Week 2 Flashcards
What are the key features of prokaryote chromosomes?
Usually just 1, circular, from 0.5 to 14 Mbps in size –> prokaryotes are haploid
What are the key features of extrachromsomal DNA?
Generally non essential, small self-replicating molecules that confer adaptive advantages –> plasmids
Where is prokaryote chromosome?
Their chromosome is free in the cytoplasm but very densely packed. The region of the cytoplasm that contains this genetic material is called NUCLEOID.
How can the size of prokaryote chromsomes differ?
A classic example of small genome is Mycoplasma genitalium (human pathogen), which has a 580 kb genome
Symbiotic bacteria - Carsonella ruddii, which lives off sap-feeding insects, has taken the record forsmallest genomewith just 159,662 bp
Among the biggest genomes Sorangium cellulosum, with 13 Mb
What are operons?
Genes are usually close to each other which are under under the control of a single regulatory sequence.
What does an operon do?
One operon is transcribed into a single mRNA molecule that encodes several proteins (polycistronic)
Do porkaryotic mRNAs undergo posttranscritional modifications?
Prokaryotic mRNAs do not undergo posttranscriptional modifications (they don’t have introns either)
What does the abscence of posttranscriptional modifcations mean for translation?
The absence of posttranscriptional modifications, plus the lack of a nuclear membrane barrier allow for transcription and translation to occur almost simultaneously –> Both process are coupled
What is the overall coding to noncoding relationship in prokaryotes?
Most of the prokaryotic genetic material is coding DNA, unlike in eukaryotes
What is the overview of prokaryote operons and transcription?
Polycistronic operons (several genes per mRNA)
Genes with no introns
No posttranscriptional modification of mRNA
Transcription and translation coupled in the cell
What is the overview of eukaryote operons and transcription?
Monocistronic operons (one gene per mRNA)
Genes have introns (non-coding sequences within the gene)
Posttranscriptional modification of mRNA is required for translation
Transcription and translation are uncoupled (mRNAs need to leave nucleus to be translated)
What is horizontal gene transfer?
Movement of genetic material between two cells that are not necessarily related, as opposed to the transfer from mother to daughter cells, known as vertical gene transfer
What is the advanatage of horizontal gene transfer?
The prokaryotic genome is extremely adaptable and quickly evolving, thanks mostly to horizonal gene transfer
What is the key feature of horizontal gene transfer?
HGT is, alongside the occurrence of spontaneous mutations, the main generator of genetic diversity in bacteria that allows them to adapt to new, often hostile environments.
What are the 3 types of horizontal gene transfer?
Transformation
Conjugation
Transduction
How has horizontal gene transfer shown to be a quick driving force in prokaryote evolution?
Ability to use new substrates as food sources
Antibiotic resistance
Ability to detoxify harmful chemicals (heavy metals, salts)
Virulence
What are landmark evolution stages involving acquistition form distant organisms?
Archaea establishing symbiosis and internalising bacteria as the source of eukaryotic organisms)
What is transformation?
Direct uptake of extracellular “naked” DNA by the bacterial cells
Where does the DNA uptaken by transformation come from?
Acquired DNA usually comes from nearby degraded cells.
This DNA can correspond to fragments of chromosome or to extrachromosomal DNA such as plasmids.
What can happen to the uptaken DNA when in the bacteria?
The acquired material can be degraded by the cell and used as building blocks for its own metabolism, or maintained stably, either by integration in the chromosome. Replicating independently from chromosome (only plasmids)
What is the name of the physiological state that allows for the acquisition of exogenous DNA?
Competence
What are the two types of competence?
Natural and Induced
What is natural competence?
Genetically encoded capability to uptake and incorporate exogenous DNA, some prokaryotes have genes encoding the machinery necessary to capture, transport and incorporate into their chromosome.
How frequent is natural competance?
More than 80 species including both Gram-positive and Gram-negative bacteria are naturally competent
When was natural competance first described?
This phenomenon was firstly described by Frederick Griffith in 1928, after observing the transference of virulence between Streptococcus pneumoniae strains.
What was Griffith’s experimental setup which natural competance was first described?
Griffith’s experimental setup, led him to the existence of thetransformation principle: non-virulentStreptococcus pneumoniae(a rough strain) could convert to a virulent pathogen when co-inoculated with a heat-killed virulentS. pneumoniae(a smooth strain) and injected into mice.
What is the reasoning behind Griffiths experiments?
The absence of disease symptoms after monoculture inoculation due to the lack of a specificvirulence factorin the rough strain and theheat inactivationin the smooth strain. However, in addition to the ability of themixed cultureto kill the mice, Griffith was also able to re-isolate live virulent (smooth) bacteria from the infected animals
What is induced competence?
Technique carried out in laboratory settings to introduce DNA in non naturally competent cells. This is one of the essential techniques in molecular biology
What is required for induced competence?
Requires the destabilization of the bacterial membranes
What are 2 techniques for induced competence?
By treating with chemicals (such as CaCl2): chemically competent cells –> transformation by heat shock
By applying electric shock: electrocompentent cells –> transformation by electroporation
What is conjugation?
Mechanism of DNA transfer involving direct cell to cell contact via the formation of a structures called conjugal pili
What is the overview of conjugation?
Conjugation is the main transfer mechanism for plasmids.
