MICROBIOLOGY Study Guide - 2012

CHAPTER 12. HEREDITY AND VARIABILITY IN MICROORGANISMS

Genetics is the science that studies the regularities and material foundations of heredity and Variability in organisms.

Heredity is the ability of organisms to pass on their inherent traits to their offspring.

Variability is the phenomenon wherein differences arise among individuals across a range of traits or properties. In other words, it is the property of organisms to acquire new traits during individual development.

Bacterial genetics is the most extensively studied branch, focusing on microorganisms characterized by small size and rapid reproduction rates, which allows genetic changes to be tracked across vast populations over short periods. The bacterial chromosome, which carries The Cell's primary Genetic information, is approximately 1000 µm long and has a Molecular Weight of 1.5–2.0 × 10⁹ Da. It is supercoiled, circular, and contains between 3,000 and 5,000 genes. Extrachromosomal hereditary factors are also located in the bacterial Cytoplasm: Plasmids, Transposons, and IS elements.

Plasmids are small, extrachromosomal DNA molecules responsible for specific traits. For instance, the fertility factor, or F factor (from fertility), is a plasmid that contains genes controlling conjugation. The R factor, or resistance factor (from resistance), carries genes responsible for bacterial resistance to antimicrobial drugs. Plasmids can also determine bacterial virulence, such as in the causative agents of plague, tetanus, gas gangrene, and others.

Many Bacteria synthesize Proteins that inhibit the growth of related species or competitors. These specific-action proteins are called bacteriocins. Their synthesis is encoded by specialized plasmids or bacteriocinogenic factors. Bacteriocins have been isolated from Escherichia coli Cells (colicins), Pseudomonas aeruginosa (pyocins), and Lactobacillus acidophilus (lactocins). Both Chromosomes and plasmids are capable of autonomous self-Replication, which is why they are referred to as replicons.

Transposons and IS elements are Mobile Genetic Elements that facilitate The transfer of genetic information from one replicon to another. Transposons can exist freely within the cell, but they do not replicate independently; they do so only after integrating into the bacterial chromosome. IS elements are DNA fragments about 1,000 nucleotide pairs in length. They have not been found in a free state and function as mobile elements, also known as "insertion sequences." The translocation of IS elements to various Regions of the DNA regulates the interactions among transposons, plasmids, and temperate phages—both with each other and with the bacterial chromosome—and induces Mutations upon chromosomal integration.

The properties of microorganisms are determined by their genotype, which is the complete set of genes possessed by a given individual. The phenotype represents the result of the interaction between the genotype and the environment, manifested as the expression of the genotype under specific living conditions.

Variability is driven either by Changes in the genotype's response to environmental factors or by alterations in the genotype itself resulting from Gene Mutations or recombination. Prokaryotes, much like eukaryotes, exhibit two MAIN TYPES OF variability: genotypic (hereditary) and phenotypic (modificational):

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Phenotypic variability arises as an Organism's adaptive response to changing environmental conditions and is manifested across the majority of individuals within a population. A unique adaptive response to various stressors has been discovered in several bacteria. Stressful conditions in a bacterial cell inhibit the synthesis of standard proteins while inducing The production of a small group of stress-response proteins.

A modification is an alteration in microorganisms induced by environmental conditions. Only phenotypic traits are affected. Bacterial modifications are strictly temporary—occurring only during the direct exposure to the factor—and disappear once the factor is removed. The Role of phenotypic variability is to ensure the survival of the microbial population in altered environments. Modifications are expressed through changes in morphological, physiological, and other traits, with a return to the original phenotype once the triggering factor is eliminated.

Genotypic variability manifests as mutations and recombination, driven by alterations in the Introduction/19.html">Primary Structure of the genetic apparatus. Genotypic changes occur naturally as rare events in the life of a microbial population; they are non-directional and arise spontaneously.

