MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 4. BACTERIAL GENETICS

GENETIC RECOMBINATIONS

Genetic recombination involves the partial integration of genomes from two Cells, meaning Bacteria exhibit a process analogous to sexual reproduction. It differs from Sexual reproduction in eukaryotic forms in that bacteria do not form Gametes, nor do they undergo Cell Fusion or complete genome pooling. Only a portion of the donor cell's genetic material is transferred to the recipient cell, resulting in an incomplete zygote known as a merozygote. The genetic material of the recipient cell is referred to as endogenous, whereas the transferred fragment is exogenous. However, the core event of the sexual process—the EXCHANGE OF GENETIC material—does take place, and this phenomenon is termed genetic recombination. The replacement process involves Enzymes that cut the DNA molecule, namely Restriction Endonucleases (restriction enzymes), and enzymes that join the new DNA strands together, known as ligases. The DNA formed As a result of genetic recombination is called recombinant DNA. Descendants of recombinants exhibit notable trait diversity driven by Gene mixing. The Diversity of traits arising in recombinants is crucial for evolution and represents the primary advantage of sexual reproduction.

In bacteria, The formation of recombinant Chromosomes occurs through three Mechanisms of Genetic information transfer: transformation, conjugation, and Transduction.

Transformation (from Latin transformatio meaning transformation) is the process by which genetic material is transferred from a donor (from Latin donare meaning to give) to a recipient (from Latin recipiens meaning to receive) via an isolated DNA fragment.

The phenomenon of transformation was first described in 1928 by F. Griffith, who worked with pneumococci, the causative agents of Pneumonia. The Streptococcus pneumoniae culture in the R-form (serotype II, unencapsulated strain) was avirulent, whereas the same culture in the S-form (serotype III, encapsulated strain) caused disease. Upon infecting mice with a mixed culture (live R-form cells + heat-killed S-form cells), the mice died of pneumonia, and virulent pneumococci with S III type capsules were isolated from the Blood of the dead animals (Fig. 4.10). This demonstrated that the heat-killed S III type cells transferred the hereditary capacity to form S III type capsules to the R-cells, which, in turn, passed it on to their offspring.

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Fig. 4.10. Schematic representation of transformation in pneumococci

The Nature of the factors inducing Bacterial Transformation remained unknown for a long time. It was not until 1944 that O. Avery, C. MacLeod, and M. McCarty determined the Chemical Nature of the pneumococcal transforming factor, identifying it as DNA. This discovery served as decisive proof that hereditary information is localized in DNA.

The possibility of trait transfer via naked DNA was subsequently discovered in certain Representatives of the genera Haemophilus, Neisseria, Rhizobium, Bacillus, and other bacteria. Traits such as drug resistance, synthesis of type-specific capsular Polysaccharides, specific protein Antigens, prototrophy for specific Amino Acids and Vitamins, nodule-forming ability, pigment production, and others are transmitted in this manner. However, it is extremely rare for a cell to acquire more than one new trait through transformation.

Not all bacterial cells are capable of taking up donor DNA. Cells that exhibit this ability are called competent. Competency is the ability of cells to take up DNA and integrate it into their own genome. The mechanisms of competence are not yet fully understood, but it has been proven that competent cells synthesize a protein whose function is also not completely established. It may be a membrane component that catalyzes The entry of DNA into The Cell, or an enzyme that cleaves Cell wall components and exposes receptor sites for DNA. This protein has been named competence factor (cpf). Other factors associated with competence have also been identified, such as the iF-factor, which exhibits nuclease activity. Both factors are inhibited by erythromycin, an inhibitor of Protein Synthesis. The state of competence varies in duration among different bacterial species, but averages ~15 min.

Certain bacteria, such as Escherichia coli, are non-competent. To reproduce transformation in such bacteria, preliminary Treatment is required (such as exposure to Ca²⁺ ions, deep freezing and thawing, etc.) to increase cell wall permeability to the transforming DNA.