DNA is transferred unidirectionally from one donor cell (F+) to a recipient cell (F-)
When was conjugation first described?
This phenomenon was first described by Joshua Lederberg and Edward Tatum in 1946
How was conjugation discovered?
They discovered that E. coli mutant cells impaired in the production of some essential nutrients (auxotrophs) recovered the ability to produce them (prototrophs) after being in direct contact with other E. coli cells possessing the biosynthetic capabilities they were missing
How did they prove it wasnt through transformation?
Naked DNA didn’t have the same effect, and it was required that the cells were in direct contact.
What is the mechanism for conjugation?
The donor cell produces a pilus that attaches to receptors in the recipient cell.
After contact, the pilus retracts, bringing the cells closer and establishing a relaxosome bridge.
The plasmid is nicked at specific location called origin or transference (oriT) and one of its strands transfers through the relaxosome. It starts replicating in the donor cell. Once in the recipient cell, the other strand replicates too.
After reconstitution of the plasmid, the recipient cell becomes a potential donor (F+) .
What are plasmids?
Plasmids are extra chromosomal DNA molecules that are physically separated from chromosomal DNA and can replicate independently. They are mostly circular, double stranded DNA molecules. Very diverse in size.
What are episomes?
When a DNA plasmid is integrated into the chromosome
What are the key function of plasmids?
Plasmids are not essential, but often carry genes that confer multiple advantages to their hosts
What are examples of key function plasmids bring to their hosts?
Ability to conjugate (F factor in E. coli).
Antibiotic resistance and/or biosynthesis.
Heavy metal resistance.
Nitrogen fixation and nodulation (symbiosis with legumes).
Utilization of uncommon substrates as nutrients.
Degradation of toxic molecules.
Virulence.
What happens if a bacteria has several types of different plasmids?
Several different plasmids can coexist in the same cell, unless they have the same replication machinery, in which case they are mutually exclusive and they are classified as part of the same incompatibility group
What are the key section of a DNA plasmid?
origin of transference (oriT)
origin of replication
Cargo (antibiotic resistance gene / virulence/ degradation / biosynthesis genes)
Mobilisation genes
How diverse is conjugation?
Conjugation can happen between cells of different species, or even different kingdoms of life.
What is an example of cross kingdom conjugation?
Agrobacterium tumefaciens, a plant pathogen responsible for crown gall in over 140 species of plants.
How does Agrobacterium tumefacians cause crown gall?
A. Tumefaciens contains a virulence plasmid (Tumor inducing or pTi plasmid). This plasmid contains all the genes necessary for conjugation and a region called T-DNA, that contains genes for the synthesis of plant hormones and nutrients for the bacterium.
What happens when the Agrobacterium tumefacians plasmid is introduced to the plant?
After penetrating the plant through a wound and establishing a conjugation pilus, the plasmid excises the T-DNA and it is transferred to the plant cell nucleus, where it integrates in the plant genome.
The T-DNA is then expressed and the resulting hormones stimulate cell growth and tumour formation.
What is the use of Agrobacterium conjugation into plants?
The ability of Agrobacterium to introduce DNA in plants has been harnessed for biotechnology applications, and is a commonly used tool in labs to genetically manipulate plants
How can conjugation be used on bacteria?
Interspecies conjugation is a technique routinely used in the lab to transfer plasmids between different bacteria, especially to those that are difficult to transform.
How are plasmids and conjugation useful for experiments?
Thousands of artificial plasmids have been generated for multiple molecular biology and biotechnology purposes (gene and protein expression, mutant generation). Carrying human-designed cargo and selectable markers (e.g antibiotic resistance genes) to select for cells containing the plasmid
The manipulation and transfer of plasmids are also basic molecular biology and biotechnology techniques
Whats transduction?
Transduction can happen when a phage or prophage initiate a lytic cycle and bacterial DNA is packed in the newly formed viral particles. This new particles can then infect other bacteria and transfer them this DNA.
What is the advantage of transduction?
This mechanism doesn’t require direct contact between the cells, as it is the phage the one that carries the DNA
What happens to the bactrial DNA?
Phages infect bacteria and inject their own genetic material into the bacterial cytoplasm
What are the different outcomes of the phages and bacteria DNA which has been inserted?
The viral genetic material is recognized and destroyed by bacterial immunity systems.
The phage genome hijacks the cell machinery and starts multiplying, eventually killing the cell and releasing all the new phage particles –> lytic cycle.
The phage genome integrates in the bacterial chromosome becoming a prophage and stays dormant or expressing at low level –> lysogenic cycle.
What happens to the Lytic cycle?
Phage attaches to a host cell and injects its DNA
Phage DNA circularises
Certain factors induces lytic cycle
New phage DNA and proteins are synthesised and assembled into phages
The cell lyses, releasing phages
What happens in the Lysogenic cycle?
Phage DNA integrates into the bacterial chromosomes, becoming a prophage
The bacterium reproduces normally, copying the prophage and transmitting it to daughter cell
Many cell divisions produce a large population of bacteria infected
Occasionally, a prophage exits the bacterial chromosomes initiating a lytic cycle
What are the different transduction types?
Generalised transduction
Specialised transduction
Lateral transduction
What is generalised transduction?
Transfer of DNA from any part of the host genome to the recipient cell
What is specialised transduction?
Transfer of a few specific sets of genes between cells