Specific transduction in bacteria. Bacteriophages, structural features and practical application. Features of the physiology of anaerobic bacteria

Specific transduction was discovered in 1956 by M. Morse and spouses E. and J. Lederberg. characteristic feature specific transduction is that each transducing phage transmits only a certain, very limited region of the bacterial chromosome. If in generalized transduction the phage acts as a “passive” carrier of the genetic material of bacteria, and genetic recombination in the transduced bacteria occurs according to the general patterns of the recombination process, then in the case of specific transduction, the phage not only transfers the genetic material, but also provides it incorporation into the bacterial chromosome. The best known example of specific transduction is the transduction performed by the λ phage, which is capable of infecting E. coli bacterial cells with subsequent integration of its DNA into the bacterial genome. During the lysogenization of bacteria, the temperate phage λ as a result of site-specific recombination (break and cross reunion of DNA strands) is integrated into their chromosome only in one place: in the area between the bio and gal loci. This area is called attλ. Excision (excision) of the prophage from the chromosome during the induction of the prophage is also carried out according to the mechanism of site-specific recombination. Site-specific recombination occurs accurately, but not without error. Approximately once per million events during prophage excision, recombination occurs not in the attλ site, but captures the gal or bio regions. It is believed that this is due to the “wrong” formation of the loop during the disintegration of the prophage. As a result, the region of the bacterial genome adjacent to the prophage is cleaved from the chromosome and becomes part of the free phage genome. The region of the prophage genome corresponding to its location in the loop remains in the bacterial chromosome. Thus, a genetic exchange takes place between the prophage and the bacterial chromosome. The bacterial genetic material that integrates into the phage genome can replace up to 1/3 of the phage genetic material. After packaging of phage DNA, part of which is replaced by bacterial DNA, defective phage particles are formed into the phage head. The phage is defective due to the fact that the volume of the head is limited and when a bacterial DNA fragment is included in its genome, a part of the phage genome remains in the bacterial chromosome. If the defect is insignificant, then the phage remains viable, since its protein coat remains intact and ensures adsorption on cells. Such a defective phage can infect other cells, but cannot cause a reproductive infection, since the genes responsible for reproduction are absent. If in such a defective phage DNA sticky ends are preserved, which ensure its transformation into a circular form, then the DNA of the defective phage, together with a fragment of bacterial DNA, can integrate into the DNA of recipient bacteria and cause their lysogenization. defective particles containing the genes of the gal locus are formed. Such defective particles are designated λdgal (phage λ, defective, gal). If the genome of phage λ contains the gene responsible for biotin synthesis, then λdbio. Therefore, if recipient cells are treated with bio– or gal– with a phagolysate obtained after infection of donor bacteria with phage λ, which contains defective particles, transductants bio+ or gal+ are formed with a frequency of 10–5–10–6. Specific transduction in E. coli is carried out not only by the λ phage, but also by related phages called lambdoid phages, which include φ80, 434, 82, etc. In particular, the φ80 phage is incorporated into the chromosome near the genes encoding the formation of enzymes responsible for the synthesis of tryptophan. For this reason, the φ80 phage is suitable for the transfer of trp genes. It was found that the P22 phage of S. typhimurium, in addition to general transduction, can also carry out specific transduction. During the lytic cycle of development, bacteriophage P22 can carry out general transduction, while during lysogenization, it can carry out specific transduction. P22 phage DNA is integrated into a region of the chromosome next to the genes responsible for proline synthesis. Prophage integration dramatically stimulates the formation of specific transducing particles. Thus, specific transduction requires preliminary lysogenization of donor bacteria and subsequent induction of prophage from cells. The resulting defective transducing phage particles infect the cells of the recipient strain, they are lysogenized and the prophage is inserted with a portion of the bacterial genome of the donor into the recipient's chromosome. Transduction can be used in the following directions: transduce plasmids and short fragments of the donor chromosome; for the construction of strains of a given genotype, in particular isogenic strains. Here, the small size of the transferred fragments provides the advantage of transduction over conjugation. Isogenic strains constructed using generalized transduction differ only in the chromosome region carried by the transducing phage; for accurate mapping of bacterial genes, establishing the order and their location in operons, and fine structure individual genetic determinants, which is carried out using a complementation test. It is known that the synthesis of a certain group of products requires the functioning of several genes. Let us assume that the synthesis of some enzyme is determined by the products of genes a and b. Let there be two phenotypically identical mutants incapable of synthesizing the enzyme, but it is not known whether they are genetically identical or different. To identify the genotype, transduction is carried out, i.e., the phage is propagated on the cells of one population, and then the cells of the second population are infected with the phagolysate. If both large colonies of true transductants and small colonies of abortive transductants are formed upon inoculation on a selective medium, it is concluded that the mutations are localized in different genes.