A mutation (from Latin mutatio — change) is a stable, heritable alteration in The structure of the genetic apparatus. The mechanism involves the deletion, insertion, or substitution of a single nucleotide pair or a group of nucleotide pairs within the DNA molecule, as well as shifts in their sequential arrangement. Based on their origin, mutations are classified as spontaneous or induced.

Spontaneous mutations occur within a microbial population without apparent external influence and manifest as self-directed alterations in the cell's genome. Common types of spontaneous mutations include phage resistance, auxotrophy, and Antibiotic Resistance. The frequency of spontaneous mutations ranges from 1 in 10⁵ to 10⁶ cells within a population. In nature, spontaneous mutations serve as the primary source of natural microbial variability and form the foundation of the evolutionary process in both pro- and eukaryotes. Spontaneous mutations typically affect a single trait and are generally stable.

Induced mutations are triggered experimentally through exposure to physical, chemical, or biological agents.

Physical agents include various types of radiation (X-rays, ultraviolet, γ-radiation). The possibility of induced mutations was first demonstrated in 1925 by G. A. Nadson and G. S. Filippov, who exposed Yeast cells to X-rays. The frequency of induced mutations is significantly higher than that of spontaneous ones (ranging from 1 in 10³ to 10⁴) and depends on The Nature and dosage of the mutagenic agent. UV irradiation with a wavelength of approximately 260 nm is the most accessible mutagenic agent. The Mechanism of damage involves The formation of thymine dimers in the DNA molecule, which blocks normal replication. X-ray and γ-irradiation cause Various Forms of damage in bacterial DNA, including strand breaks and chemical modifications of NUCLEOTIDES. The damage effect is directly proportional to the radiation dose.

Chemical Mutagens possess high mutagenic potency. Their Mechanisms of action on microbial cell DNA vary considerably. Some act by altering the DNA Structure. For example, 5-bromouracil (5BU)—a thymine analogue that differs from thymine solely by the presence of a bromine atom in place of the CH₃ group—pairs with guanine instead of adenine during replication, resulting in an A-T to 5BU-G base pair substitution. Treatment with nitrous acid, which causes deamination of nitrogenous bases, converts cytosine into uracil and adenine into hypoxanthine. Uracil pairs with adenine rather than guanine (like cytosine), triggering a G-C to A-T mutation. Acridine Dyes induce insertions or deletions of nitrogenous bases in DNA. Nitroso compounds exhibit pleiotropic effects, inducing a high mutation frequency, and are therefore termed supermutagens. The most potent mutagens include nitrosomethylurea, ethylenimine, alkylating agents (such as sulfur mustard, nitrosoguanidine, and ethyl methanesulfonate). Nitrous acid (HNO₂) is considered a relatively mild mutagen.

Biological factors primarily include mobile elements: transposons, IS elements, and viral DNA. The translocation of mobile elements (transposition) occurs at a frequency ranging from 10^-4 to 10^-6. IS elements do not encode any phenotypic traits; they contain only the information necessary for their intracellular movement. More complex transposons carry genes responsible for cellular resistance to Antibiotics, heavy Metal Ions, and other inhibitors.

Temperate phages and specific plasmids act as vectors for mobile elements between cells. The integration of mobile elements into the bacterial chromosome generates mutant cells with altered nucleotide triplets within DNA codons, thereby disrupting METABOLISM/31.html">Transcription. The mutagenic effects of Viruses and live viral Vaccines on mammals have also been well-documented.

Depending on the number of mutated genes and The Nature of the alterations in the primary DNA structure, mutations are classified as gene or chromosomal mutations.

Gene mutations affect only a single gene and are most often point mutations. The latter involve the deletion, insertion, or substitution of a single nucleotide pair.

Chromosomal mutations span multiple genes. They involve major structural rearrangements in specific DNA segments and manifest as deletions (losses), inversions (180° rotations), or duplications (repetition of a DNA fragment). One mechanism driving chromosomal mutations involves the movement of IS elements and transposons from one DNA region to another or between replicons.