Transformation occurs most efficiently in experiments involving bacteria of the same species with different genotypes. Thus, by Means of transformation in various strains of the same bacterial species, wild-type genes can be replaced with mutated ones. Transformation can also be observed between bacteria of different species; however, the more genetically distant they are, the more difficult it is to reproduce this phenomenon, although the existence of interspecies transformation is unquestionable.

Transforming activity is exhibited by double-stranded DNA fragments with a Molecular Weight of at least 0.5–1.0 × 10⁶. The transformation process can be divided into several stages (Fig. 4.11):

- adsorption of the transforming DNA molecule onto the bacterial cell, lasting 5–10 min. If DNase is applied during this time, transformation does not occur. Enzyme treatment 10 min after THE START OF the experiment does not interrupt transformation;

- penetration of DNA into the recipient cell;

- pairing of the transforming DNA with the homologous region of the recipient chromosome, followed by recombination.

Fig. 4.11. One of the possible pathways of transformation:

1 - donor DNA; 2 - active uptake; 3 - donor DNA becomes single-stranded; 4 - donor DNA strand replaces the original, non-identical recipient DNA strand; 5 - displaced recipient DNA fragment is eventually degraded; 6 - DNA Hybridization; 7 - Replication of hybrid DNA

Thus, the STUDY OF BACTERIAL transformation revealed The Significance of Selection/9.html">Nucleic Acids AS carriers of hereditary information.

Conjugation (from Latin conjugatio meaning connection or pairing) is The process of genetic material exchange between two cells via direct physical contact. This phenomenon was first described in 1946 by J. Lederberg and E. Tatum. The authors drew Conclusions regarding the possibility of genetic exchange between bacteria using a model of auxotrophic mutants of Escherichia coli, each of which individually was incapable of synthesizing two different factors necessary for survival. One double mutant

was unable to synthesize the vitamin biotin (bio^-) and The amino acid Methionine (met^-), while the second was incapable of synthesizing the amino acids Threonine (thr^-) and leucine (leu^-). Neither of these mutants could grow on a minimal medium. When a mixture of both mutants was plated onto the same minimal medium, colonies appeared, and the cells of these colonies exhibited the hereditary ability to synthesize all the growth factors necessary for them, meaning they belonged to the bio⁺met⁺thr⁺leu⁺ type. Consequently, some form of Genetic information exchange had taken place. Subsequently, Electron Microscopy established that E. coli cells can come into direct contact with one another, i.e., conjugate (Fig. 4.12). Such cells appeared at a frequency of ~1 × 10⁶.

Fig. 4.12. Recombination resulting from conjugation between two complementary mutants of Escherichia coli

These were genetic recombinants that arose through the combination of genetic material from two reciprocally defective (complementary) parental cells. The Use of double mutants as parental forms prevented the appearance of revertants, since the probability of simultaneous reversion at two genes is ~10^-14-10^-16 per generation.

Analogous crossing experiments, in which one of the parental strains was streptomycin-resistant, led to the Conclusion that genetic material is transferred directionally. When cells were plated on streptomycin-containing medium after mating, recombinants appeared only if the recipient strain was streptomycin-resistant and survived. The behavior of the other strain was not critical—as long as it successfully performed its function of transferring genetic material.

Fig. 4.13. Electron micrograph of two conjugating cells

From this, it followed that genetic material is transferred in one direction—from the donor ("male" strain) to the recipient ("female" strain); the entire process of recombination and trait segregation occurs within the recipient cells.

The donor ability of cells is determined by genes localized in a small circular DNA molecule called the sex factor, or F-factor (from English *fertility*). Its size is approximately equivalent to phage DNA. This is a unique plasmid that encodes the protein for specific pili (F-pili or sex pili). F-pili facilitate cell contact. Cells lacking the F-factor function as recipients (F^- cells) and conjugate exclusively with male cells. Cells possessing the sex factor (F⁺ cells) are capable of functioning as both Donors and recipients, meaning that conjugation can occur via F⁺ x F^- or F⁺ x F⁺ cell interactions, but is impossible in the presence of F^--type cells alone.

