Lecture 12 - Plasmids, Phages & Gene Transfer

Pregunta	Respuesta
How do plasmids replicate themselves?	- Similar way to the chromosome within bacteria. - Some either replicate autonomously or they require host functions to do so. - From location oriV. - Bacteria tend to choose one plasmid to replicate within its cell and will not keep all types.
How many plasmids can be carried by one bacteria?	More than one, however, they must be compatible.
What types of genes do plasmids do not/ do carry?	- Do not carry any essential/ house keeping genes (these are contained within the chromosome). - Genes for antibiotic resistance. - Ancillary genes that may be useful at times.
Are plasmids acquired by the bacterium for a purpose?	No; they are acquired randomely/ by chance, and then propagated if useful. They are then most likely LOST if proven unuseful.
What is rather annoying if a plasmid is useless?	They can not always be rid of that easily - they are SELFISH, and some can have negative effects on the bacterium if not careful, since plasmid genes are so easily integrated into the host genome.
What is meant by conjugation between plasmids and bacteria?	The mobilisation of plasmids e.g. F plasmid between hosts.
Describe the plasmid movement between bacteria, and how they are extremely selfish.	- Plasmid movement is encoded by the plasmid itself. - Their aim is to get around and replicate in as many bacteria as possible. - They convince the bacteria that they have some useful genes so that the bacteria keep them and propagate them. - Sometimes the bacteria can NOT get rid of them anyway. - When they do, a bridge is made between 2 adjacent bacteria (donor and recipient) = Tra+ (transfer). - Then, Mob+ function causes the DNA of the plasmids to be replicated by Rolling Circle Replication.
What is the name of the component of the plasmid used to build the pillus between the 2 bacteria during conjugation? Describe its structure.	- Tra+ - Initiates transfer by constructing a stable, transient bridge made up of a series of proteins - This bridge however only last approx. an hour.
What is the name of the component of the plasmid used to initiate replication of the plasmid's DNA within the donor?	- Mob+ - Initiates Rolling Circle Replication
Describe Rolling Circle Replication.	- The tube between the 2 bacteria is very narrow. - You cannot force a plasmid down this tube and therefore the idea is to produce a narrow, linear strand of the plasmid to thread through the tube. - The Mob+ machinery nicks the DNA of the plasmid at a given point - origin of transfer. - The origin of transfer recruits a series of enzymes, encoded by the plasmid to copy the DNA whilst the donor strand is being pushed out across the pillus. - Once the donor strand is inside the recipient, the second strand is then copied onto that, and the plasmid is formed again inside a new bacterial cell.
What is the name of the plasmid first ever to be discovered in E.coli ?	The F plasmid.
What is meant by the terms F+ and F- in conjugation of the F plasmid?	F+ = Bacteriam which carries the F plasmid and is the donor. F- = Bacterium which does not yet carry the F plasmid and is the recipient. ** Both can be entirely different species.
What vital trait do many bacterial cells have as a result of conjugation?	Antibiotic resistance.
What is meant by a chimeric plasmid?	When 2 plasmid cross over at regions of homology - i.e. a combination of 2 plasmids.
What is meant by a 'wonder' plasmid?	When a chimeric plasmid is formed, and the recombination events which take places at these homologous regions initiate a significant mutation which allows the plasmids to combine their resources to carry more functions which are useful to them.
Why is recombination of a plasmid into a chromosome of the bacterium not really an advantage to the plasmid?	Because this means the plasmid cannot move like a plasmid should be able to.
Why are plasmids' sequences always read as direct repeats within their genomes?	Since they can turn around and are in loop form, so are easily read as having the same orientation as the sequence in question.
Describe a way in which plasmids can effectively 'steal' genes from the bacterial host. Giving examples where necessary.	- Plasmids integrate into the chromosome of the host via homologous recombination. - i.e. Hfr bacterial cell - Spontaneous disintegration can then add surrounding chromosomal genes to another/ remaining plasmid if another homologous region was to be found on the chromosome.
If spontaneous disintegration between a plasmid and a chromosome takes place, when can this go terribly wrong for the bacteria?	If the gene taken is really important for the chromosome. The plasmid as a result, will leave the cell to die.
What is the F plasmid?	An episome - a form of plasmid which can integrate itself into the bacteria chromosome.
What is the Hfr bacterial cell?	A high-frequency recombination bacterial cell >> a bacterium with a conjugative plasmid integrated into its genomic DNA.
How are F' plasmids formed from Hfr bacterial cells?	When the plasmid disintegrates from the bacterial chromosome; normally including part of the bacterial chromosome.
