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Bacteriophage Genetics And Molecular Biology Pdf

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Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering, Volume 421

In molecular biology and genetics , transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane s. For transformation to take place, the recipient bacterium must be in a state of competence , which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Transformation is one of three processes for horizontal gene transfer , in which exogenous genetic material passes from one bacterium to another, the other two being conjugation transfer of genetic material between two bacterial cells in direct contact and transduction injection of foreign DNA by a bacteriophage virus into the host bacterium. As of about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria ; the number might be an overestimate since several of the reports are supported by single papers.

Transformation in bacteria was first demonstrated in by the British bacteriologist Frederick Griffith. However, he discovered that a non-virulent strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some " transforming principle " from the heat-killed strain was responsible for making the harmless strain virulent.

They isolated DNA from a virulent strain of S. It was originally thought that Escherichia coli , a commonly used laboratory organism, was refractory to transformation. Transformation using electroporation was developed in the late s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed. Transformation is one of three forms of horizontal gene transfer that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by homologous recombination ; the other two are transduction , carried out by means of a bacteriophage , and conjugation , in which a gene is passed through direct contact between bacteria.

Competence refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory. It appears to be an ancient process inherited from a common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair of DNA damage, especially damage acquired under stressful conditions.

Natural genetic transformation appears to be an adaptation for repair of DNA damage that also generates genetic diversity. Transformation has been studied in medically important Gram-negative bacteria species such as Helicobacter pylori , Legionella pneumophila , Neisseria meningitidis , Neisseria gonorrhoeae , Haemophilus influenzae and Vibrio cholerae. Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane s. The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system , as well as DNA translocase complex at the cytoplasmic membrane.

Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins.

The translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA -dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence, but its role is uncertain.

Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be responsible for this process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state.

Competence development in Bacillus subtilis requires expression of about 40 genes. The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species in seven different phyla are known to undergo this process. Transformation in Haemophilus influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase.

By releasing intact host and plasmid DNA, certain bacteriophages are thought to contribute to transformation. Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in Streptococcus pneumoniae by the DNA damaging agents mitomycin C a DNA cross-linking agent and fluoroquinolone a topoisomerase inhibitor that causes double-strand breaks.

Of these, only six, all DNA damaging agents, caused strong induction. These DNA damaging agents were mitomycin C which causes DNA inter-strand crosslinks , norfloxacin, ofloxacin and nalidixic acid inhibitors of DNA gyrase that cause double-strand breaks [38] , bicyclomycin causes single- and double-strand breaks [39] , and hydroxyurea induces DNA base oxidation [40]. UV light also induced competence in L.

Charpentier et al. Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication.

The process of homologous recombinational repair HRR is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome.

During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation. To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using B.

The particular process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes see Evolution of sexual reproduction ; Meiosis.

Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature. Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell.

Cells that are able to take up the DNA are called competent cells. It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to- lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. The surface of bacteria such as E. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.

DNA entry into E. Their role was established when cobalamine which also uses these channels was found to competitively inhibit DNA uptake. In this poly HB is envisioned to wrap around DNA itself a polyphosphate , and is carried in a shield formed by Ca ions. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

Electroporation is another method of promoting competence. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. Most species of yeast , including Saccharomyces cerevisiae , may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab. Efficiency — Different yeast genera and species take up foreign DNA with different efficiencies.

Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. A number of methods are available to transfer DNA into plant cells. Some vector -mediated methods are:. There are some methods to produce transgenic fungi most of them being analogous to those used for plants.

However, fungi have to be treated differently due to some of their microscopic and biochemical traits:. Introduction of DNA into animal cells is usually called transfection , and is discussed in the corresponding article. The discovery of artificially induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins.

Typically plasmids are used for transformation in E. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication , which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.

The cells are incubated on ice with the DNA, and then briefly heat-shocked e. This method works very well for circular plasmid DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA. The transformation efficiency using the CaCl 2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA.

Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid.

Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow.

Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.

In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones. Other commonly used reporter genes are green fluorescent protein GFP , which produces cells that glow green under blue light, and the enzyme luciferase , which catalyzes a reaction with luciferin to emit light.

The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods. From Wikipedia, the free encyclopedia. Not to be confused with an unrelated process called malignant transformation which occurs in the progression of cancer.

Main article: Natural competence. Further information: Transformation efficiency. Nature Reviews. Molecular Biology of the Cell. New York: Garland Science. The Journal of Hygiene. Journal of Molecular Biology. Bibcode : PNAS Bibcode : Natur. The American Phytopathological Society. Retrieved 14 January Access Excellence.

Bacterial and Bacteriophage Genetics (4th edn)

In molecular biology and genetics , transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane s. For transformation to take place, the recipient bacterium must be in a state of competence , which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory. Transformation is one of three processes for horizontal gene transfer , in which exogenous genetic material passes from one bacterium to another, the other two being conjugation transfer of genetic material between two bacterial cells in direct contact and transduction injection of foreign DNA by a bacteriophage virus into the host bacterium. As of about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria ; the number might be an overestimate since several of the reports are supported by single papers. Transformation in bacteria was first demonstrated in by the British bacteriologist Frederick Griffith.


Bacteriophage: Genetics and Molecular Biology Library recommendation (​email) (pdf). (viruses that infect bacteria) are fascinating organisms that have played and continue to play a key role in bacterial genetics and molecular biology.


Transformation (genetics)

Bacteriophages phages are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases.

Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome , and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes e. MS2 and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.

Genetic Engineering of Bacteriophages Against Infectious Diseases

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Bull, M. Badgett, H. Three different point mutations were identified that each recovered growth at high temperature.

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Genetic Engineering of Bacteriophages Against Infectious Diseases

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