The plasmid DNA molecule consists of two strands. During conjugation, one of the strands of the double-stranded F-factor DNA passes through a sex pilus from the donor cell (F⁺) into the recipient cell (F^-). This factor is retained in the donor cell while a copy of its own is synthesized in the recipient cell. Thus, the population of F^- cells gradually acquires the F⁺ type. Donor cells can spontaneously lose the F-factor and thereby become F^- cells.

In approximately one out of 10⁵ cases, the F-factor is capable of integrating into the host cell's chromosomal DNA molecule (Fig. 4.14). In this case, not only the F-factor but also a portion of the chromosomal DNA is transferred during conjugation. This process takes ~90 min, but cells may separate earlier, prior to complete DNA exchange. Such donor strains continuously transfer all or most of their DNA to other cells and are termed Hfr strains (from English *high frequency of recombination*), meaning that genes localized in the donor cell chromosome are transferred to recipient cells with high frequency.

Fig. 4.14. Conversion of F⁺ bacteria into Hfr:

the circular F-factor DNA carrying the O site (a) pairs with the circular bacterial chromosome DNA between the ton and lac genes (b). Reciprocal Crossing-over between the F factor and the bacterial chromosome results in the Integration of the F factor into the chromosome (c), accompanied by the formation of an Hfr strain which, upon conjugation with an F^- cell, transfers the ton gene first and the lac gene last

During conjugation, a break occurs in the chromosome at the F-factor integration locus; it becomes linear and is transferred in this state into the recipient cell starting from the proximal O-end, which is opposite to the site of F-factor integration. Marker transfer proceeds sequentially along the length of the chromosome, with the sex factor transferred last. During this period, the integrity of the chromosome or the conjugation bridge is frequently disrupted (broken), and the F-factor is rarely transferred from Hfr cells to F^- bacteria.

F' cells are intermediate "male" cells in which the sex factor exists in an autonomous state and carries various segments of the bacterial chromosome, such as the lactose Fermentation trait F' lac (see Fig. 4.15). Gene transfer mediated by the sex factor is termed sexduction.

Fig. 4.15. Formation of the F' sex factor carrying a bacterial chromosome lac gene, or F-lac:

the F factor integrated into the Hfr chromosome between the ton and lac genes (a) forms an aberrant loop (b), as a result of which the lac region of the chromosome is integrated into the loop (c) carrying the fertility factor genes. Reciprocal crossing-over leads to the formation of a circular F factor harboring the lac gene (d)

The conjugation bridge is unstable and easily breaks without compromising the viability of the conjugating cells. Consequently, chromosomal integrity may be impaired during transfer. All of this explains the relatively infrequent transfer of the F factor from Hfr cells to F^- bacteria, since the latter must receive both the initial and terminal segments of the donor chromosome for this to occur.

The process of genetic material transfer during conjugation has been studied primarily in members of the family Enterobacteriaceae: the genera Escherichia, Shigella, Salmonella, as well as in Pseudomonas aeruginosa. As established, conjugation can be:

- intraspecific – between strains of the same species;

- interspecific – between species of the same genus (among shigellae);

- intergeneric – between species of different genera, for example, between Escherichia coli and salmonellae.

Transduction is the process of genetic material transfer from a donor cell to a recipient cell mediated by a bacteriophage. In other words, the phage acts as a "gamete," transferring a fragment of the donor cell DNA into the recipient cell.

Three MAIN TYPES OF transduction are known: generalized (nonspecific), localized (specific), and abortive. In nonspecific transduction, any DNA fragments can be transferred from donor to recipient cells with the participation of temperate transducing phages. The donor DNA fragment carried by the phage is capable of integrating into the homologous region of the recipient cell via recombination.