The textbook consists of seven parts. Part one - "General Microbiology" - contains information about the morphology and physiology of bacteria. Part two is devoted to the genetics of bacteria. The third part - "Microflora of the biosphere" - considers the microflora of the environment, its role in the cycle of substances in nature, as well as the human microflora and its significance. Part four - "The doctrine of infection" - is devoted to the pathogenic properties of microorganisms, their role in the infectious process, and also contains information about antibiotics and their mechanisms of action. Part five - "The Doctrine of Immunity" - contains modern ideas about immunity. The sixth part - "Viruses and the diseases they cause" - provides information about the main biological properties of viruses and the diseases they cause. Part seven - "Private medical microbiology" - contains information about the morphology, physiology, pathogenic properties of pathogens of many infectious diseases, as well as modern methods of their diagnosis, specific prevention and therapy.

The textbook is intended for students, graduate students and teachers of higher medical educational institutions, universities, microbiologists of all specialties and practitioners.

5th edition, revised and enlarged

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It differs from nonspecific in that in this case, transducing phages always carry only certain genes, namely, those that are located on the chromosome of the lysogenic cell to the left of attL or to the right of attR. Specific transduction is always associated with the integration of the temperate phage into the chromosome of the host cell. When leaving (exclusion) from the chromosome, the prophage can capture the gene from the left or right flank, for example, either gal or bio. But in this case, it must lose the same size of its DNA from the opposite end, so that its total length remains unchanged (otherwise it cannot be packed into the phage head). Therefore, with this form of exclusion, defective phages are formed: ?dgal or ?dbio.

specific transduction at E. coli carries out not only the lambda phage, but also related lambdoid and other phages. Depending on the location of attB sites on the chromosome, when they are excluded, they can turn on various prophage-linked bacterial genes and transduce them into other cells. The material incorporated into the genome can replace up to 1/3 of the genetic material of the phage.

The transducing phage, in the case of infection of the recipient cell, integrates into its chromosome and introduces a new gene (new trait) into it, mediating not only lysogenization, but also lysogenic conversion.

Thus, if during nonspecific transduction the phage is only a passive carrier of genetic material, then during specific transduction, the phage includes this material in its genome and transfers it, lysogenizing bacteria, to the recipient. However, lysogenic conversion can also occur if the temperate phage genome contains its own genes that are absent in the cell but are responsible for the synthesis of essential proteins. For example, only those pathogens of diphtheria possess the ability to produce exotoxin, in the chromosome of which a moderate prophage carrying the tox operon is integrated. It is responsible for the synthesis of diphtheria toxin. In other words, the temperate tox phage causes lysogenic conversion of non-toxigenic diphtheria bacillus to toxigenic.

The agar layer method is as follows. First, a layer of nutrient agar is poured into the dish. After solidification, 2 ml of 0.7% agar, melted and cooled to 45 °C, is added to this layer, to which a drop of concentrated bacterial suspension and a certain volume of phage suspension are first added. After the top layer hardens, the cup is placed in a thermostat. Bacteria multiply inside the soft layer of agar, forming a continuous opaque background against which phage colonies are clearly visible in the form of sterile spots (Fig. 84, 2). Each colony is formed by the multiplication of one parent phage virion. The application of this method allows: a) by counting colonies, to accurately determine the number of viable phage virions in a given material;

b) by characteristics(size, transparency, etc.) to study hereditary variability in phages.

According to the spectrum of their action on bacteria, phages are divided into polyvalent(lyse related bacteria, for example, the polyvalent Salmonella phage lyses almost all Salmonella), monophages(they lyse bacteria of only one species, for example, the Vi-I phage lyses only pathogens typhoid fever) And type-specific phages that selectively lyse individual variants of bacteria within a species. With the help of such phages, the most subtle differentiation of bacteria within a species is carried out, with their division into phage variants. For example, using a set of Vi-II phages, the causative agent of typhoid is divided into more than 100 phage variants. Since the sensitivity of bacteria to phages is a relatively stable trait associated with the presence of the corresponding receptors, phage typing is of great diagnostic and epidemiological significance.

Rice. 84. Detection of bacteriophages in the test material:

1 - spot test; 2 - titration according to Grazia

In general transduction, phage particles containing segments of the host cell's DNA transfer relatively long stretches of genomic DNA from one bacterial cell to another. Transducing phage particles are formed during certain infectious processes, when the DNA of the cell is effectively degraded and fragments

cellular DNA, approximately the size of the phage genome, by chance packed into mature bacteriophage particles. As a result of the subsequent infection of bacterial cells with a population of phage particles, including those containing transducing phages, with the help of the latter, the DNA of donor cells is transferred to these infected cells. Recombination between the introduced fragments of the donor DNA and the DNA of the recipient cell leads to a change in the genotype of the latter.

