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Genetics of bacteria. Hereditary apparatus of bacteria

Modern biology and genetics owe their outstanding achievements to microbiology, which provided them with microorganisms as experimental subjects. The importance and relevance of research in the field of genetics of microorganisms lies primarily in the fact that they were the first to create methods for controlling heredity.

In 1865, Czech scientist Gregor Mendel discovered the existence of genes as discrete units of heredity. In 1928, F. Griffiths was the first to transform nonvirulent pneumococci into virulent ones. He infected white mice with a mixture of bacteria consisting of heat-killed capsular pneumococci, which therefore lost their virulence, and live acapsular, nonvirulent pneumococci. In control experiments, these groups of bacteria, introduced separately, did not kill the mice. However, in the experimental group, the mice died and live pneumococci were isolated from their blood, having acquired the capsule of killed pneumococci. Consequently, killed capsular pneumococci contained a substance capable of transmitting the sign of capsule formation (virulence) to living pneumococci. The mechanism of transformation remained unknown.

In 1953, F. Crick and D. Watson determined the gene structure based on the double helix of DNA. This discovery showed how the gene performs its three most important functions:

  • 1) continuity of heredity - semi-conservative mechanism of DNA replication;
  • 2) control of the structures and functions of the body - using a genetic code that uses a reserve of only four bases (letters) - adenine (A), thymine (T), guanine (G), cytosine (C);
  • 3) the evolution of organisms due to mutations and genetic recombinations.

Through the works of F. Crick, S. Brenner, M. Nirenberg, S. Ochoa, X. Korana, using microbial objects, by 1966 the genetic code was deciphered, its triplicity, non-overlapping and universality for all living organisms were shown.

Scientists appreciated the ease of working with bacteria and viruses due to their properties: short generation period, rapid accumulation of populations with a huge number of individuals, ease of cultivation and use. In bacterial objects, messenger, ribosomal, and transfer RNAs were discovered and the mechanism of protein synthesis was established. D. Lederberg and E. Tatum discovered conjugation in bacteria, V. Hayes discovered a plasmid that determines the sexual polarity of bacteria. F. Jacob and E. Wolman created the theory of plasmids. The concept of the operon, developed on the Escherichia coli model by F. Jacob and J. Monod, has become a universal concept for the genetic control of gene expression.

In 1972, genetic engineering arose and is rapidly developing. Of key importance for its occurrence and control of heredity was the discovery in 1970 by G. Temin, S. Mizutani, D. Baltimore of the enzyme reverse transcriptase (revertase, RNA-dependent DNA polymerase) in some oncogenic viruses. This made it possible to obtain a copy DNA gene on a messenger RNA matrix and use it in genetic engineering. Biotechnology based on genetic engineering widely uses bacterial enzymes, plasmid and viral vectors, as well as bacteria as the main producers of biological products.

Prokaryotes (bacteria) have morphologically distinct cellular structures containing genetic information - nucleoids. The nucleoid consists of one coiled chromosome, which is freely located in the cytoplasm, but is associated with certain receptors on the cytoplasmic membrane. Therefore, a bacterial cell, unlike eukaryotes, is haploid, i.e. contains one set of genes.

Under some conditions, bacterial cells can contain copies of a chromosome that are completely identical in their set of genes, and the bacteria remain haploid. Unlike all other organisms, bacteria have the unique property of changing the mass of their DNA, regulating the content of copies of their genes depending on living conditions, equivalent to the mass of 2, 4, 6, 8 chromosomes. This allows them to regulate the rate of their own reproduction - one of the main conditions that ensure the survival of bacteria in the environment, and therefore the preservation of the species in nature.

The bacterial chromosome is a double-stranded DNA molecule (circular chromosome) containing genes arranged in a linear order. Since the length of the chromosome (y E. coli about 1000 µm) is many times longer than the length of a bacterium (1.5-3.0 µm on average), the chromosome is compactly packed in a supercoiled form in the form of 12-80 loops associated with a core structure represented by a special class of RNA - 4.5 S RNA. The genome (the entire set of nucleotides contained in the chromosome of a given individual) and genotype (the entire set of individual genes in a given individual) in bacteria are not unambiguous, but are close, since most genes are contained in the chromosome in the singular, unlike eukaryotes containing up to 30-50% of repeated nucleotide sequences in the genome. Therefore, the size of genomes in bacteria, viruses, and plasmids is expressed either in molecular weight, or in the number of nucleotide pairs of the genomic nucleic acid, or in the number of genes. These values ​​are comparable, since on average each gene consists of 1000 nucleotide pairs, and the mass of one DNA nucleotide is 500 daltons. Yes, chromosome E. coli has a molecular weight of 2.8 x 10 9 daltons, the number of nucleotide pairs is 3.8 x 10 6 and contains 2500-3000 genes.

The bacterial chromosome consists of two types of genes: structural (cistrons), encoding the synthesis of a specific polypeptide chain, and regulatory (or acceptor), regulating the activity of genes (regulators, operators, promoters, attenuators, terminators, etc.). The main structural and functional unit of a chromosome is the operon. This is a group of structural cistron genes physically linked to each other and to the operator gene that controls their expression. In turn, an operon or a group of them is under the control of one gene-regulator, which represents a more complex structural and functional unit - a regulon.

Genes on a chromosome are arranged linearly, so you can study their sequence and draw up a chromosomal (genetic) map. To do this, the time of transfer of the corresponding genes during bacterial conjugation is studied. The localization of genes on the chromosome is determined in minutes of their transfer, in particular for Escherichia coli - from 0 to 100 minutes.

Currently, the main method for studying the organization of genomes of living organisms is sequencing - determining the sequence of nucleotides in the DNA of genes. First, using the cloning technique, a large number of necessary DNA fragments are obtained. The nucleotide sequence of DNA chromosomes of 20 most important bacteria (K pest is, E. coli, P. aeruginosa etc.).

Some genes and gene groups of bacterial chromosomes and bacterial plasmids belong to transposable genetic elements, i.e. genetic structures capable of moving in an intact form within a given genome or moving from one genome to another, for example, from plasmid to bacterial and vice versa. Transposable genetic elements are represented by IS elements and transposons. IS elements, or insertion sequences, are usually small in size, not exceeding two thousand base pairs. They carry only one gene encoding a protein transposase, with the help of which IS elements are integrated into various parts of the chromosome. They are designated: IS 1, IS2, IS3, etc. Transposons (Tp) are larger segments of DNA flanked by inverted IS elements. In addition to the genes that enable their transposition, they contain various other genes. Within a large transposon there may be smaller transposons.

