Genetic recombination through sexual reproduction is an important means of genetic variation in eukaryotes. Although bacteria have no exact equivalent to sexual reproduction, they exhibit a primitive means for sharing or recombining parts of their genomes. An event in which one bacterium donates DNA to another bacterium is a type of genetic transfer termed recombination, the end result of which is a new strain different from both the donor and the original recipient strain. In general, any organism that has acquired genes that originated in another organism is called a recombinant.
Recombination in bacteria depends largely on their extreme versatility in acquiring and expressing the genetic material of other bacteria and even other organisms. This capacity for genetic sharing has tremendous effects on the diversity and evolution of bacteria. Unlike spontaneous mutations, recombinations are generally beneficial. They can provide additional genes for resistance to drugs and metabolic poisons, new nutritional and metabolic schemes, increased virulence, and adaptations to changing environ mental conditions.
Transmission of Genetic Material in Bacteria
DNA transfer between bacterial cells typically involves small pieces of DNA in the form of plasmids or chromosomal fragments. Plasmids are small, circular pieces of DNA that contain their own origin of replication and therefore can replicate independently of the bacterial chromosome. Plasmids are found in many bacteria (as well as some fungi) and typically contain a few dozen genes. Although plasmids are not necessary for bacterial survival, they often carry adaptive genes, such as those for drug resistance.
Chromosomal fragments that have escaped from a lysed bacterial cell are also commonly involved in the transfer of genetic information between cells. An important difference between plasmids and fragments is that, while a plasmid can be stably replicated and inherited, chromosomal fragments must integrate themselves into the bacterial chromosome in order to be replicated and eventually passed to progeny cells. The rate of natural genetic recombination varies a great deal among bacteria, but its frequency can be increased in the laboratory, where the ability to transfer genes between organisms has become an important tool of research and technology.
Depending upon the mode of transmission, the means of genetic recombination in bacteria is called conjugation, transformation, or transduction. Conjugation requires the attachment of two cells and the formation of a bridge that can transport DNA. Trans formation entails the transfer of free DNA and requires no special vehicle. Transduction is DNA transfer mediated through the action of bacterial viruses (table1).
Table1. Types of Genetic Recombination in Bacteria
Conjugation: Genetic Transmission through Direct Contact
Conjugation is a mode of genetic recombination in which a plasmid or fragment of DNA is transferred from a donor cell to a recipient cell via a direct connection. Both gram-negative and gram-positive bacteria can conjugate, but only gram-negative cells operate with a specialized plasmid called a fertility, or F, factor. This plasmid directs the synthesis of a unique pilus, also called a sex pilus,8 that functions in most conjugation transfers. The recipient cell is usually a related bacterium with a recognition site on its surface for inter acting with the pilus (process figure1, step 1).
Fig1. Process Figure 1 Conjugation: Genetic transmission through direct contact between two cells.
A cell’s role in conjugation is denoted by F+ for the cell that has the F plasmid and by F− for the cell that lacks it. Contact is made when a pilus grows out from the F+ cell, attaches to the sur face of the F− cell, contracts, and draws the two cells together (process figure 1, step 1). At the site where the pilus attaches the two cells, a mating or conjugative bridge is formed that serves as a transfer system for the plasmid. The latest reliable information indicates that the pilus becomes part of a complex secretory system that opens a gateway between the cell walls and membranes of the two cells and serves as a passageway for the plasmid.
There are hundreds of conjugative plasmids with some variations in their properties. One of the best understood plasmids is the F factor in E. coli, which exhibits these patterns of transfer:
1. Because bacteria tend to conserve plasmids, the donor cell first makes a copy of the F factor, which is simultaneously transferred across the bridge (process figure1, step 2). This way, it retains the original plasmid, remains F+, and can continue to conjugate with other bacteria. The F− cell has been changed into an F+ cell capable of producing a pilus and conjugating with other cells. No additional donor genes are transferred during this transfer.
2. In high-frequency recombination (Hfr) donors, the fertility factor has been integrated into the F+ donor chromosome.
The term high-frequency recombination denotes that a cell with an integrated F factor transmits its chromosomal genes at a higher frequency than other cells. This is because the F factor can direct a more comprehensive transfer of part of the donor chromo some to a recipient cell. This transfer occurs through replication of the donor chromosome along with the F factor. One strand of DNA is retained by the donor, and the other strand is transported across to the recipient cell (process figure 1, step 3). The F factor may not be transferred during this process. The transfer of an entire chromosome takes about 100 minutes, but the pilus bridge between cells is ordinarily broken before this time, and rarely is the entire genome of the donor cell transferred.
