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Bacteriophages, structural features and practical application. Transduction

Transduction– transfer of genes from one bacterial cell to another using a bacteriophage. One gene is transduced, rarely 2 and very rarely 3 linked genes. When genetic material is transferred, a section of the phage DNA molecule is replaced. The phage then loses its own fragment and becomes defective. The inclusion of genetic material into the chromosome of the recipient bacterium is carried out by a mechanism such as crossing over. An exchange of hereditary material occurs between homologous regions of the recipient chromosome and the material introduced by the phage. There are three types of transduction: nonspecific, specific and abortive. At 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. At specific transduction The prophage is inserted into a certain place on the bacterial chromosome and transduces certain genes located in the chromosome of the donor cell next to the prophage. Abortive transduction - a fragment of a donor chromosome transferred into a recipient cell is not always included in the recipient chromosome, but can be stored in the cytoplasm of the cell (only in one of the daughter cells).

The transmission of traits by transduction has been found in many bacteria, including species Salmonella, Escherichia, Shigella, Bacillus, Pseudomonas, Staphylococcus, Vibrio And Rhizobium. But not all phages can carry out transduction, and not all bacteria can transfer DNA in this way.

Nonspecific transduction

The transfer of bacterial chromosome regions by phages was discovered in 1951 by Lederberg and Zinder Salmonella typhimurium. In a crucial experiment, a B+ donor strain was infected with the temperate bacteriophage P22. After lysis of the host cells, free phages were isolated and incubated together with a recipient strain B~, which was genetically different from the B+ strain in at least one trait. The authors found that after seeding incubated cells on a suitable medium, recombinants appeared that had characteristics of the B + donor strain.

The processes occurring during such nonspecific DNA transfer are very complex. During the reproduction of phage P22 in the cells of the B + donor strain, fragments of the bacterial chromosome may be included in the capsids instead of phage DNA. Thus, the phagolysate contains a mixture of normal and defective phages. Infection of the recipient strain B with a normal phage leads, as a rule, to cell lysis. However, some cells are penetrated by defective transducing phages, the DNA of which is capable of recombining with the recipient chromosome. An exchange of homologous DNA sections occurs, which can lead to the replacement of the defective recipient gene with an intact gene donor.

Since only small fragments of DNA are transduced, the probability of recombination affecting a particular trait is very small: it ranges from 10 ~ 6 to 10 ~ 8. It becomes clear that with the help of one particle of phage P22 Salmonella or nonspecifically transducing phage PI Escherichia coli in each case, only one gene (or several very closely located genes) can be transduced. The amount of bacterial DNA comparable to the phage genome is only 1-2% of the total amount of DNA contained in the bacterial cell. The exception is bacteriophage PBS 1 Bacillus subtilis, which can transduce up to 8% of the host genome.

Specific transduction

The best known example is transduction carried out by a phage. X(see section 4.2.2). It usually transduces only certain genes, namely gal And Yo. As already mentioned, this phage, upon transition to the prophage state, is included in a certain region of the chromosome of the host bacterium - between the genes gal And Yo. The separation of phage DNA from the bacterial chromosome (for example, as a result of UV irradiation) may not occur accurately, i.e. some fragment of it will remain in the chromosome, and closely located genes of the host cell will be captured by phage DNA. Apparently, the reason for this may be incorrect recombination.

In the case of infection with a transducing phage of cells defective in a certain gene, for example gal~, recombination can occur with the replacement of the bacteria’s own defective gene with an intact transduced gene; in this case, recombinants (transductants) are formed gal + .

Gene transfer occurs in the same way by bacteriophage Phi 80. Its DNA is included in the chromosome near the genes encoding enzymes responsible for tryptophan biosynthesis. For this reason, Phi 80 is particularly suitable for gene transfer trp.

A prerequisite for successful gene transfer during specific transduction (as opposed to nonspecific) is the integration of the phage into the genome of the host cell.

In some cases, it has been shown that the transduced DNA fragment does not recombine with the recipient chromosome, but remains outside the chromosome. In this case, the cell becomes heterozygous for the transferred genes. The transferred DNA is transcribed (indicated by the synthesis of the corresponding gene product), but is not replicated. This leads to the fact that during cell division the donor fragment passes into only one of the daughter cells (abortive transduction). If the recipient is auxotrophic, and the transferred fragment corrects the corresponding defect, then only those cells that inherited this fragment can grow; when sown on agar they form tiny colonies.

While studying bacteriophages, a phenomenon was discovered called transduction.

Transduction(from lat. transductio- movement) - the process of transferring bacterial DNA from one cell to another by a bacteriophage.

There are two types of transduction:

1. specific

2. nonspecific (general).

Nonspecific (general) transduction:

It is carried out by phage P1, which exists in the bacterial cell in the form of a plasmid, and by phages P22 and Mu, which integrate into any part of the bacterial chromosome. After prophage induction, with a probability of 10−5 per cell, erroneous packaging of a bacterial DNA fragment into the phage capsid is possible; in this case, there is no phage DNA in it. The length of this fragment is equal to the length of normal phage DNA, its origin can be anything: a random part of the chromosome, a plasmid, other temperate phages.

Once in another bacterial cell, a fragment of DNA can be incorporated into its genome, usually by homologous recombination.

Plasmids transferred by the phage are able to close into a ring and replicate in a new cell. In some cases, a DNA fragment is not integrated into the recipient chromosome and is not replicated, but is stored in the cell and transcribed. This phenomenon is called abortive transduction.

Specific transduction:

Specific transduction has been best studied using the example of phage λ. This phage is integrated into only one site (att-site) of the chromosome E. coli with a specific nucleotide sequence (homologous to the att region in the phage DNA). During induction, its exclusion may occur with an error (probability 10−3-10−5 per cell): a fragment of the same size as the phage DNA is cut out, but with the beginning in the wrong place. In this case, some of the phage genes are lost, and some of the genes E. coli is captured by him.

Each temperate phage specifically integrated into the chromosome is characterized by its own att site and, accordingly, genes located next to it that it is capable of transmitting. A number of phages can integrate into any place on the chromosome and transfer any genes through a specific transduction mechanism.