Transposons are capable of inserting into different parts of a chromosome or moving from one genome to another. Very often, transposons are contained in R-plasmids. Transposons have been found in the genomes of bacteria, plasmids, viruses, and eukaryotes. They play an important role in the variability and evolution of living matter. Transposons are designated by serial number: Tn1, Tn2, Tn3, etc.

All known plasmids are small, covalently closed in a ring, supercoiled double-stranded DNA molecules, the sizes of which vary from 1.5 to 200 MD (from 1500 to 400,000 nucleotide pairs). The greater the molecular weight, the more complex the set of genes and the more diverse the functions of plasmids. Plasmids contain self-replication genes; genes that control self-transfer or mobilization for transfer; other genes that determine the specific functions of the plasmid itself. For example, F-plasmids determine the donor functions of the cell and its ability to conjugate; Ent-plasmids - synthesis of enterotoxins; biodegradative plasmids - destruction of various organic and inorganic compounds.

Plasmids are characterized by the following properties:

  • self-regulated replication;
  • the phenomenon of surface exclusion (a mechanism that does not allow another, related plasmid to enter a cell that already contains a plasmid);
  • the phenomenon of incompatibility (two closely related plasmids cannot stably coexist in one cell, one of them is removed);
  • control of the number of plasmid copies per cell chromosome (there are low-copy - 1-4 copies and high-copy - from 12 to 38 copies of the plasmid);
  • control of stable preservation of plasmids in the host cell;
  • control of uniform distribution of daughter plasmids into daughter bacterial cells;
  • ability to self-transfer (in conjugative plasmids);
  • ability to be mobilized for transfer (in non-conjugative plasmids);
  • the ability to provide the host cell with additional important biological properties that promote the survival of bacteria and plasmids in nature.

Plasmids spread among bacteria in two ways: vertically - by transfer from the parent cell to daughter cells during the process of bacterial cell division; horizontally - by transfer between cells in a bacterial population regardless of cell division. The transfer of plasmids between bacterial cells is carried out by the mechanism of self-transfer by conjugation controlled by the tra-operon of the plasmid. Depending on the presence of this operon, plasmids are divided into conjugative and non-conjugative. Other mechanisms of plasmid transfer are also possible (mobilization for the transfer of non-conjugative plasmids using conjugative plasmids, transformation, transduction).

The classification of plasmids is based on their unique property of incompatibility, i.e. the inability of related plasmids to stably coexist in the same cell. It manifests itself after the plasmid penetrates into a cell that already contains a closely related plasmid. Plasmids that are incompatible with each other, but compatible with others, are combined into one Inc group. For example, 39 Inc-group plasmids were found in enterobacteria. Plasmids belonging to the same Inc group have many common characteristics.

Plasmids have important medical significance, as they control the synthesis of various pathogenicity factors of bacteria and their drug resistance. The general biological significance of plasmids is that they are a unique means of bacterial self-defense and favor the preservation of bacteria in nature.

The transfer of genetic information to offspring (vegetative DNA replication) occurs in bacteria and plasmids according to a universal mechanism - semi-conservative DNA replication. In this case, daughter cells receive chromosome DNA, in which one strand is parental (conservative), the other DNA strand is newly synthesized on its matrix, which ensures very accurate transmission of genetic information (heredity). Vegetative DNA replication in bacteria begins from a strictly fixed chromosomal site (oriC), which is recognized by replication initiating enzymes. It is semi-conservative in nature, goes simultaneously in two directions, and ends at a strictly fixed point - terminus. Since the DNA chains are antiparallel (if one strand starts from the 5th end, the other from the 3rd end), and DNA polymerase III carries out DNA synthesis only in the 5-3 direction, replication occurs differently on each strand: on one of the untwisted strands (“straight”, “leader”) it goes continuously, and on the other (“lagging”) DNA polymerase III must return to grow the strand also in the 5-3 direction, intermittently, through the formation of Okazaki segments with a length of about 1000 in bacteria nucleotides.

The rate of DNA replication in E. coli at 37 °C corresponds to the incorporation of 2 x 10 3 nucleotide pairs per second. DNA replication involves a complex of enzymes that form a single structure - the replisome. Genetic control of DNA replication is exercised by a large group of chromosomal genes.

In addition to the vegetative type, bacteria have conjugative and reparative types of DNA replication. Conjugative replication occurs during the conjugative method of exchange of genetic material and is controlled by plasmid genes (tra-operon). In this case, the second strand of DNA is completed, the complementary strand transferred from the donor to the recipient. Reparative replication serves as a mechanism for eliminating structural damage from DNA or occurs at the final stage of genetic recombination. It is controlled by chromosomal and plasmid genes.

The information contained in the bacterial genome is deciphered, materialized and implemented through protein biosynthesis. The universality of the genetic code corresponds to the universality of its decoding and implementation (expression). However, protein biosynthesis in bacteria has some features at the transcription stage. The genes of bacteria, unlike the genes of eukaryotes and viruses, do not contain nitrones, so bacteria do not have a splicing process during the synthesis of mRNA. RNA splicing is a process in which introns (non-coding sequences in genes with an intron-exon structure) are excised from primary RNA transcripts and exons are joined together, resulting in the formation and then translation of mature mRNA. The lack of RNA splicing in bacteria is a natural genetic barrier in the implementation of eukaryotic genetic information in bacteria (prokaryotes). Overcoming this barrier led to the creation of genetic engineering on bacterial objects.

Genetic information is realized by microorganisms very “economically”, in accordance with the specific conditions of their existence. Only genes necessary to ensure cell viability under given conditions “work” (are expressed). Self-regulation of the genetic information system is ensured by the presence in it, in addition to structural genes encoding proteins and other macromolecules, of special nucleotide sequences (acceptor or regulatory genes) that do not have coding functions, but control the operation of structural genes. As already mentioned, a set of structural and regulatory genes located nearby constitutes an operon, the unit of genetic regulation. The classic model of an operon is the lactose operon. Let us consider, using the example of the lactose operon of Escherichia coli, its structure and method of regulating the activity of structural genes encoding the synthesis of enzymes involved in the absorption of lactose.