Conjugation has great biomedical importance. Special resistance (R) plasmids, or factors, that bear genes for resisting antibiotics and other drugs are commonly shared among bacteria through conjugation. Transfer of R factors can confer multiple resistance to antibiotics such as tetracycline, chloramphenicol, sulfonamides, and penicillin. Other types of R factors carry genetic codes for resistance to heavy metals (nickel and mercury) or for synthesizing virulence factors (toxins, enzymes, and adhesion molecules) that increase the pathogenicity of the bacterial strain. Conjugation studies have also provided a means to map the bacterial chromosome.
A few gram-positive bacteria (Bacillus and Streptococcus, for example) are known to recombine through conjugation. Because they lack pili, they must transport the plasmids via specialized proteins activated during adhesion of the two cells. The mechanisms involved are yet to be fully worked out.
Transformation: Capturing Free DNA from Solution
One of the cornerstone discoveries in microbial genetics was made in the late 1920s by the English physician and bacteriologist Frederick Griffith working with Streptococcus pneumoniae and laboratory mice (figure 2). The pneumococcus exists in two major strains based on the presence of the capsule, colonial morphology, and pathogenicity. Encapsulated strains bear a smooth (S) colonial appearance and are virulent; strains lacking a capsule have a rough (R) appearance and are nonvirulent. (Recall from chapter 4 that the capsule protects a bacterium from the phagocytic host defenses.) Griffith first showed that when mice were injected with a live, virulent (S) strain, they soon died. Mice injected with a live, nonvirulent (R) strain remained alive and healthy. Next he tried a variation on this theme. He heat-killed an S strain and injected it into mice, which remained healthy. Then came the ultimate test: Griffith injected both dead S cells and live R cells into mice, with the result that the mice died from pneumococcal blood infection. If killed bacterial cells do not come back to life and the nonvirulent live strain was harmless, why did the mice die? Although he did not know it at the time, Griffith had demonstrated that dead S cells, while passing through the body of the mouse, broke open and released some of their DNA (by chance, a segment containing the genes for making a capsule). A few of the live R cells subsequently picked up this loose DNA and were transformed by it into virulent, capsule-forming strains.
Fig2. Griffith’s classic experiment in transformation. In essence, this experiment proved that DNA released from a killed cell can be acquired by a live cell. The cell receiving this new DNA is genetically transformed—in this case, from a nonvirulent strain to a virulent one. Barry Chess/McGraw Hill
Later studies supported the concept that a chromosome released by a lysed cell breaks into fragments small enough to be accepted by a recipient cell and that DNA, even from a dead cell, retains its genetic code. This nonspecific acceptance by a bacterial cell of small fragments of soluble DNA from the surrounding environment is termed transformation (figure 3). Transformation is facilitated by special DNA-binding proteins on the cell wall that capture DNA from the surrounding medium. Cells that are capable of accepting genetic material through this means are termed competent. The new DNA is passed through a DNA up take system in the cell wall and membrane. Within the cytoplasm, the donated strand is inserted into the chromosome of the recipient cell, now said to be transformed. Natural transformation is found in several groups of gram-positive and gram-negative bacterial species. In addition to genes coding for the capsule, bacteria exchange genes for antibiotic resistance and bacteriocin synthesis in this way.
Fig3. Steps in bacterial transformation as seen in S. pneumoniae. This shows how genes coding for capsule formation are brought into the cell and inserted into the chromosome. Cap+ indicates genes for forming a capsule.
Because transformation requires no special appendages, and the donor and recipient cells do not have to be in direct contact, the process is useful for applications in recombinant DNA technology. With this technique, foreign genes from a completely unrelated organism are inserted into a plasmid, which is then introduced into a competent bacterial cell through transformation. These recombinations can be carried out in a test tube, and human genes can be experimented upon and even expressed outside the human body by placing them in a microbial cell. Transformation is often used in the production of genetically engineered organisms such as yeasts, plants, and animals.
Transduction: Viruses as Vectors
Bacteriophages (bacterial viruses) have been previously described as destructive bacterial parasites. Viruses can also serve as genetic vectors (an entity that can bring foreign DNA into a cell). The process by which a bacteriophage serves as the carrier of DNA from a donor cell to a recipient cell is transduction. Although it occurs naturally in a broad spectrum of bacteria, the participating bacteria in a single transduction event must be the same species because of the specificity of viruses for host cells.