When a temperate phage carrying bacterial genes is integrated into the chromosome of a new host bacterium, it already contains two identical genes - its own and those brought from outside. Since the phage lacks part of its own genes, it often cannot be induced and reproduce. However, when the same cell is infected with a “helper” phage of the same species, the induction of a defective phage becomes possible. Both the DNA of the normal “helper” phage and the DNA of the defective phage emerge from the chromosome and replicate, along with the bacterial genes it carries.

24 . Classification of viruses

It has been established that all studied organisms are affected by viruses. Many different viruses cause disease or latently infect vertebrate and invertebrate animals, as well as protozoa, plants, fungi and bacteria. More than 4,000 different viruses are known, of which several hundred infect humans and animals.

ICTV classification:

In 1966, the International Committee on Taxonomy of Viruses adopted a system of classifying viruses based on the difference in type (RNA and DNA), number of nucleic acid molecules (single- and double-stranded) and the presence or absence of a core envelope. The classification system is a series of hierarchical taxa:

Squad ( -virales)

Family ( -viridae)

Subfamily ( -virinae)

Genus ( -virus)

View ( -virus)

Baltimore classification of viruses:

Nobel laureate biologist David Baltimore proposed his own scheme for classifying viruses based on differences in the mechanism of mRNA production. This system includes seven main groups:

(I) Viruses that contain double-stranded DNA and do not have an RNA stage (for example, herpesviruses, poxviruses, papovaviruses, mimivirus).

(II) Double-stranded RNA viruses (eg rotaviruses).

(III) Viruses containing a single-stranded DNA molecule (eg, parvoviruses).

(IV) Viruses containing a single-stranded RNA molecule of positive polarity (for example, picornaviruses, flaviviruses).

(V) Viruses containing a single-stranded RNA molecule of negative or double polarity (for example, orthomyxoviruses, filoviruses).

(VI) Viruses containing a single-stranded RNA molecule and having in their life cycle the stage of DNA synthesis on an RNA template, retroviruses (for example, HIV).

(VII) Viruses containing double-stranded DNA and having in their life cycle the stage of DNA synthesis on an RNA template, retroid viruses (for example, hepatitis B virus).

Currently, both systems are used simultaneously to classify viruses, as complementary to each other.

Modern classification:

The modern classification of viruses is universal for viruses of vertebrates, invertebrates, plants and protozoa. It is based on the fundamental properties of virions, of which the leading ones are those characterizing nucleic acid, morphology, genome strategy and antigenic properties. Fundamental properties are put in first place, since viruses with similar antigenic properties also have a similar type of nucleic acid, similar morphological and biophysical properties.

An important feature for classification, which is taken into account along with structural features, is the strategy of the viral genome, which is understood as the method of reproduction used by the virus, determined by the characteristics of its genetic material.

The modern classification is based on the following main criteria:

Type of nucleic acid (RNA or DNA), its structure (number of strands).

The presence of a lipoprotein membrane.

Viral genome strategy.

Size and morphology of the virion, type of symmetry, number of capsomeres.

Phenomena of genetic interactions.

Range of susceptible hosts.

Pathogenicity, including pathological changes in cells and the formation of intracellular inclusions.

Geographical distribution.

Transfer method.

Antigenic properties.

Human and animal viruses:

The modern classification of human and vertebrate viruses covers more than 4/5 of the known viruses, which are divided into 17 families; of these, 6 are DNA genomic viruses and 11 are RNA genomic viruses.

25 . In general, a mature viral particle (virion) consists of nucleic acid, proteins and lipids - complex viruses (clothed), or it contains only nucleic acids and proteins - simple viruses (naked).

A protein whose main role is to form a protective cover for nucleic acid. Based on the fact that the amount of genetic information in viruses is limited, Crick and Watson (1956) suggested that the protein covers of simple viruses consist of repeating subunits. Sometimes the viral protein is represented by a single type of polypeptide, but more often there are two or three. Proteins on the surface of the virion have a special affinity for complementary receptors on the surface of sensitive cells.

Lipids are found in complexly organized viruses and are mainly found in the lipoprotein shell (supercapsid), forming its lipid bilayer into which supercapsid proteins are inserted.

All complexly organized RNA-containing viruses contain a significant amount of lipids (from 15 to 35% of dry weight). Of the DNA-containing viruses, lipids contain smallpox, herpes and hepatitis B viruses. Approximately 50-60% of the lipids in viruses are phospholipids, 20-30% are cholesterol.

The lipid component stabilizes the structure of the viral particle.

The carbohydrate component of viruses is found in glycoproteins. The amount of sugars in the composition of glycoproteins can be quite large, reaching 10-13% of the virion mass. Their chemical specificity is completely determined by cellular enzymes that ensure the transfer and addition of the corresponding sugar residues. Common sugar moieties found in viral proteins are fructose, sucrose, mannose, galactose, neuraminic acid, and glucosamine. Thus, like lipids, the carbohydrate component is determined by the host cell, due to which the same virus grown in cells of different species can vary significantly in sugar composition.

26 . The genetic information encoded in a single gene can be thought of as instructions for producing a specific protein in a cell. Such an instruction is perceived by the cell if it is sent in the form of mRNA. cells whose genetic material is represented by DNA must “rewrite” this information into a complementary copy of mRNA.

First stage of replication viruses is associated with the penetration of viral nucleic acid into the host cell. This process is facilitated by special enzymes that are part of the capsid or outer shell of the virion, and the shell remains outside the cell or the virion loses it immediately after penetration into the cell. The virus finds a cell suitable for its reproduction by contacting individual sections of its capsid with specific receptors on the cell surface like a “key and lock.” If there are no specific (“recognizing”) receptors on the cell surface, then the cell is not sensitive to viral infection: the virus does not penetrate it.

In order to realize its genetic information, the viral DNA that has entered the cell is transcribed by special enzymes into mRNA. The resulting mRNA moves to the cellular “factories” of protein synthesis – ribosomes, where it replaces the cellular “messages” with its own “instructions” and is translated (read), resulting in the synthesis of viral proteins. The viral DNA itself doubles (duplicates) many times with the participation of another set of enzymes, both viral and those belonging to the cell.

The synthesized protein, which is used to build the capsid, and the viral DNA, multiplied in many copies, combine and form new, “daughter” virions. The formed viral offspring leaves the used cell and infects new ones: the virus reproduction cycle repeats.