Describe the conjugation events that occur once the F' plasmid has been disintegrated from the bacterial chromosome.	- Pilus connects the bacterial cell (containing the F' plasmid) to a neighbouring bacterial cell. - F' plasmid DNA is replicated and transferred into the neighbouring cell. - Now 2 cells contain this plasmid which contains chromosomal DNA.
What is meant by a virulent phage?	- A bacteriophage is a virus which infects bacteria, i..e is virulent.
How do virulent phages cause damage to bacteria?	- Inject their DNA into the cell. - Replicate their genome. - Package & lyse the cell. - Sometimes cuts up host chromosome & packages pieces of that.
What is meant by generalised transduction?	The process by which any bacterial gene may be transferred to another bacterium via a bacteriophage >> carrying only bacterial DNA and NO viral DNA (only a viral envelope).
When phages inject bacterial DNA into neighbouring bacteria via generalised transduction, what can happen to this DNA?	It can integrate into the chromosome by homologous recombination. (However, there must be a double cross-over event for this to occur!)
Give an example of a virulent bacterophage.	Phage P1 that infects E.coli.
Briefly describe Generalised Transduction by Phage P1.	- Bacteriphage attaches to the bacterial cell wall & injects bacterial DNA. - Host DNA is destructed. - Viral DNA and coat proteins are synthesised within the bacterium. - The virus capsids are synthesised, and the virual particles assembled inside the cell - these particles are known as transducing particles. - The bacterial cell is lysed and phage particles are released. - These phages can then go on to infect other bacteria.
What are transducing particles?	Particles synthesised during generalised transduction and they contain both viral and bacterial DNA.
When new DNA synthesised from generalised transduction has been released in phages from the bacterium and then used to infect neighbouring bacteria; what are the three fates of the bacteria?	- Stable gene transfer = The donor DNA will be integrated into the recipient chromosome & recycled by the cell i.e. used to produce phages if activated. - Unsuccessful gene transfer = The donor DNA will be degraded. - Abortive transduction = The donor DNA survives and could become a plasmid again.
What is meant by temperate phages?	- As oppose to virulent phages. - They still inject their DNA into recipient; replicate their genome & package and lyse the cells, HOWEVER... - They are phages which go on to have the ability to display a lysogenic life cycle. - They can sometimes integrate their genome into the host via SS- recombination.
What is meant by specialised transduction?	A form of transduction whereby one, or only some of the host bacterial genes are transferred with the bacteriophage.
What type of phage undergoes specialised transduction?	Temperate phage.
How does lysogen formation lead to mutations?	- Temperate phages undergo specialised transduction within the host bacterium - taking bacterial genes with it. - Upon entry into the recipient, the phage undergoes lysogen formation and integrates these foreign genes into the host genome. - This is a form of mutation.
Discuss what is meant by a prophage.	If a temperate phage, undergoes specialised transduction (phage carries host genes with it to an adjacent bacterial host), undergoes lysogen formation and then integrates foreign genes into the chromosome, the mutation which takes place causes stable integration.
What is meant by stable integration of foreign genes into the host genome from phages?	The genetic material from a bacteriphage can be incorporated into the genome of the recipient and then able to produce phages if activated.
Give an example of a prophage.	Phage lambda (infects E.coli)
Briefly describe the Lambda lifecycle (both lytic and lysogenic).	LYTIC - Phage DNA is copied using rolling circle replication process. - Phage heads, tails & DNA assemble into PROGENY phage. - Cell lysis. - Phages re-infect an adjacent E.coli. - Phage infects the E.coli through adsorption & penetration. - Thus, phage DNA has cyclized. LYSOGENIC - Phage DNA is integrated into host genome to form a prophage. - The cell divides to form many lysogenic clones. - This continues to happen and if one of these cells undergoes UV light induction, it is activated and the phage can separate from the host genome and the phage DNA has cylisized.
Briefly describe the process of Specialised Transduction by Lambda.	- You start with a lysogenized cell with prophage. - The DNA is induced i.e. by UV. - In lambda, there is then rare de-integration whereby some bacterial genes separate from the chromosome with the phage. - There is then replication of the defective virus DNA with incorporated host genes. - There is then assembly and release of transducing phage particles. - These phage particles can then go on to infect recipient cells. - The phage is integrated as a prophage into the host genome. - The final bacterial cell has a chromosome containing both viral and donor DNA.
What is meant by a lysogen?	A lysogenic bacterium whereby a phage exists as DNA in its dormant stage (prophage) i.e. either integrated into the host bacteria's chromosome or more rarely as a stable plasmid within the host.

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