Bacteria do not have an obligate sexual reproductive stage in their life cycle, but they can be very active in the exchange of genetic information. The genetic information carried in the DNA can be transferred from one cell to another; however, this is not a true exchange, because only one partner receives the new information. In addition, the amount of DNA that is transferred is usually only a small piece of the chromosome. There are several mechanisms by which this takes place. In transformation, bacteria take up free fragments of DNA that are floating in the medium. To take up the DNA efficiently, bacterial cells must be in a competent state, which is defined by the capability of bacteria to bind free fragments of DNA and is formed naturally only in a limited number of bacteria, such as Haemophilus, Neisseria, Streptococcus, and Bacillus. Many other bacteria, including E. coli, can be rendered competent artificially under laboratory conditions, such as by exposure to solutions of calcium chloride (CaCl2). Transformation is a major tool in recombinant DNA technology, because fragments of DNA from one organism can be taken up by a second organism, thus allowing the second organism to acquire new characteristics. Transduction is the transfer of DNA from one bacterium to another by means of a bacteria-infecting virus called a bacteriophage. Transduction is an efficient means of transferring DNA between bacteria because DNA enclosed in the bacteriophage is protected from physical decay and from attack by enzymes in the environment and is injected directly into cells by the bacteriophage. However, widespread gene transfer by means of transduction is of limited significance because the packaging of bacterial DNA into a virus is inefficient and the bacteriophages are usually highly restricted in the range of bacterial species that they can infect. Thus, interspecies transfer of DNA by transduction is rare. Conjugation is the transfer of DNA by direct cell-to-cell contact that is mediated by plasmids (nonchromosomal DNA molecules). Conjugative plasmids encode an extremely efficient mechanism that mediates their own transfer from a donor cell to a recipient cell. The process takes place in one direction since only the donor cells contain the conjugative plasmid. In gram-negative bacteria, donor cells produce a specific plasmid-coded pilus, called the sex pilus, which attaches the donor cell to the recipient cell. Once connected, the two cells are brought into direct contact, and a conjugal bridge forms through which the DNA is transferred from the donor to the recipient. Many conjugative plasmids can be transferred between, and reproduce in, a large number of different gram-negative bacterial species. Plasmids vary in size, from a few thousand to more than 100,000 base pairs; the latter are sometimes called megaplasmids. The bacterial chromosome can also be transferred during conjugation, although this happens less frequently than plasmid transfer. Conjugation allows the inheritance of large portions of genes and may be responsible for the existence of bacteria with traits of several different species. Conjugation also has been observed in the gram-positive genus Enterococcus, but the mechanism of cell recognition and DNA transfer is different from that which occurs in gram-negative bacteria. Horizontal gene transfer (HGT) is the movement of genetic material between organisms. It plays a key role in bacterial evolution and is the primary mechanism by which bacteria have gained antibiotic resistance and virulence. Scientists have studied how HGT occurs in nature and have learned how to introduce genetic materials into cells in the lab. The introduction of foreign DNA or RNA into bacteria or eukaryotic cells is a common technique in molecular biology and scientific research. There are multiple ways foreign DNA can be introduced into cells including transformation, transduction, conjugation, and transfection. Transformation, transduction, and conjugation occur in nature as forms of HGT, but transfection is unique to the lab. Let’s take a look at these different methods of DNA insertion.  TransformationTransformation is the uptake of genetic material from the environment by bacterial cells. In nature, this genetic material often comes from adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment. Various factors promote natural transformation in different bacteria such as growth phase of the cells (Baltrus and Guillemin, 2006) or the presence of specific substances (Meibom et al., 2005). Though not all bacteria are naturally competent to take up DNA, they can be made competent through chemical manipulation in the lab. This is commonly done using calcium chloride which permeabilizes the cell membrane so the bacteria can easily uptake your plasmid of interest. Scientists can also use electroporation, the application of an electrical charge to cells, to increase cell membrane permeability and thus transformation efficiency. Check out Addgene’s blog to learn about making your own competent cells and our protocols page to learn about bacterial transformation in the lab.
