Characterization Of The Genetic Recombination Process Between Different B. Subtilis Strains And The Defective Phage Induced From Bacillus

Horizontal gene transfer (HGT), the transfer of genes between different species, is an important mean for bacteria acquiring diverse genetic materials. There are three traditional modes of HGT:transformation, transduction, and conjugation. These three pathways are distinguished in the dependence of direct contact between donor and recipient cells and the DNase sensitivity. Natural transformation process is recognized as an important mechanism for HGT. Its occurrence involves dozens of related genes. While conjugation and transduction only related to mobile plamids and phages respectively. In the past decades, natural transformation was used as genetic tools to resolve other biology problems, and most studies of natural transformation focused on the mechanism of competence establishment and foreign DNA uptake of recipient cells. There were few reports on the functions of DNA donor in HGT. However, the free DNA fragments in the environment are limited, and dozens of genes participate in the DNA uptake process. It is incredible that recipient cell establishes complex competence to untake the limited DNA in the surrounding environment. There may exist other forms of donor DNA. To investigate the donor DNA souce and its function is essential for the interpretation of the mechanism of HGT.We have found that the genetic recombination process could occur between two different B. subtilis strains without the addition of free DNA. In this study, we further characterized the genetic recombination process between B. subtilis strains. The results showed that genetic recombination process occurred through the transfer of chromosomal DNA fragments, and this process was universal in B. subtilis strains. Besides, the genetic recombination process between B. subtilis strains was sensitive to DNase, which resembled transformation. So we further searched for the transformable DNA souce. Indeed, a13kb DNA fragment, which was considered as the donor DNA, was detected in the supernatant of donor culture. The13kb DNA fragment was produced by the defective phage PBSX, which was resident in the donor cell chromosome. PBSX can be induced by various DNA-damaging agents such as mitomycin C (MMC), ultraviolet light, and hydrogen peroxide. The release of PBSX is accompanied by the lysis of host cells. Mature phage particles have a small head and long contractile tails. DNA contained in the phage particles, which is uniform in size, consists of a group of random sequence derived from various sites of the host chromosome. Thus, PBSX cannot propagate normally, but can attack sensitive strains which do not contain the homologous phage. The trait of PBSX is similar to gene transfer agents (GATs) which could mediate HGT. Therefore, the relationship between PBSX and genetic recombination process was investigated through the over expression of PBSX and PBSX gene knock-out. The results showed that PBSX did not mediate the genetic recombination process between B. subtilis strains. Besides, the direct contact between donor and recipient cells was essential for the genetic recombination process, which made this process similar to conjugation. In addition, the viable donor cells ensured the high-efficiency of genetic recombination process. And the chromosomal DNA existed in donor cells possessed higher transformable efficiency compared with the artificially exacted chromosomal DNA. The genetic recombination process between B. subtilis strains both possessed the traits of transformation and conjugation and was designated "cell-to-cell transformation process relying on living donor cells".The genetic recombination process between B. subtilis strains was occurred on solid substrate. The number of recombinants is too enormous to count when the mixture of donor and recipient culture was directly plated on selected plates. And the number of recombinants was not proportional to the dilution when the diluted mixture was plated. To further investigate the mechanism of genetic recombination process between B. subtilis strains, we established the genetic recombination system occurred in microporous filtering film. In this syetem, mixture of donor and recipient cells was spotted on the microporous filtering film which was placed on selected plates and cultured for enough time. When the recombination process was completed, the mixture on filter film was washed and diluted to spread corresponding solid plates. The ratio of recombinants and recipient cells was designated recombination frequency which was used to estimate the recombination process. And we also detected the viable count change recipient and recombinant cells in their mixture in the culture process. The recombinants were detected after about10h, and more recombinants were detected along with the delay of culture time. The recombination frequency kept stable after20h.Although PBSX did not mediate the recombination process between B. subtilis strains, it played an important role for the reservation of bacterial DNA. To further detect the universial of PBSX-like defective phage resident in B. subtilis. We investigated39B. subtilis strains preserved in China Center for Type Culture Collection. The results showed that above60%B. subtilis strains harboured PBSX-like defective phages. Besides, diversity of the defective phages was showed in industry strains and wild type strains isolated from natural envioroment.In the above investigation process, we induced a defective phage PBP180from B. pumilus AB94180. PBP180only packaged8kb DNA fragment which was smaller than that in PBSX. PBP180had similar morphology and DNA packaging to PBSX, but their killing range was different. In addition, comparative genome analysis of PBP180, PBSX, and other PBSX-like elements in B. pumilus AB94044and SAFR-032revealed that their overall architectures were very similar, but significant differences existed in the open-reading frames encoding tail proteins that are responsible for sensitive strain recognition.In conclusion, the investigation of genetic recombination process between B. subtilis strains showed that the DNA donor cell functioned in the HGT process. It is helpful for the deeply investigation of natural HGT process to reveal the mechanism of genetic recombination process. In addition, the characterization of PBP180provides more evidence for the research of defective phage evolution and the relationship of defective phages resident in different Bacillus species.