Virus replication stages:

1. Attachment to the cell membrane-adsorption. In order for a virion to be adsorbed on the surface of a cell, it must have a protein (often a glycoprotein) in its plasma membrane - a receptor specific for a given virus. The presence of the receptor often determines the host range.

2. Penetration into the cell. At the next stage, the virus needs to deliver its genetic information inside the cell.

3. Cell reprogramming. When a cell is infected with a virus, special antiviral defense mechanisms are activated. Infected cells begin to synthesize signaling molecules - interferons, which transfer surrounding healthy cells into an antiviral state and activate the immune system. Damage caused by the virus multiplying in a cell can be detected by internal cellular control systems, and the cell will have to “commit suicide” through a process called apoptosis. Its survival directly depends on the ability of the virus to overcome antiviral defense systems.

4. Persistence. Some viruses can enter a latent state, weakly interfering with the processes occurring in the cell, and become activated only under certain conditions.

5. Creation of new viral components. Reproduction of viruses in the most general case involves three processes - 1) transcription of the viral genome - that is, the synthesis of viral mRNA, 2) its translation, that is, the synthesis of viral proteins and 3) replication of the viral genome. Many viruses have control systems that ensure optimal consumption of host cell biomaterials.

6. Maturation of virions and exit from the cell., newly synthesized genomic RNA or DNA is dressed with appropriate proteins and leaves the cell.

27 .Rhabdoviruses– a family of viruses containing a non-segmented single-stranded RNA molecule of linear form. They cause infectious diseases in vertebrates, invertebrates and plants. Viruses that infect animals are bullet-shaped, while plants are bacilli-shaped. The nucleocapsid is double-stranded, helical, in a lipoprotein shell. The virus is sensitive to the action of fat solvents, acids, and heat. Rhabdoviruses include 2 genera - vesiculoviruses and lyssaviruses. The former include viruses of the vesicular stomatitis group, the latter - viruses of the rabies group. The rhabdovirus family also includes fever viruses. Vesicular stomatitis is a viral disease of animals, sometimes affecting humans and manifesting itself as an acute self-limited influenza-like infection. Virions are bullet-shaped. The outer shell is formed by a lipid bilayer. Vesicular stomatitis virus is transmitted by mosquitoes. The virus multiplies in the body of insects. The genus Lyssavirus includes the rabies virus and rabies-like viruses (Mokola, Duvenhage - pathogenic for humans and animals;). Rabies- infectious disease of viral etiology. It is characterized by damage to the central nervous system and leads to death. People become infected by being bitten, salivated, or scratched. The incubation period ranges from 10 days to 3-4 (but more often 1-3) months. There are 3 periods of the disease: 1. period of precursors Lasts 1-3 days. Accompanied by an increase in temperature to 37.2-37.3 °C, a depressed state, poor sleep, pain at the site of the bite.2. Heightened stage (hydrophobia) Lasts 1-4 days. It is expressed in sharply increased sensitivity to the slightest irritation of the sensory organs, noise causes muscle cramps in the limbs, Patients become aggressive.3. The period of paralysis (the stage of “ominous calm”) Paralysis of the eye muscles and lower extremities occurs, lasting 5-8 days. Replication of rhabdoviruses occurs in the cytoplasm of infected cells and can occur even in cells lacking a nucleus. Replication RNA is provided by the enzymatic activity of L+ NS proteins and proceeds to form the plus strand and the replicative precursor. There are mechanisms for regulating synthesis, as a result of which minus strands of RNA are formed many times more often than plus strands, and different proteins are synthesized in different quantities. During RNA synthesis, different classes of DI particles are formed. Assembly of nucleocapsids occurs in the cytoplasm, and virions are formed on cell membranes, leaving the cell through budding.

28 . In a nucleocapsid, the interaction of nucleic acid and protein occurs along the same axis of rotation. Each virus with helical symmetry has a characteristic nucleocapsid length, width, and periodicity. Nucleocapsids Most human pathogenic viruses have helical symmetry (for example, coronaviruses, rhabdoviruses, para- and orthomyxoviruses, bunyaviruses and arenoviruses). This group also includes the tobacco mosaic virus. The organization based on the principle of helical symmetry gives viruses a rod-shaped shape. With spiral symmetry the protein cover better protects hereditary information, but requires a large amount of protein, since the coating consists of relatively large blocks.

Tobacco mosaic virus was the first virus isolated in pure form. When infected with this virus, yellow specks appear on the leaves of a diseased plant - the so-called leaf mosaic. Viruses spread very quickly either mechanically when diseased plants or plant parts come into contact with healthy plants, or through the air through smoke from cigarettes made from infected leaves.

29 . Acquired immune deficiency syndrome (AIDS) is a condition that develops against the background of HIV infection and is characterized by a decrease in the number of lymphocytes, multiple opportunistic infections, non-infectious and tumor diseases. AIDS is terminal stage HIV infection. To date, no vaccine against HIV has been created; treatment of HIV infection significantly slows down the course of the disease, but only a single case of complete cure of the disease as a result of a modified stem cell transplant is known. Routes of transmission of HIV infection: 1. Sexual 2. Injection and instrumental - when using syringes, needles, catheters contaminated with the virus 3. Hemotransfusion (after transfusion of infected blood or its components - plasma, platelet, leukocyte); 4. Perinatal (antenatal, transplacental - from an infected mother); 5. Transplantation (transplantation of infected organs, bone marrow, artificial insemination with infected sperm); 6. Milk (infection of a child with infected mother’s milk); 7. Professional and household - infection through damaged skin and mucous membranes of people in contact with blood. HIV is not transmitted through casual contact. Stages of HIV development: 1The incubation stage lasts from the moment of infection until the body reacts in the form of manifestations of an acute infection or the production of antibodies (from 3 weeks to 3 months, but in some cases it can last up to a year). Stage 2 of primary manifestations has an additional set of characteristics: acute infection, asymptomatic infection, persistent generalized lymphadenopathy (enlargement of at least two lymph nodes in two different groups, excluding inguinal lymph nodes. At the stage of acute infection, a transient decrease in T-lymphocytes is often noted, which is sometimes accompanied by the development of manifestations of secondary diseases (candidiasis, herpetic infection). These manifestations are mild, short-term and respond well to therapy (treatment). Usually the duration of the acute infection stage is 2-3 weeks, after which the disease becomes an asymptomatic infection 3). The stage usually begins to develop 3-5 years after infection. It is characterized by bacterial, fungal and viral lesions of the mucous membranes and skin, and inflammatory diseases of the upper respiratory tract. At the stage (5-7 years from the moment of infection), skin lesions are more profound and tend to be protracted. The stage (after 7-10 years) is characterized by the development of severe, secondary diseases, their generalized (general) nature, and damage to the central nervous system.

