Bacterial Genetics 2

Question 1

Conjugation

Definition

Answer 1

sexual process in bacteria

Question 2

Davis U-Tube Experiment

Answer 2

• U shaped tube with a fine pore filter half way along it
• strain A put into the right hand side and cotton wool put in right end of the tube
• strain B put into the left hand of the tube
• bung with a tube through it put in the left end so that pressure or suction could be applied
• showed that physical contact was a requirement between the two strains of the Lederberg and Tatum experiment for exchange/conjugation to occur

Question 3

Molecular Explanation for the Lederberg & Tatum Experiment

Answer 3

• donor strains possess a small DNA molecule, an F (fertility) plasmid so they are F+
• recipient strains don’t have the F plasmid so are F-
• the donor strains (F+) can transfer F plasmids to recipient strains (F-)

Question 4

Conjugal Plasmid

Definition

Answer 4

• a plasmid is a supernumerary DNA molecule capable of replicating autonomously, they are transferred vertically through populations by asexual division
• conjugal plasmids also code for transfer apparatus, e.g. pilli, and can be transferred horizontally through cell to cell contact

Question 5

Horizontal Transmission of Conjugal Plasmids

Answer 5

• pillus synthesised
• pillus makes contact with recipient cell and draws it closer
• one strand of the plasmid is copied and passed through the pillus
• inside the recipient cell, the complimentary strand is synthesised
• both cells end up with a complete copy of the plasmid

Question 6

F Plasmid

Answer 6

• a typical conjugal plasmid
• codes for everything needed to pass DNA between two bacterial cells
• can be transferred horizontally/laterally

Question 7

How can F plasmids transfer bacterial chromosomal DNA?

Answer 7

• transfer of bacterial chromosomal genes is much rarer than the transfer of F plasmids
• F plasmids can become incorporated into the genome of their host cell by homologous recombination
• the F plasmid still encodes for all of the same machinery to enable cell to cell transfer of DNA
• so when cell to cell contact is made and the plasmid is transferred, it is still attached to the bacterial chromosome so part / all of the bacterial chromosome is transferred with it
• this means that the recipient cell is now a partial diploid

Question 8

What does HFr mean?

Answer 8

• strains of bacteria which have the F plasmid integrated into the bacterial genome are called HFr strains
• on contact with another cell, DNA is transferred unidirectionally from a single point in the plasmid sequence into the donor cell
• different HFr strains can have the F plasmid incorporated at different point in their genome

Question 9

Where in bacterial chromosomes is it possible for an F plasmid to be integrated?

Answer 9

• integration of F plasmids into bacterial chromosomes happens by homologous recombination
• this means that part of the F plasmid sequence also has to be found in the bacterial chromosome
• IS elements are transposable elements that are very common in all bacterial DNA
• the F plasmid will contain IS elements that match with ID elements in the bacterial chromosome sequence
• this means that there are many possible sites for integration within any bacterial chromosome

Question 10

Merozygote

Answer 10

• a partial zygote

- name given to the partial diploid formed after transfer of DNA from an HFr donor strain

Question 11

Why are merozygotes unstable?

Answer 11

• transfer of complete chromosome from HFr is very rare
• recipient remains F- since not all of the F plasmid genes are received
• the linear transferred DNA strand can be combined into the circular bacterial genome by homologous recombination
• crossing over at two points allows aa section of the bacterial chromosome to be swapped out for the donor strand
• so a bacterial cell with DNA from two different parents is produced, i.e. a sexual process in bacteria

Question 12

Can conjugation occur between different bacterial species?

Answer 12

• IS elements are common to all bacteria
• this means that it is possible for a sexual process to occur by conjugation between cells of different bacterial species that last shared a common ancestor

Question 13

Using HFr strains to determine the order of genes in E.coli

Answer 13

• as the plasmid is always transferred from the same point and in the same direction, it is posible to cut off the DNA transferred at different points by interrupting the mating between the HFr strain and the F- strain at different times
• or the longer the transfer time required for a particular gene to be visible in the progeny the further from the transfer point the gene is
• this allows the order of the genes to be determined

Question 14

plasmids

Answer 14

• can be rapidly transmitted from bacterium to bacterium

- they can code for characteristics that are clinically significant e.g. antibiotic resistance

Question 15

How do plasmids become antibiotic resistant?