The operon begins with the “site of attachment of the activator protein” - the product of the upstream regulon (Cap protein, without which RNA polymerase cannot contact the operon and begin transcription). Next on the chromosome is a promoter - a site for recognition by RNA polymerase and its attachment. Then comes the operator - the site to which a special transcription-inhibiting regulatory protein binds. After the operator, the structural genes z, y, and a are located sequentially, encoding, respectively, the synthesis of three enzymes involved in the digestion of lactose - R-galactosidase, galactoside permease, thiogalactoside transacetylase. The lac operon ends with a terminator - a small section of DNA that serves as a stop signal that stops the progression of RNA polymerase and transcription of the operon. Outside the 1ac operon, at another place on the chromosome, there is a special regulator gene that encodes the continuous synthesis of a regulatory protein. When there is no lactose in the environment, the regulatory protein attaches to the operon and, like a “barrier,” prevents the movement of RNA polymerase from the promoter to the structural genes, repressing transcription and, ultimately, the synthesis of enzymes. Lactose, if present in the nutrient medium, binds to the regulator protein and allosterically changes its configuration, as a result of which the regulator protein can no longer attach to the operator. As a result, the “barrier” is opened, RNA polymerase transcribes structural genes into the corresponding mRNA, on the matrix of which enzymes that digest lactose are synthesized.

Thus, lactose induces the synthesis of enzymes necessary for its absorption. Such enzymes are called adaptive or inductive. The type of regulation of gene activity considered is called negative, since it is based on repression of the operon by a regulatory protein. There are two variants of this type of regulation: negative induction, which we looked at, and negative repression.

In the latter case, in the initial position, the regulatory protein cannot bind to the operator, and enzyme synthesis occurs, and in the presence of an effector, usually the final product of the action of anabolic enzymes, the regulatory protein, under its influence, binds to the operator and enzyme synthesis is repressed. In addition to the negative, a positive type of genetic regulation of protein synthesis is also known. Its difference from the negative type is that the protein product of the gene regulator does not prohibit the transcription of the operon, but, on the contrary, activates it. This type of regulation is also found in bacteria in two variants - positive induction and positive repression. For example, the ara operon, which controls the uptake of arabinose, works in Escherichia coli through a positive induction mechanism.

The manifestation of the characteristics of living organisms, controlled by the genotype, depends on the conditions of existence of the organism. The set of characteristics of an organism in the specific conditions of its existence is called a phenotype. Depending on the conditions, microorganisms of the same genotype can have different phenotypes, since different parts of the genetic information of the genotype are implemented or implementation occurs in a different range of the genotype reaction norm. The change of phenotypes when the conditions of existence of an organism change is called modification. In other words, modifications are phenotypic differences caused by external factors in hereditarily identical microorganisms.

The distinctive signs of modification in bacteria are (three “Os”):

  • certainty of variability (a certain environmental factor or conditions causes a change in a strictly defined characteristic);
  • community of changes (changes in a trait simultaneously in all or most individuals of a genetically homogeneous population);
  • reversibility of changes (changes are not inherited and disappear after the cessation of the external factor).

In some cases, so-called long-term modifications are observed in bacteria, when a change in a characteristic persists for several generations after the cessation of the action of the factor. This is due to the fact that during cell division, not only the structures of the genotype are transferred, but also the contents of the cell, which partially retain the remains of substances formed under previous conditions.

Let's consider an example of modification variability of bacteria. When sowing a diluted bacterial culture Proteus vulgaris Colonies of bacteria grew on the nutrient agar within 24 hours, each of which was surrounded by a swarming zone. After reseeding the colonies on nutrient agar with bile, all colonies grew within 24 hours without swarming. After reseeding these colonies onto the original nutrient agar, all grown colonies again had swarming zones.

Changes in the phenotype of Proteus on a medium with bile should be considered a modification, since all three distinctive features are present: certainty of variability (the connection between the absence of swarming and the factor - bile), general variability (changes in all colonies of the population), reversibility (in the absence of bile in the nutrient medium, return bacteria to the original phenotype).

The possibility of bacterial modification should be constantly taken into account in the practical work of microbiologists. For accurate taxonomy and identification of bacteria, standard (unified) conditions for studying the properties of bacteria (standard nutrient media, tests, reagents, temperature and other cultivation conditions) must be strictly observed.

The primary source of new genes in nature is mutations. There is no generally accepted definition of a mutation. Mutation from lat. mutatio is a change in the genetic structures present in the cell at a given moment, which is stably inherited. There are two groups of mutations: chromosomal aberrations, including 3 types (changes in the number of sets of chromosomes, changes in the number of individual chromosomes, chromosome rearrangements) and gene mutations. Bacteria can have mutations such as chromosome rearrangements and gene mutations. In this case, chromosome rearrangements are carried out through divisions (loss of a chromosome fragment), inversion (rotation of a chromosome section by 180°), transpositions (insertion of small DNA fragments into some place on the chromosomes, for example, insertion segments or transposons). Gene mutations can be single-site (in one region of the gene) or multi-site. For a mutation to appear in a gene, a single point change in one pair of nucleotides is sufficient. The direction of mutation can be direct or reverse. Direct mutations cause changes in the characteristics of a wild-type organism; back mutations are accompanied by reversion to the wild type. Restoration of the original phenotype as a result of a reverse mutation in another part of the gene or in another gene is a suppressor mutation. A mutation that changes two or more characteristics of an organism is called pleiotropic. There are also spontaneous and induced mutations. Spontaneous mutations occur spontaneously in the sense that they are determined, but we do not know their specific causes. Induced mutations are caused by exposure to certain mutagenic factors. These include various types of ionizing radiation, ultraviolet rays, and chemical mutagens.

Variability in the mutation mechanism is characterized by certain distinctive features.

  • 1. Heredity.
  • 2. Low frequency (rate) of mutation (in bacteria 1 x 10 6 - 1 x 10 7, i.e. mutation in one cell out of 1-10 million). The ability to produce mutations (mutability) is influenced by the genotype. In bacteria, mutability increases sharply if they have special genes - mutators. For example, two mutator genes were found in Escherichia coli - mut T, mut SI.
  • 3. Non-directionality of variability (i.e. uncertainty, inadequacy of changes in characteristics due to an influencing factor; the same factor causes different mutations).