30. Paramyxoviruses (Paramyxoviridae) is a family of viruses that cause measles, mumps, parainfluenza, Newcastle disease, and distemper in dogs. Possibly causing atypical pneumonia. Virions have a spherical shape. The genome is represented by single-stranded unfragmented RNA, which limits resistance to mutation. The life cycle of parainfluenza viruses takes place in the cytoplasm of the cell; paramyxoviruses do not require primer mRNA for their transcription. Classification: The family includes the following taxa: subfamily Paramyxovirinae:genus Avulavirus - Newcastle disease virus,genus Henipavirus,genus Morbillivirus - measles virus, canine distemper virus, genus Respirovirus - human parainfluenza virus, serotypes 1 and 3, genus Rubulavirus human parainfluenza virus serotypes 2 and 4, mumps, genus TPMV-like viruses;subfamilyPneumovirinae:genus Pneumovirus- respiratory syncytial virus, genus Metapneumovirus. Replication Features: The genome is represented by one linear molecule of negative polarity, single-stranded. There are 6 genes separated by conserved non-coding regions that signal the start and end of polyadenylation. Seven proteins have been found in paramyxoviruses: NP (or N), P, M, F, L, and HN (or H or G). They are common to all genera. The HN protein ensures the attachment of virions to cells and causes the formation of VNA, which prevents the adsorption of the virus on cellular receptors. The F protein is involved in the penetration of the virus into the cell. Reproduction Paramyxoviruses occur in the cytoplasm. Virions, using the HN protein, attach to the glycolipid receptors of the cell. The F protein then fuses the viral envelope with the plasma membrane of the cell. As a result, the nucleocapsid ends up in the cell with three proteins associated with it (N, P and L), after which the transcription process begins, carried out by the virion RNA-dependent RNA polymerase. The genome is transcribed to form 6-10 discrete unprocessed mRNAs as a result of sequential discontinuous synthesis from a single promoter. A full-length copy of genomic RNA (+RNA) is also synthesized and serves as a template for the synthesis of genomic RNA (-RNA). the synthesized genomic RNAs associated with N-protein and transcriptase form nucleocapsids. Virion maturation includes:
1) introduction of viral glycoproteins into altered areas of the cell plasma membrane;
2) binding of matrix protein (M) and other non-glycosylated proteins to the altered cell membrane;
3) placement of nucleocapsid subunits under the M protein;
4) formation and release of mature virions by budding.

The most important representatives: Parainfluenza viruses are very common pathogens of acute respiratory infections. human parainfluenza virus more often affects the cells of the larynx, so the disease occurs with symptoms of laryngitis (dry painful “barking cough”, hoarse voice). In children, diseases caused by HPV are more severe and they are more likely to develop intoxication. Respiratory syncytial virus The pathogen belongs to the genus Pneumovirus of the paramyxovirus family and is one of the most common causative agents of acute respiratory diseases in children in the first years of life. measles virus- a representative of the genus Morbillivirus of the paramyxovirus family. In morphology it is almost no different from other members of the family. It lacks neuraminidase. It has hemagglutinating, hemolytic and symplastic activity. The virus has hemagglutinin, hemolysin (F), nucleoprotein (NP) and matrix protein, which differ in antigenic specificity and immunogenicity. The measles virus has serovars and shares antigenic determinants with other morbilliviruses (canine distemper virus and rinderpest virus).

31 In isometric structures, the packaging of the nucleic acid of the viral genome is complex: the nucleocapsid envelope proteins are relatively weakly associated with the nucleic acid or nucleoproteins, which imposes minimal restrictions on the way the nucleic acid is packaged. In this case, the nucleoproteins of the “core” can be very complexly organized: for example, in papovaviruses, double-stranded circular DNA, binding to histones, forms structures very similar to nucleosomes.

In such viruses, the nucleic acid is surrounded capsomeres, forming the figure of an icosahedron - a polyhedron with 12 vertices, 20 triangular faces and 30 angles. Viruses with a similar structure include adenoviruses, reoviruses, iridoviruses, herpesviruses and picornaviruses. The organization based on the principle of cubic symmetry gives viruses a spherical shape. The principle of cubic symmetry is the most economical for the formation of a closed capsid, since relatively small protein blocks are used to organize it, forming a large internal space into which the nucleic acid fits freely.

32. The life cycles of most viruses are probably similar. But they apparently penetrate the cell in different ways, since, unlike animal viruses, bacterial and plant viruses also have to penetrate the cell wall. Penetration into the cell does not always occur by injection, and the protein shell of the virus does not always remain on the outer surface of the cell. Once inside the host cell, some phages do not replicate. Instead, their nucleic acid is incorporated into the host's DNA. Here this nucleic acid can remain for several generations, replicating along with the host's own DNA. Such phages are known as temperate phages, and the bacteria in which they lurk are called lysogenic. This means that the bacterium can potentially lyse, but cell lysis is not observed until

until the phage resumes its activity. Such an inactive phage

called a prophage or provirus.

33. Structure and chemical composition. Virions are spherical in shape. In the center there is a nucleocapsid with a spiral type of symmetry, surrounded by an outer shell with styloid processes. Single-stranded “–” RNA. The nucleocapsid contains several virus-specific enzymes, including RNA polymerase. It has a supercapsid and 3 virus-specific proteins: 2 – NH glycoproteins (have hemagglutinating and neuraminidase activity), 3 – F protein (participates in the fusion of cell membranes with the viral envelope).