Nonspecific transduction can be demonstrated using two Salmonella typhimurium mutants that differed in a single trait. If the donor strain (met+) is infected with temperate phage P22 and then, after lysis, free phage is introduced into a suspension of recipient strain cells (met^-), some cells acquire the met+ trait (see Fig. 4.16). In this setup, the recipient strain (L-22) is lysogenic for phage P22. If free phage P22 infects Cells of the donor strain (L-2), it lyses them, releasing normal phage particles, some of which contain donor DNA fragments. Upon incubating this phage lysate with cells of the recipient strain L-22, the phage infects these cells. However, since this strain is not lysogenic for it, lysis does not occur, and the donor DNA fragments contained within the phage are taken up by the cells. If this donor DNA carries a gene that the recipient cell lacks (or in which it is defective), complementation (mutual supplementation) may result. The donor DNA fragment transferred by the phage integrates into the homologous region of the recipient cell DNA via recombination.

Fig. 4.16. Schematic diagram of transduction using the prototrophic met⁺ strain as a donor and the met^- auxotrophic mutant as a recipient

Thus, transduction involves a donor cell, a transducing phage, and a recipient cell. The donor bacterium determines the Specificity of the transduced trait, while the phages merely transfer genetic material to the recipient cell. During generalized transduction, phages transfer various genes encoding The ability to ferment CARBOHYDRATES, form flagella in non-motile bacterial forms, Antibiotic Resistance, sporulation, synthesis of amino acids, enzymes, vitamins, etc.

Transducing phages are typically defective because a single phage particle cannot accommodate its entire own DNA alongside a fragment of the bacterial chromosome. Using heavy DNA (labeled with 5-bromouracil), it has been shown that some transducing fragments contain exclusively bacterial DNA. It is hypothesized that during phage maturation within the donor bacterial cell, fragments of the bacterial chromosome are packaged into the capsid instead of the phage DNA. Each such phage particle is capable of transducing only a single bacterial gene or a few closely linked genes. The amount of bacterial DNA corresponding to the total phage DNA is no more than 1-2% of the total DNA content of the bacterial cell. The frequency of co-transduction of two bacterial genes serves as a criterion for the distance between them and can be utilized in constructing chromosome maps.

In specialized (localized) transduction, the phage transfers only specific genes from donor cells to recipient bacteria. This occurs because the formation of the transducing phage proceeds via the integration of its DNA exclusively with specific bacterial genes located on the donor cell chromosome adjacent to the prophage (a linked gene cluster). For example, phage λ locates on the bacterial chromosome next to the gal region (galactose fermentation) and specifically transduces the gal⁺ gene into gal^- recipient cells, or the bio gene, which determines biotin synthesis, as they are linked to the phage DNA on the bacterial chromosome (Fig. 4.17). Phage φ 80 positions itself adjacent to the try region and transduces genes responsible for Tryptophan synthesis.

Fig. 4.17. Diagram of the integration of prophage λ DNA into the bacterial cell chromosome

When such phages interact with recipient cells, lysogenization occurs, and the donor cell gene enters the recipient bacterium's chromosome along with the defective phage DNA. Bacteria lysogenized by the defective phage become immune to subsequent infection by the same virulent phage.

In abortive transduction, the donor chromosome fragment introduced by the phage is not integrated into the recipient cell chromosome; instead, it remains localized in the Cytoplasm, where it can remain functional. During the division of the transduced cell, the donor DNA fragment may be passed on to only one of the two daughter cells—meaning it is inherited unilinearly and is ultimately lost in subsequent generations.

The process of trait transfer via transduction was first discovered in Salmonella typhimurium and subsequently described in numerous other bacteria, including Escherichia, Shigella, Bacillus, Pseudomonas, Staphylococcus, Vibrio, and others.

All three processes—transformation, conjugation, and transduction—despite their morphological differences, are fundamentally identical: each results in The transfer of a DNA fragment from one cell to another. In transformation, free DNA is taken up by the recipient bacterium; in transduction, a phage captures a fragment of the donor bacterium's chromosome and delivers it to the recipient; in conjugation, a segment of DNA is transferred through the formation of a cytoplasmic bridge. Across all three mechanisms of genetic recombination, typically only a fragment of the chromosome, rather than the entire molecule, is transferred.