The experiments of S. Luria and M. Delbrück (fluctuation test), Newcomb (redistribution test) showed the existence in the original populations of bacteria of spontaneous mutants that are resistant to the influencing factor - the phage - even before its influence. The fingerprint (replica) technique developed by D. Lederberg made it possible to directly isolate mutants from a population of cells before exposure to an adequate factor or after exposure to a mutagen - a variety of mutants that are not adequate in characteristics.

Bacteria have special systems for repairing mutational DNA damage. The most studied systems are: “photoreactivation”, “dark repair”, “replicative repair”. “Photoreactivation” repairs defects (thymine dimers) in DNA caused only by ultraviolet rays. During “dark repair,” a complex of enzymes operates that restores damage on one DNA strand by cutting out the damaged area and synthesizing in its place a second strand of a complementary DNA segment that replaces the defect. The “replicative repair” system replaces damage to both strands of DNA through recombination.

Another mechanism of hereditary variability is the exchange of genetic material between cells of bacterial populations (horizontally). It does not create new elementary traits, but it greatly accelerates the creation of organisms with new combinations of traits due to the redistribution of genes from different genomes, which contributes to the rapid adaptation of bacteria to environmental conditions. Bacteria are capable of extensive genetic exchange between different species and genera, as well as with bacteriophages and plasmids. In bacteria, three main forms of exchange of genetic material have been identified: transformation, transduction, conjugation (Fig. 3.2, 3.3). They differ in the way genetic material is transmitted.

Transformation is characterized by the transfer of some of the donor's genes to the recipient cell using free DNA isolated from the donor's genome. Transformation can be spontaneous or induced. Spontaneous transformation in natural conditions manifests itself in the appearance of recombinants when genetically different cells are mixed. It occurs due to DNA released by cells into the environment during their lysis or as a result of active DNA release by viable donor cells. Induced (artificial) transformation occurs when purified DNA obtained from donor bacteria is added to the bacterial culture. Successful transformation requires a number of conditions related to the DNA and recipient bacteria. The DNA must be double-stranded, have fragments of 3-5 x 10 6 daltons, and be partially or completely homologous to the recipient's DNA. The recipient cells must have competence, i.e. susceptibility, which occurs only during a certain period of the life cycle, is due to the release by the cell of a special protein “competence factor” and a specific change in the permeability of the cell wall and membrane. The transformation process consists of several stages: binding of DNA on the surface of a competent recipient to the “competence factor”, penetration of DNA by “pulling” into the cell, inclusion of DNA into the chromosome of the recipient bacterium by recombination, expression of transferred genes. The efficiency of genetic transformation increases many times if the mixture of DNA and transformed cells is treated with an electrical impulse (electrotransformation method). Under natural conditions, the efficiency of transformation is less significant than other forms of transfer of genetic material. Bacterial cells have a mechanism for protecting the genome from foreign DNA - special modification and restriction systems. These systems protect their DNA by modification (usually by methylation) and destroy foreign DNA using special enzymes - restriction endonucleases. A particular transformation option is transfection, when bacteriophage or plasmid DNA is introduced into a recipient cell lacking a cell wall.

Transduction is the transfer of genetic material from a donor cell to a recipient cell using bacteriophages. There are general (nonspecific) and specific transduction. The mechanism of general transduction is that during the intracellular reproduction of a virulent phage, a fragment of bacterial DNA equal to the length of the phage may be accidentally included in its head instead of phage DNA. This is how defective phages arise, which instead of their own genomic DNA contain a DNA fragment from the donor bacterium. Such phages retain infectious properties. They are adsorbed on the bacterial cell and introduce DNA into it, but the phage does not reproduce. In the case of genetic recombination of a donor DNA fragment introduced by a phage with the chromosome of the recipient cell, the new trait is hereditarily fixed. Thus, during general transduction, the phage is only a passive carrier of genetic material.

Specific transduction is distinguished by the transfer of a strictly defined DNA fragment of a donor bacterium by temperate bacteriophages. As is known, temperate bacteriophages are those that can integrate bacterial cells into the chromosome, causing its lysogenization. A temperate phage (prophage) integrated into the chromosome of a donor bacterium, under certain conditions, leaves the chromosome, “grabbing” the nearest DNA sections of the bacterial chromosome and leaving behind part of its genome. A defective temperate phage arises that incorporates the bacterial genes of the donor bacterium into its genome. Next, the transducing phage introduces its DNA into the cell of the recipient bacterium, where it, together with a DNA fragment of the donor bacterium, is integrated into the recipient chromosome. Subsequently, the phage can leave the recipient's chromosome, but the genes of the donor bacterium transferred by it remain in the recipient. An example is the temperate bacteriophage lambda (X), which always carries the gal operon or oneron bio. Specific and general transduction are low-frequency (10 -4 - 10 -7 per 1 phage particle). A particular variant of specific transduction is phage or lysogenic conversion. The transducing phage, integrating into the recipient chromosome, causes lysogenization of the bacterium and transfers genes for new characteristics, for example, toxin formation, to diphtheria bacteria. However, the genes that control the new trait are constantly included in the genome of such transducing phages. The appearance of these genes is not associated with the preliminary reproduction of the phage on toxigenic donors. The phage probably incorporated these genes into its genome at earlier stages of its evolution. Such transducing phages are non-defective and cause phage conversion at a very high frequency.

Conjugation is characterized by the transfer of genetic material through direct contact between cells. This process is polar - genetic material is transferred only from donor bacteria to recipient bacteria. The donor cell function and the conjugation process are controlled by conjugation transfer genes (tra-operon), localized in conjugative plasmids. Among the many conjugative plasmids, there is plasmid F, which controls only these functions. In an autonomous, extrachromosomal state, it ensures the donor cell type F+ (male), the formation of hollow villi (F-pili) and its own transfer into F- (female) recipient cells. In this case, the donor chromosome is not transferred, and recipients who received the F plasmid acquire the donor type F+. When plasmid F is integrated into the chromosome of the host bacterium, Hfr (high frequency of recombination) is formed - bacterial strains with a high frequency of transmission of chromosomal genes. However, plasmid F usually does not pass into the recipient cell, since it is located at the end of the chromosome opposite to the one from which its transition begins.