CLASSIFICATION OF FLU VIRUSES
All members of the orthomyxovirus family are influenza viruses. They are classified into influenza viruses of types A, B and C by the RNP antigen, which does not give cross-type serological reactions." A characteristic feature of type A influenza viruses is a change in the antigenic properties of both surface proteins (glycoproteins) hemagglutinin and neuraminidase. Numerous antigenic variants of influenza viruses." with different types of hemagglutinin and neuraminidase are isolated from domestic and wild animals. The presence of various antigenic variants required a unified classification of viruses based on the antigenic properties of hemagglutinin and neuraminidase. Since influenza virus type C differs from influenza viruses types A and B in a number of fundamental properties, it is classified as a separate genus. Although the influenza B virus has antigenic variants, there are not so many of them. they do not need classification. Unlike type A viruses, which circulate in both humans and animals, type B influenza viruses have been isolated only from humans.

34. The main feature of the viral genome is that the hereditary information of viruses can be recorded on both DNA and RNA. The genome of DNA-containing viruses is double-stranded (with the exception of parvoviruses, which have single-stranded DNA), non-segmented and exhibits infectious properties. The genome of most RNA viruses is single-stranded (the exception is reoviruses and retroviruses, which have double-stranded genomes) and can be segmented or non-segmented. Viral RNAs are divided into two groups depending on their functions. The first group includes RNAs that are capable of directly translating genetic information to the ribosomes of a sensitive cell, i.e., performing the functions of mRNA and mRNA. They are called plus-strand RNA. They have characteristic endings (“caps”) for specific recognition of ribosomes. In another group of viruses, RNA is not capable of translating genetic information directly to ribosomes and functioning as mRNA. Such RNAs serve as a matrix for the formation of mRNA, i.e., during replication, a matrix is initially synthesized ( +RNA) for the synthesis of -RNA. In viruses of this group, RNA replication differs from transcription in the length of the resulting molecules: during replication, the length of the RNA corresponds to the mother strand, and during transcription, shortened mRNA molecules are formed. The exception is retroviruses, which contain single-stranded +RNA, which serves as a template. for viral RNA-dependent DNA polymerase (reverse transcriptase). With the help of this enzyme, information is copied from RNA to DNA, resulting in the formation of a DNA provirus that integrates into the cellular genome.

35. DNA-containing viruses differ in their method of replication from RNA-containing viruses. DNA usually exists in the form of double-stranded structures: two polynucleotide chains are connected by hydrogen bonds and twisted in such a way that a double helix is formed. RNA, on the other hand, usually exists as single-stranded structures. However, the genome of some viruses is single-stranded DNA or double-stranded RNA. The first stage of viral replication is associated with the penetration of viral nucleic acid into the host cell. This process can be facilitated by special enzymes that are part of the capsid or outer shell of the virion, with the shell remaining outside the cell or the virion losing it immediately after penetration into the cell. The virus finds a cell suitable for its reproduction by contacting individual sections of its capsid (or outer shell) with specific receptors on the cell surface in a “key-lock” manner. If there are no specific (“recognizing”) receptors on the cell surface, then the cell is not sensitive to viral infection: the virus does not penetrate it. In order to realize its genetic information, the viral DNA that has entered the cell is transcribed by special enzymes into mRNA. The resulting mRNA moves to the ribosomes, resulting in the synthesis of viral proteins. The viral DNA itself doubles many times with the participation of another set of enzymes, both viral and those belonging to the cell. The synthesized protein, which is used to build the capsid, and the viral DNA, multiplied in many copies, combine and form new, “daughter” virions. The formed viral offspring leaves the used cell and infects new ones: the virus reproduction cycle repeats. Some viruses, during budding from the cell surface, capture part of the cell membrane into which viral proteins have been embedded “in advance”, and thus acquire an envelope. In some RNA viruses, the genome (RNA) can directly act as mRNA. However, this feature is characteristic only of viruses with a “+” strand of RNA (i.e., with RNA having positive polarity). For viruses with a “-” strand of RNA, the latter must first be “rewritten” into the “+” strand; Only after this does the synthesis of viral proteins begin and virus replication occurs. So-called retroviruses contain RNA as a genome and have an unusual way of transcribing genetic material: instead of transcribing DNA into RNA, as happens in a cell and is typical for DNA-containing viruses, their RNA is transcribed into DNA. The double-stranded DNA of the virus is then integrated into the chromosomal DNA of the cell. On the matrix of such viral DNA, a new viral RNA is synthesized, which, like others, determines the synthesis of viral proteins.

