeared at post exponential growth stage and reached at 3×10 -3 and 1.9×10 -6, respectively.

Shen Ping

Papers

The Breeding of Alkaline Protease High-Yielding Strain

Bacillus subtilis Undergo Cell-to-Cell Natural Transformation via DNA Releasing and Competence Acquiring