There are two versions of transduction. In generalized trans duction (process figure4), random fragments of disintegrating host DNA are taken up by the phage during assembly. Virtually any gene from the bacterium can be transmitted through this means. In specialized transduction (process figure 5), a highly specific part of the host genome is regularly incorporated into the virus. This specificity is explained by the prior existence of a temperate prophage inserted in a fixed site on the bacterial chromosome. When activated, the prophage DNA separates from the bacterial chromosome, carrying a small segment of host genes with it. During a lytic cycle, these specific viral-host gene combinations are incorporated into the viral particles and carried to another bacterial cell. Instances of transduction include the transfer of drug resistance seen in Staphylococcus spp. and the transmission of gene regulators in gram-negative rods such as Escherichia and Salmonella.
Process Fig4. Generalized transduction: Genetic transfer by means of a virus carrier. (1) A phage infects cell A (the donor cell) by normal means. (2) During replication and assembly, a phage particle incorporates a segment of bacterial DNA by mistake. (3) Cell A then lyses and releases the mature phages, including the genetically altered one. (4) The altered phage adsorbs to and penetrates another host cell (cell B), injecting the DNA from cell A rather than viral nucleic acid. (5) Cell B receives this donated DNA, which recombines with its own DNA. Because the virus is defective (biologically inactive as a virus), it is unable to complete a lytic cycle. The transduced cell survives and can use this new genetic material.
Fig5. Specialized transduction: Transfer of specific genetic material by means of a virus carrier. (1) Specialized transduction begins with a cell that contains a prophage (red). (2) When the virus enters a lytic cycle, it excises itself from its host cell, carrying along some host DNA. (3) Replication and assembly results in production of a chimeric virus containing viral and bacterial DNA. (4) Release of the recombinant virus and infection of a new host result in transfer of bacterial DNA between cells. (5) Recombination can occur between the new host chromosome and the virus DNA, resulting in either bacterial DNA or a combination of viral and bacterial DNA being incorporated into the bacterial chromosome.
Transposons: “The Case of the Jumping Genes” One type of genetic transferral of great interest involves transposable elements, or transposons. Transposons have the distinction of shifting from one part of the genome to another and so are termed “jumping genes.” When the idea of their existence in corn plants was first postulated by the geneticist Barbara McClintock, it was greeted with skepticism, because it had long been believed that the location of a given gene was set and that genes did not or could not move around. Now it is evident that jumping genes are widespread among prokaryotic and eukaryotic cells and viruses. Results from analysis of human DNA indicates that they make up nearly 45% of the hu man genome.
Transposons contain DNA sequences coding for two enzymes— a transposase and a resolvase—that allow the transposon to excise itself from one area of the genome and integrate itself somewhere else, akin to cutting and pasting a sentence in a word-processing document. Movement may be from one chromosomal site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome (figure 6). Because transposons can occur in plasmids, they can also be transmitted from one cell to another in bacteria and a few eukaryotes. Some transposons replicate themselves before jumping to the next location, and others simply move without replicating first.
Fig6. Transposons: Shifting segments of the genome. (1) A transposon exists as a small piece of DNA integrated into the host cell chromosome (red). (2) The transposon may excise itself and move from one location to another in the genome, maintaining itself as a single copy per cell. (3) It may also replicate prior to moving, leading to an increase in the copy number and a greater effect on the genome of the host. (4) Finally, the transposon may jump to a plasmid, which can then be transferred to another bacterial cell.
Flanking the coding region of the transposon DNA are sequences called inverted repeats, which mark the point at which the transposon is removed or reinserted into the genome. The smallest transposons consist of only these two genetic sequences and are often referred to as insertion elements. A type of transposon called retrotransposon can transcribe DNA into RNA and then back into DNA for insertion in a new location. Another example is the integron, which can carry large blocks of genetic material that are especially prominent in transmitting drug resistance.
The overall effect of transposons—to scramble the genetic language—can be beneficial or adverse, depending upon such variables as where insertion occurs in a chromosome, what kinds of genes are relocated, and the type of cell involved. In bacteria, transposons are known to be involved in
● changes in traits such as colony morphology, pigmentation, and antigenic characteristics;
● replacement of damaged DNA; and
● the transfer of drug resistance in bacteria