36. The Bunyaviridae family is considered the largest in terms of the number of viruses it contains (about 250). Transmitted by contact, airborne dust and nutritional routes. Bunyavirus virions are spherical in shape and have a diameter of 90-100 nm. The genome is formed by an RNA molecule consisting of three (L, M and S) segments. The nucleocapsid of bunyaviruses is organized according to helical symmetry. The outside of the nucleocapsid is covered with a bilayer lipid supercapsid, on which protein structures with hemagglutinating activity are located, united in the form of a surface lattice. The protein composition of different bunyaviruses varies, but all contain surface glycoproteins G1 and G2 and an internal glycoprotein associated with RNA N-protein. Most viruses contain an RNA-dependent RNA polymerase. The replication cycle of bunyaviruses occurs in the cytoplasm. Pathogens of arboviral infections: Viruses of the genus Phlebovirus cause various mosquito fevers (for example, pappataci fever, Neapolitan and Sicilian fevers, Rift Valley fever, Punta Toro fever, etc.). The genus Nairovirus includes the Crimean-Congo hemorrhagic fever virus, which causes disease in Russia, Moldova, Ukraine, the Balkans and Africa. The range of natural hosts of bunyaviruses is wide: the natural reservoir of more than half of the species is rodents, 1/4 birds and 1/4 various artiodactyls. Most bunyaviruses are transmitted by mosquitoes of the family Culicinae; over 20 types of viruses are transmitted by ticks of the families Ixodidae and Argasidae; Several viruses are carried by biting midges and mosquitoes. The Calicivirus genus of the Caliciviridae family unites viruses with a “naked” cubic capsid with a diameter of 37-40 nm. The genome of caliciviruses is formed by a +RNA molecule. Negative contrast microscopy reveals 32 cup-shaped depressions on the surface of the virions, which is why the viruses got their name [from the Greek. kalyx, bowl]. Caliciviruses do not reproduce in known cell cultures; immune electron microscopy is usually used for their diagnosis. Types of caliciviruses pathogenic to humans cause gastroenteritis and hepatitis. In addition to true caliciviruses, the genus includes the Norwalk virus and the causative agent of hepatitis E. Causative agents of gastroenteritis The pathogenesis of diseases is caused by necrotic lesions of the epithelium of the mucous membrane of the small intestine by caliciviruses, accompanied by the development of diarrhea syndrome. The incubation period of calicivirus gastroenteritis does not exceed 1-2 days; Most authors identify three main types of lesions: diseases with severe vomiting (usually observed in the winter months, more often in children); epidemic diarrhea (in adolescents and adults) and gastroenteritis (more often in children). Calicivirus gastroenteritis is accompanied by myalgia and headache; 50% of patients report moderate fever. Diarrheal syndrome with calicivirus gastroenteritis is mild - the stool is watery, without blood. After 7-10 days, spontaneous recovery occurs. Treatment of calicivirus gastroenteritis is symptomatic; There are no means of etiotropic therapy and specific prevention. The genus coronaviruses includes many important pathogenic viruses of mammals and birds that cause respiratory diseases, enteritis, polyserositis, myocarditis, hepatitis, nephritis and immunopathology. In humans, coronaviruses, together with other viruses, cause the common cold syndrome. Most coronaviruses have a pronounced tropism for epithelial cells of the respiratory tract and intestinal tract. Some coronaviruses are isolated with difficulty and only with the use of organ cultures. Representatives of the genus coronaviruses have round virions with a diameter of 80-220 nm. Coronavirus virions consist of a nucleocapsid of helical symmetry and a glycoprotein shell, on the surface of which there are characteristic, widely spaced, club-shaped protrusions 20 nm long, forming something like a solar corona. Some coronaviruses also have shortened peplomeres measuring 5 nm in length. Coronaviruses contain three or four main structural proteins: nucleocapsid protein N; major peplomeric glycoprotein S; transmembrane glycoproteins M and E. Some viruses also contain a HE protein. Toroviruses contain the same proteins as coronaviruses, but do not contain the E protein. Bovine torovirus contains HE protein (M, 65000). Among representatives of the genus coronaviruses, three antigenic groups are distinguished. The following structural proteins have been found in representatives of the genus coronaviruses. Glycoprotein S (150-180 kDa) forms large protrusions on the surface of virions. Glycoprotein S can be divided into 3 structural segments. Large outer transmembrane and cytoplasmic segments. The large outer segment, in turn, consists of two subdomains S1 and S2. Mutations in the S1 segment are associated with changes in the antigenicity and virulence of the virus. The S2 segment is more conservative. The bovine coronavirus S protein (180 kDa) is cleaved by cellular proteases into S1 and S2 during or after virion maturation, remaining non-covalently bound in virion peplomers. The breakdown of S protein in different coronaviruses depends on the cellular system. The S protein causes the formation of VNA and is responsible for the fusion of the viral envelope with the cell membrane. S protein is multifunctional.

37. A huge number of mutant forms are known for animal viruses. There are, in particular, mutants that differ in the morphology of plaques and pockmarks; host- or temperature-dependent mutants; mutants unable to induce thymidine kinase synthesis; resistant to or dependent on certain chemicals; differing in the thermosensitivity of their infectious properties or enzymatic activity, in the antigenic properties of membrane proteins, in the ability to form plaques in the presence of various inhibitors, as well as many others. For genetic studies, mutants with a clearly defined, fairly stable phenotypic trait that is easy to take into account are needed; this trait must be caused by a single mutant gene with full penetrance.

38. Temperate phages do not lyse all cells in the population; they enter into symbiosis with some of them, as a result of which the phage DNA is integrated into the bacterial chromosome. In this case, the phage genome is called a prophage. The prophage, which has become part of the cell's chromosome, replicates synchronously with the bacterial gene during its reproduction, without causing its lysis, and is inherited from cell to cell to an unlimited number of descendants. The biological phenomenon of symbiosis of a microbial cell with a temperate phage (prophage) is called lysogeny, and a bacterial culture containing a prophage is called lysogenic. This name (from the Greek lysis - decomposition, genea - origin) reflects the ability of the prophage to spontaneously or under the influence of a number of physical and chemical factors be excluded from the cell chromosome and move into the cytoplasm, i.e. behave like a virulent phage that lyses bacteria. Lysogenic cultures do not differ in their basic properties from the original ones, but they are immune to re-infection by a homologous or closely related phage and, in addition, acquire additional properties that are under the control of prophage genes. The change in the properties of microorganisms under the influence of a prophage is called phage conversion. The latter occurs in many types of microorganisms and concerns their various properties: cultural, biochemical, toxigenic, antigenic, sensitivity to antibiotics, etc. In addition, passing from an integrated state to a virulent form, a temperate phage can capture part of a cell chromosome and, when lysing the latter, transfers this part of the chromosome to another cell. If a microbial cell becomes lysogenic, it acquires new properties. Thus, temperate phages are a powerful factor in the variability of microorganisms. Temperate phages can harm microbiological production. Thus, if microorganisms used as producers of vaccines, antibiotics and other biological substances turn out to be lysogenic, there is a danger that the temperate phage will transform into a virulent form, which will inevitably lead to lysis of the production strain.

39. Retroviruses(lat. Retroviridae) - family of RNA viruses,

infecting mainly vertebrates. The most famous and active

The representative being studied is the human immunodeficiency virus. Retroviruses

with the help of which DNA is synthesized on the virion RNA matrix.

After a cell is infected with a retrovirus, synthesis begins in the cytoplasm

viral DNA-genome using virion RNA as a matrix.

All retroviruses use a reverse mechanism to replicate their genome.

transcription: viral enzyme reverse transcriptase (or revertase)

synthesizes one strand of DNA on a viral RNA template, and then on the template

the synthesized DNA strand completes the second, complementary strand.

A double-stranded DNA molecule is formed, which, having penetrated through nuclear

shell, is integrated into the chromosomal DNA of the cell and then serves as a matrix

for the synthesis of viral RNA molecules. These RNAs leave the cell nucleus and

cells are packaged in the cytoplasm into viral particles that can

infect new cells.

According to one hypothesis, retroviruses could have originated from retrotransposons-

mobile regions of the eukaryotic genome.