The passage of a chromosome through a conjugation bridge between cells through the F-pili channel is associated with its replication. In this case, only one strand of DNA of the chromosome passes through the bridge, on which a complementary second strand is synthesized in the recipient cell, and then, under the control of the recombination genes (recombination genes) of the recipient chromosome, the transferred DNA fragment is integrated into the recipient chromosome. Plasmid F can revert in Hfr strains to the original extrachromosomal state by capturing an adjacent region of the bacterial chromosome. In this way, plasmid F' (F-prim) is formed, which carries part of the chromosomal genes of the host bacterium, for example, plasmid F' lac. The transfer of donor genes into recipient cells via F' plasmids is called sexduction. In this case, plasmid F’ transfers its genome, which also contains some donor genes, with high frequency, but the chromosome of the donor bacterium is not transferred.


Transfer of other types of conjugative plasmids (R, Ent, Col, etc.) also occurs with high frequency. It should be noted that the effective transfer of plasmids by conjugation does not know “related” barriers and occurs between bacteria of different species and genera.

In any form of exchange of genetic material, the final stage is recombination between the resulting DNA and the chromosome of the recipient cell. When one DNA strand is transferred, it is first completed by a complementary strand. Only double-stranded DNA recombines with each other. General recombination, site-specific recombination and recombination controlled by transposable elements are known.

General recombination occurs between homologous DNAs. Site-specific recombination is due to the presence of specific sites in the recombining DNA molecules, for example, chromosomes E. coli and the temperate bacteriophage lambda. General and site-specific recombinations are controlled by the hea A gene. Recombinations carried out by transposable elements are also site-specific and determined by special nucleotide sequences, but do not depend on the hea A gene. The leading role in recombination processes in bacteria belongs to the gene A. Its product, the Rec A protein (molecular weight 38 kDa), has unique functions: it binds firmly to single strands of DNA; promotes the release of the broken strand from the DNA double helix; holds together the single strand of DNA and the double helix of DNA; has the property of a DNA-dependent ATPase. Gene hes A is involved not only in the process of recombination. Its product is necessary for post-replicative repair, prophage induction, cell division and other important functions of bacteria. Recessive mutations in such a gene affect all these functions, so they are called SOS functions, and their totality represents a single SOS system. The expression of any SOS function depends on the activity of the hec A gene product. The SOS system is triggered after any damaging effects on DNA. Therefore, the hec A gene is of primary importance in the self-defense of the genetic system of bacteria.

The use of genetic methods to study the bacterial genome made it possible to create a genetic taxonomy of bacteria. On its basis, the current classification of bacteria has been significantly clarified and the prerequisites have been created for the development of natural taxonomy and classification. In the interests of taxonomy, a number of methods are used. Method of DNA-DNA hybridization (detection of the level of DNA homology). A measure of 60-100% DNA homology is considered to indicate relatedness at the species level. However, there are no generally accepted criteria. The DNA-rRNA hybridization method reveals genetic connections between the region of DNA that controls rRNA synthesis and rRNA nucleotides. The cistrons responsible for rRNA synthesis are conservative and make it possible to identify relationships at the genus and family level. The most reliable method is DNA sequencing. This method reveals the nucleotide sequence in individual DNA fragments or the entire DNA of a chromosome. The best object of study (the most conservative) is the DNA region that controls the synthesis of bacterial 16S ribosomal RNA. The DNA of this fragment is cloned and then sequenced. The DNA sequencing method makes it possible to identify the relatedness of bacteria at the level of kingdom, class, family, and genus, but is not sensitive enough to establish the species. This method makes it possible to identify the evolutionary relationships of bacteria and is fundamental in gene systematics. In the most important bacteria, the complete sequence of chromosomal DNA nucleotides (Escherichia coli, Pseudomonas aeruginosa, etc.) was studied by sequencing.

The genetic mechanism of the very important medical problem of acquired drug resistance in bacteria has been revealed.

It has been established that R-plasmids carrying genes for resistance to 1-10 antibiotics in any combination are of primary importance in the rapid development of bacterial resistance to antibiotics. They can transfer antibiotic resistance genes to susceptible bacteria within minutes, rendering the entire population of bacteria in the body resistant. There is still no effective means of removing R plasmids or blocking their transmission. So far, the real achievement is the use of drugs that block the activity of enzymes that destroy antibiotics, which are controlled by R-plasmids. For example, to inactivate beta-lactamase, clavulanic acid, sulbactam, tazobactam are used in combination with antibiotics of the beta-lactam group.

Genetic control of bacterial pathogenicity factors has been established. The possibility of localizing these genes in the genomes of bacteria, bacteriophages, and plasmids has been demonstrated. The mechanisms of formation of pathogenic strains among opportunistic bacteria have been identified. This information makes it possible to identify pathogenic bacteria using molecular biological methods and purposefully obtain avirulent vaccine strains of microorganisms.

A major achievement in genetics is the development of the polymerase chain reaction (PCR) method, which was awarded the Nobel Prize (Mullis K., 1993). Based on PCR, a fundamentally new universal system for indicating microorganisms and diagnosing infectious diseases has been created - genoindication. The PCR method allows you to amplify (“multiply”, clone) certain sections of DNA in vitro, obtaining millions of copies of this DNA in 2-4 hours. The essence of the PCR method is a multiple cyclic process, alternately including three stages in each cycle - thermal denaturation of DNA (melting), its annealing (attachment of synthetic oligonucleotide primers), synthesis (completing the second strand of DNA with a thermostable DNA polymerase). The change of these stages occurs as a result of a change in the temperature of the reaction mixture in a special amplifier device (thermal cycler). Denaturation of the original and subsequent copies of DNA, leading to the division of DNA into two single strands, occurs at 90-95 ° C for 40-50 seconds. Annealing (addition of primers) occurs at a temperature of 40-65 °C. Primers - synthetic oligonucleotides (20-30 nucleotides) - are attached to a single-stranded DNA target, flanking the desired specific DNA fragment. Primers are selected so that they limit the desired fragment and are complementary to the opposite DNA strands; in this case, one primer is on one strand, the other on the opposite. Synthesis (elongation) - completion of the second strand of DNA DNA occurs at 72 °C with the participation of Tag-DNA polymerase. The completion of the second DNA strand occurs from the 5" end to the 3" end of each DNA strand, i.e. in opposite directions. As a result of the first cycle, two copies of the DNA region limited by the primer are formed. The cycle duration is 2-2 minutes or 30-40 seconds, depending on the type of thermal cycler. As a result of each subsequent cycle, the number of synthesized copies doubles exponentially (Fig. 3.4). Typically, PCR includes 20-30 cycles, which ensures the synthesis of 1 million copies of the desired DNA fragment.