Each transducing phage particle usually contains only one random fragment of the original donor chromosome. The probability of including any part of the donor genome into such a particle is approximately the same. However, due to the rather large size of the transduced DNA segments (for certain bacteriophages it is about 100 kb, or 2.5 percent of the entire E. coli chromosome), the recipient cell usually acquires a whole group of genes in one transduction event. As a result, genes that are closely linked to each other on the donor chromosome are co-transduced with a high frequency, while genes that are distant from each other are transduced independently. Determining the frequency of gene co-transduction helps refine genetic maps by allowing estimation of the relative distances between closely linked genes. 3 Specific (limited) transduction

Transduction of the second type, specific, is characteristic of temperate bacteriophages, the infectious cycle of which is interrupted as a result of the inclusion of the virus genome in a specific chromosomal DNA locus of the infected cell. Bacteria containing such integrated phage genomes are called lysogenic. They carry viral genomes as hereditary elements of their own chromosomes. In a lysogenic cell, viral and cellular genomes replicate as a single unit and are mutually compatible. Integration of the phage genome with the genome of the host cell deprives the phage of the ability to induce cell death and produce infectious progeny. For this reason, bacteriophage

capable of lysogenesis, unlike virulent phage, got the name moderate.

Under certain conditions - induction- the lysogenic state is interrupted and the viral genome is excised from the host chromosome. It replicates to form many viral particles and kills the cell. Usually, the excision of the viral genome is very precise, and the resulting phage contains a viral genome that fully corresponds to the original one.

Sometimes the phage genome is cut incorrectly and chromosomal genes are included in the daughter phage particles, adjacent to the integrated viral genome. These genes are switched on in place of some viral genes. During the next cycle of infection, the genes of the donor cell pass along with the phage genes to the recipient cells. After incorporation of the DNA of the transducing phage into the recipient genome, the cell acquires, along with the phage genome, the genetic information of the previous phage host.

Thus, in specific transduction, phage serves as a vector for gene transfer from one cell to another. Using this mechanism, only those chromosomal genes of the host cell that are closely linked to the integration site of the viral genome are transduced.

Because different temperate phages insert into different chromosomal sites, miscutting them produces phages that transduce different chromosomal genes. so phages lambda transduce the genes responsible for the metabolism of galactose, or the genes that control the synthesis of biotin, and f80 phages - a different number of genes encoding the enzymes of tryptophan biosynthesis.

The phage genome is capable of specific transduction provided that:

1 It must acquire a covalently linked non-viral DNA segment to be transduced. This segment of DNA is usually of cellular origin, but in principle it can be from any source. It can be incorporated anywhere in the viral genome, as long as it

does not affect viral DNA replication in an infected host cell or its ability to be packaged into mature phage particles.

2 The phage genome must be able to replicate after infection of the recipient cell has occurred, i.e. The viral DNA must retain the origin of replication (OP) and the genes necessary for replication to occur.

3 Phage genes encoding structural phage proteins must be functionally active.

Specific transduction is widely used in molecular genetics. Consider one example of such an application of this phenomenon. The Escherichia coli gene encoding the synthesis of the enzyme beta-galactosidase contains 3600 bp. and is one thousandth of the genome of this microorganism. If a DNA fragment of a bacterial cell encoding the synthesis of beta-galactosidase is inserted into the genome of a transducing bacteriophage lambda, it occupies one fifteenth of it, that is, the DNA of the lambda phage is enriched in the beta-galactosidase gene 100 times more than the DNA of Escherichia coli.

Transduction is the transfer of genes from one bacterial cell to another by a bacteriophage. This phenomenon was first established in 1952 by N. Zinder and J. Lederberg. They conducted studies on Salmonella typhimurium bacteria, pathogenic for mice. Two strains of these bacteria were selected: auxotrophic strain 22A, unable to synthesize tryptophan (T ~), and strain 2A, capable of synthesizing tryptophan (T 1 "). These strains were seeded in a U-shaped tube, separated at the bottom by a bacterial filter (Fig. 24 Strain 22A (T~) was inoculated into one knee of the tube, and strain 2A (T 1 ") into the other. After a certain period of incubation, the bacteria of strain 22A, when sown on a minimal nutrient medium, gave a small number of colonies (the frequency of the appearance of transduced cells was N0 ~ 5). This indicated that some cells had acquired the ability to synthesize tryptophan. How could bacteria acquire this property? Research

Rice. 24. Scheme of experiment on transduction

showed that strain 22A was lysogenic for P-22 phage. This
the phage was released from the lysogenic culture, passed through
filter and lysed strain 2A. By adding part of the genetic
of strain 2A, the Bacterial phage came back and transferred this genetic material to strain 22A. Strain 22A at
acquired specific hereditary properties of strain 2A,
in this case, the property to synthesize tryptophan. Other traits can be transduced in a similar way, including the ability
fermentation, antibiotic resistance, etc.