TransductionTransduction occurs when foreign DNA or RNA is introduced into bacterial or eukaryotic cells via a virus or viral vector. One example are bacteriophages that attach to bacterial membranes and inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles: lytic or lysogenic. Lytic phages hijack the bacterial hosts machinery to make more viral particles. Eventually the cell lyses releasing the newly formed viral particles that can infect other bacteria. In the lysogenic cycle, the phage’s genetic material is incorporated into the host’s genome at a particular integration site. The integrated phage remains dormant until it is triggered to enter the lytic cycle. During both of these life cycles bacterial DNA can be accidentally packaged into the newly created phages. Transfer of this DNA to another cell is referred to as transduction. Transferred DNA once inside the infected bacterium can either exist as transient extrachromosomal DNA, like a plasmid, or it can integrate into the host bacterium’s genome through homologous or site directed recombination. Transduction is a common tool used by scientists to introduce different DNA sequences of interest into a bacterial cell or a host’s genome. To do this scientists commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties. The phagemids are packaged into replication-incompetent phage particles with assistance from a ‘helper’ phage prior to transduction. Scientists also use transduction to introduce foreign DNA into eukaryotic cells, like mammalian cell lines. This can be done with lentiviral and Adeno Associated Viruses (AAV). Lentiviral and AAV can be used to create both transient cell lines, where a gene of interest is expressed but not integrated into the genome and stable cell lines, where foreign DNA is incorporated into the cell’s genome and is thus passed down through cell division. You can find all kinds of different lentiviral and AAV plasmids as well as ready-to-use viral preparations at Addgene. For more information on viral vectors, including transduction download our Viral Vectors 101 eBook. Bacterial conjugationConjugation was the first extensively studied method of gene transfer and was discovered in 1946 by Joshua Lederberg and Edward Tatum when they observed genetic recombination between two nutritional deficient E. coli strains that resulted in a wild type E. coli (Griffiths et al., 2000). During conjugation, genetic material is transferred from a donor bacterium to a recipient bacterium through direct contact. The donor bacterium contains a DNA sequence called the Fertility factor (F-factor). The F-factor is found on an episome, a piece of DNA that can replicate on its own or be integrated within a bacterial chromosome and allows the donor bacterium to make a small “bridge” or sex pilus that attaches to the recipient cell drawing it close. Once in contact the donor can transfer genetic material to the recipient bacterium. The genetic material transferred is commonly a plasmid and can infer genetic advantages such as antibiotic resistance.
TransfectionUnlike the last three methods which can be used in prokaryotes, transfection is only done in eukaryotic cells. Transfection is the process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab. Chemicals like calcium phosphate and diethylaminoehtyl (DEAE)-dextra neutralize or even impart an overall positive charge on DNA molecules so that it can more easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection actually pokes holes in the cell membrane so DNA can be introduced directly into the cell. Microinjection requires the use of a fine needle to deliver nucleic acids to individual cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell membrane that genetic material can pass through. If you are starting any molecular biology experiment check out Addgene’s protocol page, which has both protocols and videos for techniques in basic molecular biology, plasmid cloning, and viruses. References Baltrus, David A., and Karen Guillemin. "Multiple phases of competence occur during the Helicobacter pylori growth cycle." FEMS microbiology letters 255.1 (2006): 148-155. PubMed PMID: 16436074. Griffiths AJF, Miller JH, Suzuki DT, et al. Bacterial Conjugation. In An Introduction to Genetic Analysis. 7th edition (2000). Meibom, Karin L., et al. "Chitin induces natural competence in Vibrio cholerae." Science 310.5755 (2005): 1824-1827. PubMed PMID: 16357262. Additional resources on the Addgene blog Resources on Addgene.org |
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