Classification of retroviruses

Family Retroviridae includes three subfamilies:

Oncovirinae(oncoviruses), the most important representative of which is human T-lymphotropic virus type 1;

Lentivirinae(lentiviruses), which includes HIV; And

Spumavirinae(spumaviruses, or foaming viruses).

FEDERAL AGENCY FOR EDUCATION
STATE EDUCATIONAL INSTITUTION OF HIGHER
PROFESSIONAL EDUCATION
IRKUTSK STATE UNIVERSITY
(GOU VPO ISU)
Faculty of Biology and Soil Science
Department of Microbiology

Abstract
cytology of microorganisms
Transduction and transformation in bacteria

Completed:
student gr.04331-DS
Kuznetsova E.A.
Checked: k.b. n
Makarova A.P.

Irkutsk 2012
Content

2.1 History of the study………………………………………………………………..9
2.2 Transformation in prokaryotes…………………………………….9
2.3 Stages of bacterial transformation……………………………11

Transduction in bacteria
Transduction (from Latin transduct io - movement) is the transfer by a bacteriophage into the infected cell of fragments of the genetic material of the cell that originally contained the bacteriophage. A transducing bacteriophage usually transfers only a small fragment of host DNA from one cell (donor) to another (recipient).
Both temperate phages and virulent ones are capable of transduction; the latter, however, destroy the bacterial population, so transduction with their help is not of great importance either in nature or in research.

History of the study
Esther Lederberg was the first scientist to isolate bacteriophage lambda, a DNA virus, from Escherichia coli K-12 in 1950.
The actual discovery of transduction is associated with the name of the American scientist Joshua Lederberg. In 1952, he and Norton Zinder discovered general transduction. In 1953, Lederberg et al. showed the existence of abortive transduction, in 1956 - specific.
Behavior of phages in a bacterial cell
Phages are capable of implementing two development paths in a bacterial cell:

Transfer of DNA fragments by bacteria
Nonspecific transduction.
The transfer of bacterial chromosome regions by phages was discovered in 1951 by Lederberg and Zinder in Salmonella typhimurium. In a crucial experiment, the B+ donor strain was infected with the temperate bacteriophage P22. After lysis of the host cells, free phages were isolated and incubated with a recipient B− strain, which was genetically different from the B+ strain in at least one trait. The authors found that after seeding incubated cells on a suitable medium, recombinants appeared that had characteristics of the B + donor strain.
The processes occurring during such nonspecific DNA transfer are very complex. During the reproduction of phage P22 in the cells of the B + donor strain, fragments of the bacterial chromosome may be included in the capsids instead of phage DNA. Thus, the phagolysate contains a mixture of normal and defective phages. Infection of the recipient strain B with a normal phage leads, as a rule, to cell lysis. However, some cells are penetrated by defective transducing phages, the DNA of which is capable of recombining with the recipient chromosome. An exchange of homologous DNA sections occurs, which can lead to the replacement of the defective recipient gene with an intact gene donor.
Since only small fragments of DNA are transduced, the probability of recombination affecting a particular trait is very small: it ranges from 10-b to 10-8. It becomes clear that with the help of one particle of the Salmonella phage P22 or the nonspecifically transducing phage PI of Escherichia coli, in each case only one gene (or several very closely located genes) can be transduced. The amount of bacterial DNA comparable to the phage genome is only 1-2% of the total amount of DNA contained in a bacterial cell. An exception is the Bacillus subtilis bacteriophage PBS 1, which can transduce up to 8% of the host genome.

Specific transduction.
The best known example is transduction carried out by phage X. As already mentioned, this phage, upon transition to the prophage state, is included in a certain region of the chromosome of the host bacterium. The separation of phage DNA from the bacterial chromosome (for example, as a result of UV irradiation) may not occur accurately, i.e. some fragment of it will remain in the chromosome, and closely located genes of the host cell will be captured by phage DNA. Apparently, the reason for this may be incorrect recombination.
In case of infection of cells defective in a certain gene, for example gal, by a transducing phage, recombination can occur with the replacement of the bacteria’s own defective gene with an intact transduced gene; in this case, recombinants (transductants) gal + are formed.
Gene transfer occurs in the same way by bacteriophage Phi 80. Its DNA is included in the chromosome near the genes encoding enzymes responsible for tryptophan biosynthesis. For this reason, Phi 80 is particularly suitable for trp gene transfer.
A prerequisite for successful gene transfer during specific transduction (as opposed to nonspecific) is the integration of the phage into the genome of the host cell.
In some cases, it has been shown that the transduced DNA fragment does not recombine with the recipient chromosome, but remains outside the chromosome. In this case, the cell becomes heterozygous for the transferred genes. The transferred DNA is transcribed (indicated by the synthesis of the corresponding gene product), but is not replicated. This leads to the fact that during cell division the donor fragment passes into only one of the daughter cells (abortive transduction). If the recipient is auxotrophic, and the transferred fragment corrects the corresponding defect, then only those cells that inherited this fragment can grow; when sown on agar they form tiny colonies.

Abortive transduction
During abortive transduction, the introduced donor DNA fragment is not integrated into the recipient chromosome, but remains in the cytoplasm and functions there independently. Subsequently, it is transmitted to one of the daughter cells (i.e., inherited unilineally) and then lost in the offspring.
The properties of transducing phage particles are as follows:
The particles carry only part of the phage DNA, that is, they are not functional viruses, but rather containers carrying fragments of bacterial DNA.
Like other defective viruses, the particles are unable to replicate.
Transducing phages may contain any part of the host chromosome with genes that give the recipient bacterium some advantages (for example, antibiotic resistance genes or genes encoding the ability to synthesize various substances). This acquisition of new properties by bacteria is called the phenomenon of lysogeny.
The transduction phenomenon can be used to map the bacterial chromosome if the same principles are followed as for mapping using the transformation phenomenon.

