(A) transformation (transformation)
Transformation is the earliest form of genetic material transfer found in bacteria. 1928 Griffith's experiment of infecting mice with Streptococcus pneumoniae, the realization of Avery's transformation process in vitro after 10 years, and the confirmation of transforming factor DNA are important starting points for the development of modern life science.
1. Several Concepts
The transformed recipient bacteria directly absorb DNA fragments from the donor bacteria and integrate them into their own genome through exchange, thus obtaining new genetic characteristics.
After replication and division, the recipient cells of the transformant have the offspring of donor characteristics.
Competency: the physiological state in which bacteria can absorb DNA molecules from the surrounding environment for transformation.
(2) Transduction
1952 zinder and lederberg discovered the transduction phenomenon when verifying whether Salmonella typhimurium also has conjugation phenomenon.
The phenomenon that DNA fragments of donor cells are carried to recipient cells through completely defective or partially defective phages, and the latter obtains some genetic traits of the former through exchange integration is called transduction. Receptor cells that acquire new characteristics are called transducing cells. Phage carrying part of donor genetic material (DNA fragment) is called transduction phage. Phages containing only the DNA of donor bacteria are called completely defective phages; Phage containing both donor DNA and phage DNA is called partially defective phage (part of phage DNA is replaced by donor DNA).
According to the different ways of phage and DNA transduction, transduction can be divided into universal transduction and local transduction.
1. General transduction
The universal transduction phenomenon is realized by "packaging" any small DNA fragment of donor bacteria by completely defective phage and transferring its genetic characteristics to recipient bacteria.
The universal transduction mechanism-"packaging selection model", when phage infects sensitive bacteria and replicates and multiplies in bacteria, it also degrades host DNA into many small fragments. In the process of assembly, a few phages (10 -6- 10 -8) mistakenly packaged the host DNA fragments, which can form "phages", which are called universal transduction phages (completely defective phages). With the lysis of bacteria, a large number of transduced phages are also released. When these transduced phages infect the recipient bacteria again, the donor DNA fragments are injected into the recipient bacteria. If the DNA fragment can be paired with the homologous fragment of the DNA of the recipient strain, the gene recombination can be carried out through the double exchange of genetic material to form a stable transducer, which is called complete universal transduction. Such as P22 phage of Salmonella typhimurium, P 1 phage of Escherichia coli and PBS 1 and SP 10/phage of Bacillus subtilis. If the DNA fragment cannot be exchanged, integrated and replicated with the DNA of the recipient strain, it only exists in a free and stable state, and only carries out transcription, translation and character expression, which is called abortion. After the cell division of abortion transduction, this foreign DNA can only be distributed to one daughter cell, while the other daughter cell only obtains a small amount of products-enzymes formed by transcription and translation of donor genes, so it can still have the slight characteristics of donor bacteria in phenotype, and every division is "diluted". Therefore, the ability to form micro-colonies on selective media has become a feature of flow sensors.
2. Specialized transduction
The expression transduction phenomenon was obtained by carrying some specific genes from donor bacteria into recipient bacteria through partially defective temperate phage. The recombinant receptor cells that acquire some genetic characteristics of the donor after transduction are called localized transduction cells.
Mechanism of (1) Limited Transduction —— "Hybrid Formation Model" λ phage has a single strand of 12 nucleotides at both ends (sticky end cos site). In lysogenic state, the former phage state exists on the cell chromosome. After being induced, when the bacteria are lysed, the circular molecules formed at the sticky end form a DNA concatemer containing multiple genomes through rolling circle replication, and the size of its packaging fragment is determined by the distance between two cos sites that cut and package, and finally a transduction phage is formed. In rare cases (about 10 -5), a part of the DNA of the protophage and a section of the DNA of the bacterial host chromosome with the corresponding length are lost in the circular DNA formed by the wrong cutting of the adjacent sites at both ends of the protophage and the bacterial chromosome, so that the hybrid DNA formed in this way can be packaged and replicated normally. The newly formed transduction phage is called partially defective phage. Because both ends of λ pre-phage site are gal+ (fermentation galactose gene) and bio+ (using biotin gene) of bacterial chromosome, the formed transduction phage usually carries gal+ or bi0+ gene, so these partial defective phages are λ dga 1 (defective galactose transduction phage) or λ dbio (defective biotin transduction phage). These transduced phages can re-invade the recipient bacteria. After the invasion, phage DNA was paired with homologous fragments of recipient bacteria and integrated into the genome of recipient bacteria through double exchange, so that recipient bacteria obtained this part of the genetic characteristics of the donor.
(2) Low-frequency transduction and high-frequency transduction (LFT) in localized transduction: Because the frequency of abnormal cleavage on the host chromosome is extremely low, the proportion of some defective phages in the lysate is extremely low (10-4- 10-6), and this lysate is called LFT lysate. When LFT lysate infects the host with low m.o.i (multiplicity of infection), a very small number of transducers can be obtained. High Frequency Transduction (HFT): The frequency of transducer formation is very high, up to 50% in theory, so it is called high frequency transduction. The reason is that the donor bacterium is a diplogenic bacterium, which integrates two kinds of bacteriophages on the chromosome of the bacterium at the same time. For example, Escherichia coli K 12 strain, whose diplolytic bacteria are Escherichia coli K 12( λ/λ dg), that is, its pre-phage has λ and λ dg as defective phage, and has donor gal+ gene, but some DNA of the phage itself is lost; λ phage is a normal phage without gal gene, but it plays an auxiliary role, which can make up for the deficiency of λ dg and make λ dg a "complete phage" and be released. In this way, a bacterium can release λ dg and λ phage at the same time, and the lysate at this time is called HFT lysate. When another Escherichia coli recipient bacterium is infected with low m. o. I. HFT lysate, it can be transformed into an Escherichia coli gal+ transducer which can ferment lactose at high frequency. This method is called high frequency transduction.
When a temperate phage infects its host and makes it lysogenic, because the gene of the phage is integrated into the genome of the host, the latter gains novelty except immunity, so it is called lysogenic transformation.
(3) Conjugation
Refers to the phenomenon that the complete cells of donor bacteria and recipient bacteria directly contact to realize large-scale DNA transmission.
Iederberg and Tatum designed a famous experiment in 1946 to prove the conjugation phenomenon of prokaryotes. They screened out two kinds of K 12 mutant strains of E.coli with different auxotrophs, in which strain A was met- and bio- and strain B was thr- and Leu-, which were mixed and cultured on complete medium and then coated on basic medium. The results showed that the original colonies (about 10 -7) of met+, bi0+, thr+ and 1eu+ appeared on the basic medium, but no colonies appeared in the control group of the two parent strains coated separately. Further experiments confirmed that the formation of the above gene recombination was the result of gene recombination after the combination of two parent cells. Among bacteria, the most clearly studied conjugation phenomenon is Escherichia coli. It is found that Escherichia coli has sex differentiation, and a plasmid, namely factor F, plays a decisive role.
④ Protoplast fusion.
The protoplasts of two cells with different genetic traits were fused by artificial means, and then genetic recombination was carried out to produce a fusant with both parental traits and genetic stability. There are a wide range of cells that can fuse protoplasts, including not only prokaryotes, but also various eukaryotic cells.