Rice. 3.4.

Electrophoresis or other detection systems are used to detect PCR products.

PCR is adapted to detect most microorganisms of medical importance. It allows you to quickly detect the desired microorganism directly in the material under study without identifying a pure culture, and has high sensitivity (1 x 10 1 - 1 x 10 3 microorganisms per 1 g of material). PCR has found wide application for the detection of difficult-to-cultivate and slow-growing microorganisms (viruses, chlamydia, rickettsia, mycoplasmas, spirochetes, mycobacteria).

Genetics of bacteria and viruses.

Formation of new knowledge. Lecture block

Topic study plan:

1. Genetic material of bacteria.

2. Classification and biological role of plasmids.

3.Virus genetics

4. Biotechnology, genetic engineering.

5. Antimicrobials

Molecular biology, the study of the fundamental principles of life, is largely an outgrowth of microbiology. It uses viruses and bacteria as basic objects of study, and the main direction - molecular genetics - is based on the genetics of bacteria and phages.

Bacteria are convenient material for genetics. What distinguishes them is:

Relative simplicity of the genome (combination of chromosome nucleotides);

Haploidy (one set of genes), excluding the dominance of characters;

Various chromosome-integrated and isolated DNA fragments;

Sexual differentiation in the form of donor and recipient cells;

Ease of cultivation, rapid accumulation of biomass.

General ideas about genetics.

A gene is a unique structural unit of heredity, a carrier and guardian of life. It has three fundamental functions.

1. Continuity of heredity - ensured by the mechanism of DNA replication.

2. Control of the structures and functions of the body is ensured using a single genetic code of four bases (A-adenine, T-thymine, G-guanine, C-cytosine). The code is triplet because a codon, a functional unit encoding an amino acid, consists of three bases (letters).

3.Evolution of organisms - due to mutations and genetic recombinations.

In a highly specialized sense, a gene most often represents a structural unit of DNA, the location of codons in which determines the primary structure of the corresponding polypeptide chain (protein). The chromosome consists of special functional units - operons.

The main stages of development (complication) of the genetic system can be represented in the following diagram:

codon à gene à operon à genome of viruses and plasmids à prokaryotic chromosome (nucleoid) à eukaryotic chromosome (nucleus).

1. Nuclear structures of bacteria - chromatin bodies or nucleoids (chromosomal DNA). Bacteria have one closed ring-shaped chromosome (up to 4 thousand individual genes). The bacterial cell is haploid, and chromosome duplication (DNA replication) is accompanied by cell division. Vegetative replication of chromosomal (and plasmid) DNA determines the vertical transfer of genetic information from the parent cell to the daughter cell. The horizontal transfer of genetic information is carried out by various mechanisms - as a result of conjugation, transduction, transformation, sexduction.

2. Extrachromosomal DNA molecules are represented by plasmids, migrating genetic elements - transposons and conservation (insertion) or IS sequences.

Plasmids are extrachromosomal genetic material (DNA), simpler organisms than viruses, which provide bacteria with additional beneficial properties. The molecular weight of plasmids is much smaller than chromosomal DNA and contains from 40 to 50 genes.

Their separation into a separate class is determined by significant differences from viruses.

1. Their habitat is only bacteria (among viruses, in addition to bacteriophage viruses, there are plant and animal viruses).

2.Plasmids coexist with bacteria, giving them additional properties. In viruses, these properties occur only in moderate phages during bacterial lysogeny; most often, viruses cause negative consequences, cell lysis.

3. The genome is represented by double-stranded DNA.

4. Plasmids are “naked” genomes that do not have any shell; their replication does not require the synthesis of structural proteins and self-assembly processes.

Plasmids can spread vertically (during cell division) and horizontally, primarily through conjugative transfer. Taking into account the dependence on the presence or absence of a self-transfer mechanism (it is controlled by the tra-operon genes), conjugative and non-conjugative plasmids are distinguished. Plasmids can be integrated into the bacterial chromosome—integrative plasmids—or present as a separate structure—autonomous plasmids (episomes).

Genetic material of bacteria. - concept and types. Classification and features of the category "Genetic material of bacteria." 2017, 2018.

Molecular biology, the study of the fundamental principles of life, is largely an outgrowth of microbiology. It uses viruses and bacteria as the main objects of study, and the main direction is molecular genetics based on the genetics of bacteria and phages.

Bacteria are convenient material for genetics. They are distinguished by:

Relative simplicity of the genome (the totality of chromosome nucleotides);

Haploidy (one set of genes), excluding the dominance of characters;

Various chromosome-integrated and isolated DNA fragments;

Sexual differentiation in the form of donor and recipient cells;

Ease of cultivation, rapid accumulation of biomass.

General ideas about genetics.

Gene- a unique structural unit of heredity, the carrier and guardian of life. It has three fundamental functions.

1. Continuity of heredity - ensured by the mechanism of DNA replication.

2. Control of the structures and functions of the body is ensured using a single genetic code of four bases (A - adenine, T - thymine, G - guanine, C - cytosine). The code is triplet because a codon, a functional unit encoding an amino acid, consists of three bases (letters).

3.Evolution of organisms - due to mutations and genetic recombinations.

In a highly specialized sense, a gene most often represents a structural unit of DNA, the location of codons in which determines the primary structure of the corresponding polypeptide chain (protein). A chromosome consists of special functional units - operons.

The main stages of development (complication) of the genetic system can be represented in the following diagram:

codon à gene à operon à genome of viruses and plasmids à prokaryotic chromosome (nucleoid) à eukaryotic chromosome (nucleus).

Genetic material of bacteria.

1.Nuclear structures of bacteria- chromatin bodies or nucleoids (chromosomal DNA). Bacteria have one closed ring-shaped chromosome (up to 4 thousand individual genes). The bacterial cell is haploid, and chromosome duplication (DNA replication) is accompanied by cell division. Vegetative replication of chromosomal (and plasmid) DNA determines the vertical transfer of genetic information - from the parent cell to the daughter cell. The horizontal transfer of genetic information is carried out by various mechanisms - as a result of conjugation, transduction, transformation, sexduction.

