Microbial Genetics Flashcards
Name five types of plasmids
Fertility plasmids- direct conjugation
Resistance plasmids- confer resistance to antibacterial agents
Col plasmids- genes for colicins
Degradative plasmids- genes for metabolism of unusual molecules
Virulent plasmids- confer pathogenicity on the host bacterium
What is a plasmid, in general?
They vary in size, are mostly circular ds DNA, and have a specific copy number. They encode nonessential but beneficial products for bacterial cells. They are fully transmitted to daughter bacterial cells.
What are some functions of plasmids?
They usually give bacteria a selective advantage und only some conditions.
They carry nonessential genes
Genes products are the utilisation of unusual carbon sources (toluene), resistance to heavy metals and antibiotics and synthesis of toxins.
Describe the structure of a plasmid
It is covalently closed and most circular (can be linear ds but rare)
The nucleotides in each strand are joined covalently which prevents the strand from separating
This causes DNA to coil up (internal tension)- negative supercoiling
The remaining stress helps separation of strands for replication or transcription
Covalently closed hairpins at the ends protects against exonucleases and are important for replication.
What is a replicon?
Wen a plasmid replicates independently in the cell. It must have an origin of replication
Name two types of replication mechanisms and an exception
Theta replication
Rolling circle replication
Integrative plasmids (episomes) can replicate by inserting themselves into the bacterial chromosome.
Do bacteria exchange genetic information?
In 1946, Lederberg and Tatum mixed two auxotrophic strains of E. coli
(Y10: thr- leu- thi- bio+ phe+ cys+)
(Y24: thr+ leu+ thi+ bio- phe- cys-)
They were plated on minimal medium and neither auxotroph grew. The mixed culture was also plated and a few PROTOTROPHIC bacteria did grow - did not require any of the above factors.
The prototrophs could not be explained by mutation (why hadn’t any colonies been found on the lone Y10 or Y24 plates?) or reversion (it was a triple mutation so 3 simultaneous reversions is very rare).
Therefore, genetic recombination had occurred (the two auxotrophic strains had recombined to form a prototrophic strain)
Is physical contact necessary for the genetic exchange to take place?
A Davis U-tube has constructed- was divided into two compartments by a filter that only allowed liquid medium to flow through (no bacteria).
The two auxotrophic bacteria were placed on either side of the filter and suction was applied to one end so medium flowed between compartments.
The bacteria were plated and no prototrophic bacteria grew (therefore no genetic exchange occurred) so exchanging of genes requires direct contact between cells
What did Hayes discover in 1953?
Genetic transfer between bacteria is polar (unidirectional, not reciprocal)- one cell acts as a donor, the other as a recipient. These ‘fertility’ factors were later identified as plasmids
What is conjugation?
The process whereby plasmids transfer from one cell to another. During conjugation, the two strands of a plasmid separate and one strand moves from the donor cell to the recipient cell. In both cells, the ss plasmid DNA acts as a template to make ds DNA during replication.
Describe three ways to transfer plasmid DNA
1) non-transmissible plasmids- they lack genes for effective contact and DNA transfer.
2) self-transmissible plasmids- conjugative and mobilizable.
They encode all the functions needed to move between cells and aid in the transfer of mobilizable plasmids.
3) mobilizable plasmids - non conjugative and mobilizable
Encode some but not all of the proteins required for transfer (can prepare its DNA for transfer but needs help from self-transmissible plasmids to move)
Describe the process of the transfer of the F plasmid during conjugation (F+ x F- mating)
F+ cell provides a sex pilus to contact recipient (F-) cell. Once contact is made, the pilus retracts to bring the F- cell closer.
Pores form between F+ and F- cell membranes which allows DNA to pass through.
One of these DNA strands on the F plasmid is nicked by endonuclease at oriT and separates. The intact F plasmid is replicated in the F+ cell while the 5’ end of the nicked DNA passes into the F- cell. The complementary strand to the ss plamid is synthesised via the Rolling Circle mechanism.
As the ss DNA of the F plasmid enters the F- cell, it is replicated to become ds DNA producing a F plasmid copy. The F- cell becomes a F+ cell
What is an Hfr strain?
Bacteria with integrated plasmids (episomes) that can transfer their chromosomes. They transfer chromosomal genes with high efficiency (high frequency recombination)
How do episomes integrate into bacterial chromosomes?
Integration can occur by:
Recombination between sequences on the plasmid and on the chromosome
Recombination via insertion elements shared between the chromosome and the plasmid
Describe Hfr conjugation
An Hfr cell (with integrated F plasmid in its chromosome) connects with an F- cell via a mating bridge.
A ss of the integrated F plasmid is cut.
As transfer proceeds, the hair chromosome replicates itself. Chromosomal DNA followed by the F plasmid passes into the recipient cell (chromosomal DNA gets transferred first and the F factor DNA is last and usually gets cut off before transfer is complete because mating bridge breaks down).
The cells separate and the donor ss DNA is replicated in the recipient cell (while replication is completed in the Hfr cell). The recipient cell now carries part of the F plasmid and some of the Hfr genome so is partially diploid.
Some of the transferred DNA may be incorporated into the recipient’s chromosome by recombination at high frequency because of homologous sequences. The cell remains an F- cell because it does not have the full F factor.
DNA fragments from the donor and recipient are digested by nucleases.