Transformation in bacteria
Transformation is the process of absorption by a cell of an organism of a free DNA molecule from the environment and its integration into the genome, which leads to the appearance in such a cell of new heritable characteristics characteristic of the DNA donor organism. Sometimes transformation is understood as any process of horizontal gene transfer, including transduction, conjugation, etc.
History of the study
The transformation was discovered in 1928, when the British scientist F. Griffith showed the possibility of transforming non-pathogenic strains of Streptococcus pneumoniae into pathogenic ones (differing in the presence of a polysaccharide capsule that allows them to attach to the tissues of higher organisms) as a result of interaction with killed cells of pathogenic strains. In 1944, O. Avery (USA) showed that to transmit a trait, it is sufficient to process the DNA of a pathogenic strain of pneumococcus. This discovery was the first evidence of the role of DNA as a carrier of heredity.
In the 1960s, the study of transformation in animals began, and in the late 1970s - in plants.

Transformation in prokaryotes
In any population, only a portion of bacteria are capable of absorbing DNA molecules from the environment. The state of cells in which this is possible is called a state of competence. Typically, the maximum number of competent cells is observed at the end of the logarithmic growth phase.
In a state of competence, bacteria produce a special low-molecular protein (competence factor) that activates the synthesis of autolysin, endonuclease I and DNA-binding protein. Autolysin partially destroys the cell wall, which allows DNA to pass through it, and also reduces the resistance of bacteria to osmotic shock. In a state of competence, the overall metabolic rate also decreases. It is possible to artificially bring cells into a state of competence. To do this, use media with a high content of calcium, cesium, rubidium ions, electroporation, or replace recipient cells with protoplasts without cell walls.
The efficiency of transformation is determined by the number of colonies grown on a Petri dish after adding 1 μg of supercoiled plasmid DNA to the cells and seeding the cells on a nutrient medium. Modern methods make it possible to achieve efficiency 10 6 -10 9 .
The absorbed DNA must be double-stranded (the efficiency of transformation of single-stranded DNA is orders of magnitude lower, but increases slightly in an acidic environment), its length must be at least 450 base pairs. The optimal pH for the process to occur is about 7. For some bacteria (Neisseria gonorrhoeae, Hemophilus), the absorbed DNA must contain certain sequences.
DNA is irreversibly adsorbed on a DNA-binding protein, after which one of the strands is cut by endonuclease into fragments 2-4 thousand base pairs long and penetrates the cell, the second is completely destroyed. If these fragments have a high degree of homology with any sections of the bacterial chromosome, it is possible to replace these sections with them. Therefore, the efficiency of transformation depends on the evolutionary distance between the donor and the recipient. The total process time does not exceed several minutes. Subsequently, during division, DNA built on the basis of the original DNA strand enters one daughter cell, and DNA built on the basis of a strand with an included foreign fragment (cleavage) enters the other.

Stages of bacterial transformation
The transformation occurs in three stages:
1) adsorption of double-stranded DNA on areas of the cell wall of competent cells;
2) enzymatic cleavage of bound DNA in some randomly located places with the formation of fragments 4-5 * 10 6 D;
3) penetration of DNA fragments with a molecular weight of at least 5 * 10 6 D, accompanied by the destruction of one of the DNA chains (the last stage is energy dependent). The penetrated DNA strand recombines with the genetic material of the recipient cell.

Conclusion
Transduction serves as an active mechanism for the formation of cultures with altered properties and can play a large role in the evolution of microorganisms. The ability to transform has been found in a number of bacterial genera, but, apparently, its role in the exchange of genetic material among bacteria under natural conditions is less significant than the role of other mechanisms, because many bacteria have special restriction and modification systems.

Literature

In general transduction, phage particles containing segments of host cell 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 cell's DNA is effectively degraded and fragments

cellular DNA, approximately the size of the phage genome, accidentally packaged into mature bacteriophage particles. As a result of subsequent infection of bacterial cells with a population of phage particles, including 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 donor DNA and the DNA of the recipient cell leads to a change in the genotype of the latter.

Each transducing phage particle typically contains only one random fragment of the original donor chromosome. The probability of including any part of the donor genome in 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 act of transduction. As a result, genes closely linked to each other on the donor chromosome are cotransduced with a high frequency, while genes distant from each other are transduced independently. Determining the frequency of gene cotransduction helps refine genetic maps by allowing the relative distances between closely linked genes to be estimated. 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 viral genome in a specific chromosomal locus of the DNA 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 cause cell death and produce infectious progeny. For this reason, the bacteriophage

capable of lysogenesis, unlike virulent phage, named moderate.

Under certain conditions - induction- the lysogenic state is interrupted and the viral genome is cut out from the host chromosome. It replicates to form many viral particles and kills the cell. Typically, excision of the viral genome occurs very accurately and the resulting phage contains a viral genome that completely matches 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 instead of some viral genes. During the next cycle of infection, the genes of the donor cell are transferred along with the phage genes into the recipient cells. After the DNA of the transducing phage is incorporated into the recipient's genome, the cell acquires, along with the phage genome, the genetic information of the previous phage host.

Thus, during specific transduction, the phage serves as a vector for transferring genes 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, when they are incorrectly excised, phages are produced that transduce different chromosomal genes. So phages lambda transduce genes responsible for galactose metabolism or genes controlling biotin synthesis, and f80 phages transduce a different number of genes encoding enzymes for tryptophan biosynthesis.

The phage genome is capable of specific transduction provided:

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

does not affect the replication of viral DNA in the 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 region (OP) and the genes necessary for replication.

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

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

Specific transduction

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

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

When a recipient cell is infected, a transducing phage integrates into its chromosome and introduces a new gene (a 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 the bacteria, to the recipient. However, lysogenic conversion can also occur if the genome of a temperate phage contains its own genes that the cell does not have, but are responsible for the synthesis of essential proteins. For example, only those diphtheria pathogens that have a moderate prophage carrying the tox operon are integrated into their chromosomes to produce exotoxin. It is responsible for the synthesis of diphtheria toxin. In other words, the temperate phage tox causes the lysogenic conversion of a nontoxigenic diphtheria bacillus into a toxigenic one.

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

b) study hereditary variability in phages based on characteristic features (size, transparency, etc.).

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), monophagous(they lyse bacteria of only one type, for example, phage Vi-I lyses only the causative agents of typhoid fever) and type-specific phages that selectively lyse certain variants of bacteria within a species. With the help of such phages, the most subtle differentiation of bacteria within a species is carried out, dividing them into phage variants. For example, using the Vi-II phage set, the causative agent of typhoid fever 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 corresponding receptors, phage typing has important diagnostic and epidemiological significance.