CBG.17 - Bacterial genetics 1 (Transformation)
Describe an experiment that demonstrates the presence of an eclipse phase in bacterial transformation.
1. Mix Arg- cells with Arg+ DNA sequences. 2. Arrest the cell in different stages of transformation and treat the solution with DNase (to degrade any residual sequences so that they cannot be subsequently ingested) 3. 'Select' Arg+ cells by growing all cells on a medium without Arg supply. Only transformed Arg+ cells (prototrophs) will be able to synthesise Arg and grow on the medium, while Arg- cells that require supplements will die. 3 stages found: 1. arrest before ssDNA uptake (no Arg+ expressed -> dies) 2. Eclipse phase: arrest after ssDNA uptake but before incorporation into the host genome (no Arg+ expressed -> dies) 3. arrest after ssDNA uptake and incorporation into the host genome (Arg+ expressed -> lives)
Outline the process of competence.
1. The cell expressed genes that lead to assembly of a DNA uptake molecular machinery. 2. DNA binds to the surface of the competent cell at a DNA receptor. 3. The DNA passes through the cytoplasmic membrane via DNA translocase. Only ssDNA may pass through, will the other strand is concurrently degraded by a membrane nuclease. 4. Translocated ssDNA may then become integrated into the bacterial chromosome by a RecA-dependent process.
Describe two modes of artificial transformation.
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. *Chemical transformation* Treating bacterial cells with CaCl2 at low temperature Causes change in membrane permeability, which allows free entry of plasmid DNA (competence is not necessary) *Electroporation* Bacteria are placed in a electrical field, which changes the permeability properties of their membranes by creating holes in the membrane Membrane repair response by the bacteria restores the natural membrane permeability after the electrical field has been disrupted
Describe the experiment that identified the transforming principle first described by Griffith?
Avery, MacLeod and McCarty - identified that the transforming principle was DNA Method: 1. Lysed virulent pneumococcus bacteria and extracted the resultant solution (cytoplasmic contents of cells) 2. Treated the solution with different enzymes (protease/DNase/RNase) to destroy different molecules in order to determine which molecule was the transforming principle 3. Mixed the solutions with non-virulent bacteria and allowed time for transformation 4. Infected mice with each of the solutions treated with different enzymes Findings: the non-virulent bacteria were transformed in all cases except when the debris was treated with DNase. DNase breaks down the transforming principle, so the transforming principle must be made from DNA.
Outline the basic structure of a cloning vector
Cloning vector - small length of DNA that can be stably maintained in an organism It will not be incorporated into the genome of the recipient, but will instead exist in the cytoplasm as an independent, autonomous entity and will be expressed Origin of replication - necessary for replication and inheritance by daughter cells Antibiotic resistance - selectable marker that can be used to isolate successfully transformed individuals (the cloning vector will confer resistance against an otherwise lethal antibiotic treatment, so that all untransformed cells die and only transformed cells remain) Polylinker (cloning site) - distinct area with many unique restriction site (feature that allows a gene to be conveniently inserted into the vector or removed from it)
What is co-transformation? How can co-transformation be used to map genes?
Co-transformation - simultaneous transformation of two or more genes - Only genes in the same chromosomal vicinity can be transformed - The closer two gene loci are on the chromosome, the more likely that they will be co-transformed Using co-transformation to map bacterial genes: 1. Extract DNA from donor bacteria. 2. Break DNA up into short fragments. 3. Use the DNA fragments to transform recipient bacteria that lack the genes being mapped (engineered by knockout) (so that a recipient genotype containing the genes indicates a transformed genotype) 4. Search for transformants of each gene and cotransformants of multiple genes (by plating on media selective for genes). Calculate what percent of transformants are contransformants. If high percentage of transformants are cotransformants, then genes are closely linked on the chromosome. Rate of cotransformation inversely correlates to distance separating the loci on the chromosome. This method can be used to order and map genes on bacterial chromosomes.
What us meant by 'competent' bacteria and 'competence'?
Competent bacteria - cells that can take up DNA through their cell membranes Competence - state that bacteria enter to take up naked DNA from their environment (bacteria must enter a special physiological state (triggered by certain conditions) in order to be receptive to transformation)
Through which mechanisms can bacteria exchange genetic material? Which of these is the mechanism of antibiotic resistance?
Conjugation - when genetic material passes directly from one bacterium to another Transformation - when bacterium takes up DNA from the medium in which it is growing and incorporate it into their chromosome Transduction - when bacteriophages carry DNA from one bacterium to another Conjugation
Describe Griffith's experiment.
Experiment involving two pneumococcus strains - Virulent/smooth strain (IIIS) - smooth capsule protects the strain from the immune system - Non-virulent/rough strain (IIR) - no capsule to protect the strain from detection by the immune system Method: - Four conditions: (a) Injected virulent strain into mouse (b) Injected non-virulent strain into mouse (c) Injected heat-killed virulent strain into mouse (d) Injected mixture of heat-killed virulent strain and live non-virulent strain into mouse Findings: (a) mouse dies (b) mouse lives (c) mouse lives (d) mouse dies Autopsy of mice in condition (d) revealed that the bacteria were smooth (rough bacteria had acquired the smooth coat (virulence determining factor) merely by contact with the debris from the dead bacteria
What role does the lacZ^t gene play in the lac operon? What role does the amp^R gene play in the lac operon?
lacZ^t Will express a blue pigment unless a new sequence is integrated via transformation which interrupts its coding region (polylinker within the gene) - If the blue pigment is not expressed, this indicates that the bacteria has received n intact, functioning plasmid with the inserted gene at the correct polylinker locus amp^R Will confer ampicillin resistance on the transformed bacterium, allowing it to act as a selective marker Non-blue colonies that grow on an ampicillin-treated medium will have successfully been transformed by the lac operon.
What are the different purposes for natural transformation?
Nutrition - source of nucleotides Repair - uptake of homologous DNA would allow repair of damaged DNA by recombination and restoration of the sequence. Because bacterial genomes do not come in homologous pairs, this is a mode of homologous recombination. Diversity - increased genetic diversity by horizontal gene transfer. Lack of genetic diversity and Muller's ratchet present major barriers to asexual organisms and this helps to overcome them.
Describe the bacterial genome.
Single (typically) circular chromosome that is located inside a structure in the cytoplasm called the nucleoid
What is the mechanism by which genetic material is taken up by a bacterium and incorporated into its genome during transformation?
1. As a DNA fragment enters the cell during transformation, one strand is broken up, while the other strand moves across the membrane. 2. The ingested single strand may pair with a homologous region of the host genome. 3. Two crossover events with the recipient chromosome must occur in order for the material to be incorporated. 4. The displaced host DNA and the unincorporated foreign DNA are degraded in the cytoplasm. 5. During the next cell division, the genome is replicated, with one daughter genome containing the transformed sequence and the other containing original sequence.
What is RecA?
RecA - protein that is essential for the repair and maintenance of DNA During transformation, integration of the donor DNA into the recipient chromosome is achieved by homologous recombination - a process mediated by the RecA protein.
Distinguish between horizontal and vertical gene transfer in bacteria.
Vertical gene transfer - parent-> daughter cell through genome replication and segregation (mistakes can occur in replication) Horizontal gene transfer - transfer of genes between cells / with the environment