The phenomenon of transduction has also been established in Escherichia coli and actinomycetes. As a rule, one gene is transduced, rarely two, and very rarely three linked genes. During the transfer of genetic material, a portion of the phage DNA molecule is replaced. The phage loses its own fragment and becomes defective. The inclusion of genetic material in the chromosome of the recipient bacterium is carried out by a mechanism such as crossing over. There is an exchange of hereditary material between the homologous regions of the recipient's chromosome and the material introduced by the phage.

There are three types of transduction: general or nonspecific, specific and abortive. During nonspecific transduction during the assembly of phage particles, any of the DNA fragments of the affected bacterium can be included in their head along with phage DNA. As a result, various genes of the donor bacterium can be transferred to the recipient cells. Nonspecific transduction can be carried out by P-1 and P-22 phages in Escherichia, Shigella, and Salmonella. With specific transduction, the prophage is included in a specific place on the bacterial chromosome and transduces certain genes located on the chromosome of the donor cell next to the prophage. For example, the phage "k (lambda) in the prophage state is always included in the same place in the chromosome of Escherichia coli and transduces a locus that determines the ability to ferment galactose. When the prophages are separated from the host DNA, the bacterial genes adjacent to the prophage are cleaved along with it from the composition chromosomes, and part of the prophage genes remains in its composition.The frequency of total transduction is from 1 per 1 million to 1 per 100 million. Specific transduction occurs more often.

It has been established that a fragment of the donor's chromosome transferred into the recipient's cell is not always included in the recipient's chromosome, but can be preserved in the cytoplasm of the cell. When bacteria divide, it enters only one of the daughter cells. This condition is called abortive transduction.

Specific transduction

It differs from nonspecific in that in this case, transducing phages always carry only certain genes, namely, those that are located on the chromosome of the lysogenic cell to the left of attL or to the right of attR. Specific transduction is always associated with the integration of the temperate phage into the chromosome of the host cell. When leaving (exclusion) from the chromosome, the prophage can capture the gene from the left or right flank, for example, either gal or bio. But in this case, it must lose the same size of its DNA from the opposite end, so that its total length remains unchanged (otherwise it cannot be packed into the phage head). Therefore, with this form of exclusion, arr.

specific transduction at E. coli carries out not only the lambda phage, but also related lambdoid and other phages. Depending on the location of attB sites on the chromosome, when they are excluded, they can turn on various prophage-linked bacterial genes and transduce them into other cells. The material incorporated into the genome can replace up to 1/3 of the genetic material of the phage.

The transducing phage, in the case of infection of the recipient cell, integrates into its chromosome and introduces a new gene (new trait) into it, mediating not only lysogenization, but also lysogenic conversion.

Thus, if during nonspecific transduction the phage is only a passive carrier of genetic material, then during specific transduction, the phage includes this material in its genome and transfers it, lysogenizing bacteria, to the recipient. However, lysogenic conversion can also occur if the temperate phage genome contains its own genes that are absent in the cell but are responsible for the synthesis of essential proteins. For example, only those pathogens of diphtheria possess the ability to produce exotoxin, in the chromosome of which a moderate prophage carrying the tox operon is integrated. It is responsible for the synthesis of diphtheria toxin. In other words, the temperate tox phage causes lysogenic conversion of non-toxigenic diphtheria bacillus to toxigenic.

The agar layer method is as follows. First, a layer of nutrient agar is poured into the dish. After solidification, 2 ml of 0.7% agar, melted and cooled to 45 °C, is added to this layer, to which a drop of concentrated bacterial suspension and a certain volume of phage suspension are first added. After the top layer hardens, the cup is placed in a thermostat. Bacteria multiply inside the soft layer of agar, forming a continuous opaque background against which phage colonies are clearly visible in the form of sterile spots (Fig. 84, 2). Each colony is formed by the multiplication of one parent phage virion. The application of this method allows: a) by counting colonies, to accurately determine the number of viable phage virions in a given material;

b) by characteristic features (size, transparency, etc.) to study hereditary variability in phages.

According to the spectrum of their action on bacteria, phages are divided into polyvalent(lyse related bacteria, for example, the polyvalent Salmonella phage lyses almost all Salmonella), monophages(they lyse bacteria of only one species, for example, the Vi-I phage lyses only the causative agents of typhoid fever) and type-specific phages that selectively lyse individual variants of bacteria within a species. With the help of such phages, the most subtle differentiation of bacteria within a species is carried out, with their division into phage variants. For example, using a set of Vi-II phages, the causative agent of typhoid is divided into more than 100 phage variants. Since the sensitivity of bacteria to phages is a relatively stable trait associated with the presence of the corresponding receptors, phage typing is of great diagnostic and epidemiological significance.