Combinative variability. The second type of hereditary variability involves changes that occur in prokaryotes As a result of genetic material recombination through the partial merger of two cellular genomes. There are Three Main Mechanisms leading to prokaryotic genetic recombination: transformation, Transduction, and conjugation, which differ in how chromosomal DNA is transferred.

Transformation (from Lat. transformatio — transformation, conversion) is the direct transfer of genetic material from a donor cell to a recipient cell via the penetration of a foreign DNA fragment. The phenomenon of Bacterial Transformation was first demonstrated in 1928 by the English microbiologist F. Griffith.

The Essence of the experiment was as follows. Mice were simultaneously injected with two pneumococcal strains: a non-pathogenic, unencapsulated strain (R-strain) and a pathogenic, encapsulated strain (S-strain) that had been killed by heat. Most of the mice died of Pneumonia. Live encapsulated pneumococcal cultures were isolated from the Organs of the dead mice. It turned out that the heat-killed encapsulated pneumococcal culture induced the transformation of live unencapsulated microbes within the animal's body, endowing them with The ability to form a capsule, which accounted for their pathogenicity. In 1944, a group of scientists (O. Avery, C. MacLeod, and M. McCarty) replicated Griffith's experiment in vitro: DNA extracted from a virulent pneumococcal strain was added to a culture of a non-virulent unencapsulated pneumococcal strain. The non-virulent strain acquired pathogenic properties. Thus, the scientists proved that the transforming agent (in this case, the carrier of pneumococcal virulence) is DNA.

Bacterial transformation occurs with a frequency of 10^-2–10^-3 and depends on the microorganism species, the Properties of the transforming DNA, and the physiological state of the recipient cell. The ability of DNA to penetrate a recipient cell depends both on the Nature of the DNA itself and on the physiological state of that cell. Cells capable of taking up donor DNA are called competent. The state of competence is short-lived. It arises during a specific period of bacterial culture growth, most often at the end of the exponential phase. In the competent state, the Bacterial Cell wall becomes permeable to high-polymer DNA fragments.

A very small DNA fragment (corresponding to 0.3% of the bacterial chromosome, or roughly 15 genes) penetrates the recipient cell. Various traits can be transferred via transformation: capsule formation, the ability to synthesize Enzymes, and resistance or susceptibility to antibiotics.

During transformation, recombination occurs only if the donor and recipient bacterial DNA are related yet have different genotypes.

Transduction (from Lat. transductio — transfer, movement) is the transfer of genetic material from a donor cell to a recipient cell mediated by a temperate bacteriophage. The phenomenon of transduction was discovered in 1952 by N. Zinder and J. Lederberg using two Salmonella strains.

The mechanism of transduction is as follows. During the reproduction of certain temperate phages, a small fragment of the bacterial chromosome, containing one or several linked genes, is incorporated into the phage genome. The transducing phage transfers this DNA fragment from the previous host to another phage-susceptible cell.

There are Three types of transduction: generalized, specialized, and abortive. Generalized transduction involves the simultaneous transfer of various traits; specialized transduction provides for the transfer of only a specific trait; in abortive transduction, the piece of foreign DNA transferred by the bacteriophage into the recipient cell is not integrated into its genome, meaning no recombination occurs and the new trait is not expressed.

Under natural conditions, phage-mediated transfer of genetic material may be the most widespread mechanism of recombination in prokaryotes. In Genetic Engineering experiments, transduction opens up possibilities not only for interspecies bacterial Hybridization but also for producing hybrids among taxonomically distant prokaryotic groups.

Conjugation (from Lat. conjugatio — connection) is The process of establishing cellular contact between two sexually differentiated bacteria. This phenomenon was discovered in 1946 by D. Lederberg and E. Tatum in mutant strains of E. coli. During bacterial conjugation, There is a directed transfer of genetic material from a donor cell to a recipient cell.

The ability of bacteria to conjugate is associated with the presence of a sex factor (F-factor, from English fertility) in some of them. Such cells are designated as F⁺. The F-factor is located in the Cell Cytoplasm as circular DNA, meaning it is a plasmid. Cells lacking the F-factor act as recipients and are designated as F^-.