2.Extrachromosomal DNA molecules presented plasmids, migrating genetic elements - transposons and conservation (insertion) or IS sequences.

Plasmids are extrachromosomal genetic material (DNA), simpler organisms compared to viruses, which endow bacteria with additional beneficial properties. The molecular weight of plasmids is much smaller than chromosomal DNA and contains from 40 to 50 genes.

Their separation into a separate class is determined by significant differences from viruses.

1. Their habitat is only bacteria (among viruses, in addition to bacteriophage viruses, there are plant and animal viruses).

2.Plasmids coexist with bacteria, giving them additional properties. In viruses, these properties can only be found in moderate phages during bacterial lysogeny; most often, viruses cause negative consequences, cell lysis.

3. The genome is represented by double-stranded DNA.

4. Plasmids are “naked” genomes that do not have any shell; their replication does not require the synthesis of structural proteins and self-assembly processes.

Plasmids can spread vertically (during cell division) and horizontally, primarily through conjugative transfer. Depending on the presence or absence of a self-transfer mechanism (it is controlled by the tra-operon genes), conjugative And non-conjugative plasmids. Plasmids can be integrated into the bacterial chromosome - integrative plasmids or be in the form of a separate structure - autonomous plasmids ( episomes).

Classification and biological role of plasmids.

The functional classification of plasmids is based on the properties they impart to bacteria. Among them are the ability to produce exotoxins and enzymes, drug resistance, and the synthesis of bacteriocins.

1.F-plasmids- donor functions, induce division (from fertility - fertility). Integrated F - plasmids - Hfr - plasmids (high recombination frequency).

2.R-plasmids(resistance) - resistance to drugs.

3.Col plasmids- synthesis of colicins (bacteriocins) - factors of competition between closely related bacteria (antagonism). Colicinotyping of strains is based on this property.

4.Hly plasmids- synthesis of hemolysins.

5.Ent-plasmids- synthesis of enterotoxins.

6.Tox plasmids- toxin formation.

Closely related plasmids are not able to stably coexist, which made it possible to combine them according to the degree of relatedness into Inc-groups (incompatibility).

The biological roles of plasmids are diverse, including:

Control of genetic exchange of bacteria;

Control of the synthesis of pathogenicity factors;

Improving bacterial protection.

Bacteria for plasmids are a habitat, plasmids for them are additional genomes transferred between them with sets of genes that favor the preservation of bacteria in nature.

Migrating genetic elements- individual sections of DNA capable of determining their transfer between chromosomes or a chromosome and a plasmid using the recombination enzyme transposase. Their simplest type is insertion sequences (IS elements) or insertion elements that carry only one transposase gene, with the help of which IS elements can be integrated into different parts of the chromosome. Their functions are coordination of the interaction of plasmids, temperate phages, transposons and genophore to ensure reproduction, regulation of gene activity, and induction of mutations. The size of IS elements does not exceed 1500 base pairs.

Transposons(Tn elements) include up to 25 thousand nucleotide pairs, contain a DNA fragment carrying specific genes, and two Is elements. Each transposon contains genes that contribute important characteristics to the bacterium, as do plasmids (multiple antibiotic resistance, toxin formation, etc.). Transposons are self-integrating DNA fragments that can integrate and move among chromosomes, plasmids, temperate phages, i.e. have the potential to spread among various types of bacteria.

Hereditary apparatus of bacteria

The most important characteristics of living organisms are variability and heredity.

The basis of the hereditary apparatus of bacteria, like all other organisms, is DNA(for RNA viruses - RNA).

Along with With this is why the hereditary apparatus of bacteria and the possibilities of studying it have a number of features:

bacteria are haploid organisms, i.e. they have 1 chromosome. In this regard, when inheriting traits, there is no phenomenon of dominance;

  • bacteria have a high reproduction rate, due to With than in a short period of time (days) several dozen generations of bacteria are replaced. This makes it possible to study huge populations and quite easily identify even mutations that are rare in frequency. Hereditary apparatus bacteria is represented by a chromosome. Bacteria have only one. If there are cells with 2 or 4 chromosomes, then they are the same.

Bacterial chromosome is a DNA molecule. The length of this molecule reaches 1.0 mm and, in order to “fit” in a bacterial cell, it is not linear, like in eukaryotes, but is supercoiled into loops and folded into a ring. This ring is attached to the cytoplasmic membrane at one point. Individual genes are located on the bacterial chromosome. E. coli, for example, has more than 2 thousand of them.

Functional units of the genome

Genotype (genome) of bacteria

represented not only by chromosomal genes. Functional units of the bacterial genome, in addition to chromosomal genes, are:

  • IS sequences;
  • transposons;
  • plasmids.

IS sequences

Short DNA fragments. They do not carry structural genes (encoding a particular protein), but contain only genes responsible for transposition (the ability of IS sequences to move along the chromosome and integrate into its various sections). IS sequences are the same in different bacteria. Transposons are DNA molecules larger than IS sequences. In addition to the genes responsible for transposition, they also contain a structural gene encoding a particular trait.

Transposons move easily along a chromosome. Their position affects the expression of both their own structural genes and neighboring chromosomal ones. Transposons can exist outside the chromosome, autonomously, but are incapable of autonomous replication.

Plasmids

Circular superhelical DNA molecules. Their molecular weight varies widely and can be hundreds of times greater than that of transposons.

  • R-plasmids - drug resistance;
  • Col-plasmids - the ability to synthesize colicins;
  • F-plasmids - transmit genetic information;
  • Shu-plasmids - synthesize hemolysin;
  • Tox plasmids - synthesize toxin;
  • biodegradation plasmids - destroy one or another substrate, etc.

Plasmids can be integrated into the chromosome (unlike IS sequences and transposons, they are integrated into strictly defined areas), or they can exist autonomously. In this case, they have the ability to autonomously replicate, and that is why there can be 2, 4, 8 copies of such a plasmid in a cell.

Many plasmids contain transmissibility genes and are capable of being transferred from one cell to another through conjugation (exchange of genetic information). Such plasmids are called transmissible.

