Unformatted text preview: Topic 10: Principles of Bacterial Genetics II Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld Learning Objectives: To understand how homologous recombination takes place, and become familiar with different ways in which DNA can enter the cell. We will also discuss plasmids, conjugation, transposons and insertion restriction. Homologous recombination refers to the exchange, or crossing over, between identical or highly similar (=homologous) DNA sequences. The process of recombination (Figure 11.13) is very complex, and involves many different proteins. The most important protein involved in recombination in bacteria is RecA, which is encoded by the recA gene. In this lecture, we will go through the molecular events that take place during homologous recombination. Genetic crosses involving homologous recombination between genetically distinct sequences form the basis of classical genetics. How are genetic crosses involving recombination carried out in bacteria? First, DNA from a donor strain must enter the recipient cell (Figure 11.12). This can occur by transformation, in which naked DNA is taken up by the recipient cell, transduction, in which a phage injects DNA from the donor cell into the recipient cell, or conjugation, in which plasmid or chromosomal DNA is transferred from the donor cell to the recipient cell as a single strand. After the DNA from the donor strain enters the recipient cell, homologous recombination might occur. In order to detect a recombination event, it is necessary that the presence of the genetically distinct but homologous sequences can be differentiated by phenotype of the recombinant organism. Use of selectable markers (Figure 11.14) allows the detection of recombination events that occur at even very low frequencies. Some types of bacterial cells are naturally transformable by DNA, or competent. In organisms that are naturally competent, specific competence proteins aid in the transformation event (Figure 11.16). Some other types of bacterial cells can be made competent by physical or chemical treatment. For example, Escherichia coli cells can be made competent by treatment with calcium ions, and this has made possible the development of E. coli as the preferred host for molecular biology and genetic engineering. Electroporation, a relatively new method in which electric fields are used to create small pores in the cell membranes through which DNA molecules can enter, is gaining in popularity as a method of transformation. Bacteriophage are sometimes able to transfer genes between bacterial strains by transduction. Generalized transduction (Figure 11.17) involves the transfer of DNA from any region of the chromosome. Sometimes, when bacteriophage DNA is packaged into phage particles, some of the host cell's DNA can be accidentally packaged into some of the phage particles. These particles thus contain only host cell DNA, and no phage DNA. They are called transducing particles, because they can inject the packaged DNA from the host cell into an appropriate recipient cell, where it is free to recombine into the Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld recipient cell's genome. Specialized transduction, in contrast to generalized transduction, only involves transfer of DNA from a specific region of the chromosome (Figure 11.18). The replication of plasmids is generally carried out by host cell DNA polymerases. However, plasmids encode genes, which are involved in the regulation of the rate of plasmid replication, and thus determine plasmid copy number within the cell. If the replication of two plasmids is controlled by the same genes, then those plasmids will not be able to be maintained together in the same cell, and they are said to be incompatible. Plasmids that are incompatible with one another are closely related, and belong to the same Inc group. One of the best
studied plasmids is the F plasmid (Figure 11.19). Many plasmids are able to transfer from donor cell to recipient cell in a process called conjugation (Figure 11.21). This transfer process is mediated by the products of tra genes, some of which interact with the oriT region of the plasmid to initiate the transfer of a single strand of DNA, while others form structures such as pili, which aid in the transfer of the DNA to the recipient cell. Besides the genes involved in replication control and conjugation, many other genes can be carried by plasmids (Table 11.1). Some of the first plasmids to be studied were those that encode antibiotic resistance. These are termed resistance plasmids, which are sometimes abbreviated to R plasmids or R factors. A variety of virulence factors are encoded by plasmids. Also, some plasmids encode peptides called bacteriocins that kill closely related bacterial strains that do not carry the plasmid. As well as being able to conjugate themselves between bacterial cells, plasmids are sometimes able to integrate into the bacterial chromosome and transfer part of the chromosome between bacterial cells. Donor cells are called "males", and recipient cells are called "females". Only the donor cells have the sex pilus, which is a structure that is encoded by some of the plasmid
encoded tra genes and helps to bring the donor and recipient cells together (Figure 11.20). Once the cells have been brought together, one strand of the plasmid is nicked at the oriT by a tra
encoded endonuclease, the resulting single strand is unwound from the helix, and is transferred into the recipient cell. The strand that is transferred is replaced in the donor cell by replication, and the complementary strand is also synthesized in the recipient cell (Figure 11.21). Under appropriate conditions, plasmids can spread rapidly within and between bacterial populations by conjugation. How is it that plasmids can integrate into the chromosome and mediate conjugation of part of the chromosome? Some plasmids, such as F, have insertion sequences (IS), which are specific DNA sequences that are also found on the chromosome, and homologous recombination can therefore occur between the plasmid and the Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld chromosome at these sequences (Figure 11.22). Strains that have F integrated in the chromosome are called Hfr (for "high frequency of recombination"). A given Hfr strain will be able to transfer a particular gene at a characteristic frequency. Genes that are nearest the site of insertion in the direction of transfer are transferred at the highest frequency (Figure 11.25). This is because the genes nearest the oriT of the integrated F (Figure 11.23) will be most likely to be transferred into the recipient cell before the DNA strand is broken. The sequential nature of gene transfer can be demonstrated using interrupted mating experiments (Figure 11.27), and this property can be used to map genes. In order to detect a chromosome conjugation event between donor and recipient cells, it is necessary to use appropriate markers and selection. Usually, this entails the use of a combination of antibiotic resistance and auxotrophic markers (Figure 11.26). The conditions for selection must be such that the desired transconjugant organisms are able to grow, but the donor and recipient are not able to grow. Sometimes, an integrated F plasmid is able to excise from the chromosome, and sometimes when it does this, it brings along part of the chromosome as well. This results in a plasmid that carries chromosomal genes. Such a plasmid is called a F' plasmid. Complementation analysis can be used to determine whether two parental strains have mutations in the same gene. For example (as shown in Figure 11.28) if DNA from one Trp
mutant strain is introduced into another Trp
mutant strain, and Trp+ progeny are recovered, then this means that the two trp mutations are in different genes. If no Trp+ progeny are recovered, then the two trp mutations are in the same gene. Some genes are able to move from one part of the genome to another at low frequencies. This process is called transposition, and is carried out by transposable elements. Insertions sequences, such as those found on F, are transposable elements, as are transposons. These elements contain genes that encode transposase enzymes, which recognize terminal inverted repeat sequences (Figure 11.30). The transposition process is illustrated in Figures 11.31 and 11.32. You should note that although transposition is a recombination process, it does not involve homologous recombination, but rather site
specific recombination. Transposition is actually a very useful tool for mutagenesis. If a transposon inserts within a gene, it will disrupt the function of that gene (Figure 11.33). It is possible to set up a transposition experiment where a transposon that confers antibiotic resistance is introduced into a recipient cell, and all selected antibiotic resistant colonies contain a transposon insertion at a unique location. The insertion containing colonies can then be screened for the desired phenotype. ...
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