The conjugation process begins with the attachment of the tip of the donor cell's F-pilus to the recipient cell. Within a few minutes, the two cells draw close together, and then, via the conjugation bridge in less than 5 min, the sex F-factor is transferred independently of the bacterial chromosome from the donor cell (F⁺) to the recipient cell (F^-). At the same time, the donor cell does not lose its donor capacity, as copies of the F-factor remain within it. If the F-factor integrates into the chromosome, the bacteria acquire the ability to transfer chromosomal DNA fragments and are termed Hfr cells (from English high frequency of recombination). During the conjugation of Hfr and F^- cells, one strand of the chromosome breaks and is transferred starting from a specific region into the F^- cell. The DNA strand remaining in the donor cell serves as a template for synthesizing a second strand, while the second transferred donor DNA interacts with the recipient's DNA—resulting in Homologous Recombination. By interrupting bacterial conjugation, one can determine the relative gene order on the chromosome. Because conjugation involves only a partial transfer of genetic material, it should not be equated with the sexual process found in other organisms.

12.1. ELEMENTS OF GENETIC ENGINEERING

It is well known that crossing or hybridization among members of the eukaryotic kingdom occurs exclusively between closely related organisms. In prokaryotes, plasmid-mediated hybridization is not restricted even by major taxonomic categories. Consequently, prokaryotes offer boundless opportunities for a novel scientific field—genetic engineering—which consists of constructing hybrids from Materials of entirely disparate origins.

The prerequisites for research in genetic engineering were two discoveries made in the first half of the 20th century: first, the phenomenon of transduction, and second, the presence in bacteriophage-resistant bacteria of special enzymes that cleave double-stranded phage DNA at strictly defined sites. These enzymes were named Restriction Endonucleases (or restriction enzymes). To date, about 500 restriction enzymes have been isolated from various bacteria, exhibiting Specificity for particular DNA sequences (sites). The discovery of plasmids in bacteria laid the foundation for Recombinant DNA technology.

Genetic engineering encompasses Methods FOR PRODUCING recombinant DNA and subsequently introducing it into a recipient cell. Genetic engineering METHODS are based on the ability of restriction enzymes to cleave DNA into discrete nucleotide sequences that can be incorporated into the genomes of bacterial plasmids or phages to yield hybrid or chimeric forms composed of native DNA and additional inserted foreign DNA fragments.

To obtain recombinant DNA, it is first necessary to have a well-established host-vector system. A vector is defined as a small DNA molecule capable of accepting foreign DNA fragments and replicating within a specific organism. Animal and plant viruses, Bacteriophages, or plasmids can serve as vectors. An efficient method for introducing the vector and recombinant molecule into the microorganism is also required. Vector molecules must possess A number of properties that facilitate the convenient introduction of foreign DNA and its subsequent expression. Vector molecules contain specific sites recognized and cleaved by restriction enzymes to generate "sticky ends." Desired foreign genes bearing identical sticky ends are "spliced" into these cleaved sites using the enzyme DNA ligase. Recombinant DNA constructed in this manner is introduced into a bacterial cell or other living cells via transformation, after which the autonomous DNA acquires the ability to function independently and replicates during Cell Division.

Cosmids, which are hybrids of a plasmid and a phage, are also used as vectors. Cosmids are employed for cloning large eukaryotic DNA fragments (up to 45 kb).

Genetic engineering is of immense applied significance. Strains of microorganisms that do not exist in nature have been engineered to produce PROTEINS AND Peptides of high value to humans, notably E. coli strains that synthesize Insulin. The method of producing human insulin by cultivating a recombinant E. coli strain solved The problem of supplying diabetes patients with this medication. Genetic engineering is also utilized to manufacture products derived from scarce or expensive raw materials. Efforts are underway to improve yeast strains used in brewing and winemaking. These organisms are engineered to acquire genes that ensure the utilization of pentoses, The breakdown of Phenolic Compounds, and competitive growth under non-sterile conditions.