Fitness factor

AvailabilityF-plasmids (fertility factor, sex factor)

gives bacteria donor functions, and such cells are able to transfer their genetic information to other F cells. We can say that the presence of an F-plasmid is a phenotypic expression (manifestation) of sex in bacteria: not only the donor function is associated with the F-plasmid, but also some other phenotypic characteristics - the presence of F-pili (genital cilia) and sensitivity to L- phages. With the help of F-cilia, contact is established between donor and recipient cells. It is through their channel that donor DNA is transmitted during recombination. Receptors for male fj-phages are located on the genital cilia. F cells do not have such receptors and are insensitive to such phages.

Variability of the bacterial cell

Bacteria are distinguished 2 types of variability- phenotypic and genotypic.

Phenotypic variability - modification- does not affect the genotype, but affects the majority of individuals in the population. Modifications are not inherited and fade over time, that is, they return to the original phenotype through a larger (long-term modifications) or smaller (short-term modifications) number of generations.

Genotypic variability affects the genotype. It is based on mutations and recombinations.

Bacterial mutations are not fundamentally different from mutations in eukaryotic cells. A feature of mutations in bacteria is the relative ease of their identification, since it is possible to work with large populations of bacteria. By origin, mutations can be:

  • spontaneous;
  • induced. By length:
  • point;
  • genetic;
  • chromosomal. By direction:

- straight;

- reverse.

Recombination (exchange of genetic material) in bacteria differ from recombination in eukaryotes:

  • bacteria have several recombination mechanisms;
  • during recombination in bacteria, not a zygote is formed, as in eukaryotes, but a merozygote (carries the entire genetic information of the recipient and part of the genetic information of the donor in the form of an addition);
  • in a recombinant bacterial cell, not only the quality, but also the quantity of genetic information changes. Transformation- this is the exchange of genetic information in bacteria by introducing a ready-made DNA preparation (specially prepared or directly isolated from the donor cell) into the recipient bacterial cell. Most often, the transfer of genetic information occurs when the recipient is cultivated on a nutrient medium containing donor DNA. To perceive donor DNA during transformation, the recipient cell must be in a certain physiological state (competence), which is achieved by special methods of processing the bacterial population.

During transformation, single (usually 1) characteristics are transmitted. Transformation is the most objective evidence of the connection of DNA or its fragments with a particular phenotypic trait, since a pure DNA preparation is introduced into the recipient cell.

Transduction

Exchange of genetic information in bacteria by transferring it from donor to recipient using temperate (transducing) bacteriophages.

Transducing phages can transfer 1 or more genes (traits). Transduction happens:

  • specific - the same gene is always transferred;
  • nonspecific - different genes are transmitted.

This is due to the localization of transducing phages in the donor genome:

  • in the case of specific transduction, they are always located in one place on the chromosome;
  • when nonspecific, their localization is inconsistent. Conjugation- exchange of genetic information in bacteria by transferring it from donor to recipient during their direct contact. After the formation of a conjugation bridge between the donor and the recipient, one strand of donor DNA enters the recipient cell through it. The longer the contact, the more of the donor DNA can be transferred to the recipient.

Based on the interruption of conjugation at certain intervals, it is possible to determine the order of genes on the bacterial chromosome - construct chromosome maps of bacteria(produce mapping bacteria).

F+ cells have a donor function.

Genetics of microorganisms as a science

Note 1

Until approximately the end of the 1930s, it was believed that microorganisms did not have a nuclear apparatus. Therefore, issues of heredity and variability of microorganisms have not been carefully studied.

Only with the invention of the electron microscope did it become possible to examine the submicroscopic structure of cells in general and microorganisms in particular.

Since the early 1940s, geneticists have been turning their attention to microorganisms. Bacteria, microscopic fungi and viruses become objects of genetic research. A new branch of microbiology is being formed - the genetics of microorganisms.

The genetics of microorganisms is a section of general genetics in which the subject of study is microorganisms (bacteria, viruses, microscopic fungi) and the features of their heredity and variability.

A characteristic feature of microorganisms is a haploid set of chromosomes or a circular DNA molecule. This makes it possible for mutations to appear in the first generation of descendants.

Beginning of microbiological genetic research

Thanks to the study of the submicroscopic structure of microbial cells, it was possible to find answers to many genetic questions. American Genetics O.T. Avery, K. McLeod and M. McCarthy, conducting experiments on pneumococci, obtained the first evidence that the material carrier of heredity is the DNA molecule. Research on bread mold has made it possible to formulate the position that one gene programs the synthesis of one polypeptide chain (one protein).

But they began to study microorganisms especially intensively from the point of view of genetics after the American microbiologists S. Luria and M. Delbrock, using the example of Escherichia coli, proved the universality of the patterns of the mutation process. They proved that bacteria also obey mutational laws.

A new principle for studying variability in bacteria has appeared in science - clonal analysis. It involves a thorough study of the progeny of a single cell. This cell becomes the ancestor of the clone.

Study of bacteria

As a result of painstaking research, American geneticists J. and E. Lederberg were able to prove that mutations occur in bacteria regardless of their cultivation conditions. They developed the fingerprint method, which made it possible to greatly simplify the selection of microorganisms with desired properties for further research. They proved that in large populations of bacterial cells mutations occur in a disorderly manner—spontaneously.

In 1946, it was proven that bacteria also have a sexual process; the phenomena of chromosome conjugation and gene recombination, the transfer of genetic information from one bacterial cell to another through a bacteriophage, were discovered.

There is an opinion that in the circular nucleic acid molecule of prokaryotic cells, “reading information” depends on the place where “reading” begins. Depending on which nucleotide this process began with, the synthesis of one or another protein is determined.

Study of phages

While studying the peculiarities of the “bacterium-bacteriophage” relationship, American geneticists discovered the phenomenon of transduction (gene transfer between bacterial cells using phages) and discovered recombination in phages. This made it possible to study issues of heredity at the molecular level (molecular level of organization of matter).

German microbiologists studied the RNA molecule. A research methodology was developed for each group of microorganisms.

Genetics of fungi and algae

Lower fungi and algae have a sexual process somewhat different from the sexual process of other organisms. Thanks to their study, a new method emerged - tetrad analysis. While studying these organisms, scientists developed a technique for combining the nuclei of genetically different strains of microorganisms. All these methods can further serve to breed organisms with given qualities, to develop new generations of antibiotics and biologically active substances, as well as to combat many types of diseases of plants, animals and, of course, humans.

Note 2

But issues of genetic engineering require a careful approach to studying and applying the information obtained in practice. After all, it is not clear what consequences the appearance of genetically modified organisms in nature and in the human body may lead to.