Answer 15

• a spontaneous mutation can give rise to antibiotic resistance
• and F plasmids can pick up antibiotic resistance genes from bacterial chromosomal DNA forming F’ plasmids

Question 16

Formation of F’ Plasmids

Answer 16

• as well as be integrated into bacterial chromosomes, F plasmids can also be excised
• excision also occurs at IS elements but not necessarily with the same pair of IS elements that were used for integration
• this means that when the plasmid is removed it can have some bacterial genes attached to either or both ends of it
• this new plasmid is called an F’ plasmid
• sometimes the genes excised with the plasmid can confer antibiotic resistance to the plasmid

Question 17

How does antibiotic resistance in bacteria arise?

Answer 17

• spontaneous mutation occurs giving rise to antibiotic resistance
• if the environment contains antibiotics then this offers a selective advantage to the mutant
• cells with the mutation grow and dominate the population
• resistance genes can then be incorporated into conjugal plasmids and spread through the bacterial world

Question 18

Competent Cells

Definition

Answer 18

-cells that can pick up DNA from the environment

Question 19

Is E.coli competent?

Answer 19

-E.coli is not naturally competent, is has to be treated

Question 20

Bacteriophages P2 and P4

Answer 20

• both have broad host ranges across multiple species

- phages related to P2 and P4 are associated with the introduction of virulence genes

Question 21

Transformation

Answer 21

1) cell lysis releases bacterial DNA into the environment
2) fragment of bacterial DNA taken up from the environment by a new bacterium
3) this makes the recipient bacterium a partial diploid
4) crossing over and homologous recombination allows the fragment to be swapped out for a section of the bacterial chromosome
5) this creates a cell with genetic information from two different parents

Question 22

Evolutionary Functions and Consequences of Natural Competence

Answer 22

• selective advantage of increased genetic diversity
• source of nucleotides for other bacteria, using DNA as food when resources are low
• repair of damaged DNA by homologous recombination

Question 23

What defines a bacterial species?

Answer 23

• not reproductive isolation
• sharing ~20 morphological, biochemical etc. characteristics
• bacteria that share these properties tend to also behave similarly

Question 24

1928 Griffith - Transformation Experiment

Answer 24

• used diplococcus pneumonia
• wildtype was virulent and formed smooth colonies on a plate
• the mutant was non virulent and formed rough colonies on a plate
• a mouse injected with the wildtype, dies and smooth bacteria were recovered
• a mouse injected with the mutant, survives and no bacteria are recovered
• a mouse injected with heat-killed wildtype, dies and no bacteria are recovered
• a mouse injected with both the heat killed wildtype and mutant strains, dies and virulent smooth bacteria are recovered
• Griffith concluded that something must be being transferred from the dead S bacteria to the live R bacteria making them virulent and smooth

Question 25

What two steps are required for a sexual process in bacteria?

Answer 25

1) bringing together genetic information from two different genomes 2) exchange of genetic information to produce recombinants

Question 26

Characteristics Traditionally Analysed in Phage Genetics

Answer 26

- host range - conditional mutants (e.g. temperature sensitive mutants) - plaque morphology

Question 27

What are plaques?

Answer 27

- phages mixed with an excess of bacterial cells - spread onto a solid medium on a petri dish - as bacteria grow, each phage forms a focus of infection - successive rounds of infection lead to the formation of an area in which bacteria have been killed, a plaque

Question 28

Detecting a Plaque

Answer 28

- plaques are easy to detect - holding the plate up to the light, areas that are see through as there is no bacterial cell growth are plaques and solid areas are areas of bacterial cell growth

Question 29

RII Phenotypes of T4 Plaques on E.coli

Answer 29

- rII is a mutant phenotype that results in a more rapid lytic cycle, as a consequence larger plaques with sharper edges are formed - r+ is the wildtype phenotype, plaques are small with fuzzy edges

Question 30

rII Mutants of Bacteriophage T4

Answer 30

- rII mutants have an altered host range, can grow on E.coli strain B but not K-12 - wildtype bacteria can grow on both bacterial strains - rII mutants have a faster lytic cycle than wildtype bacteria

Question 31

Benzer - Experiment

Answer 31

- rII mutant 1 and rII mutant 2 both infect the same E.coli cell - the majority of the progeny are either rII mutant 1 or rII mutant 2 - some progeny are double mutants, DNA recombines so that they have both mutations - some progeny are wildtype, DNA recombines so that they have no mutations - incubation of progeny at 37C on E.coli B produces plaques from all 4 types - incubation of progeny at 37C on E.coli K12 produces plaques from wildtype bacteria - the total number of progeny is the number of plaques on the E.coli B plate - the number of recombinants is double the number of plaques on the E.coli K12 plate as double mutants are unable to grow on that plate as well

Question 32

Recombination | Definition

Answer 32

-process by which genetic elements in two separate genomes become physically connected via genetic exchange

Question 33

Recombination | Frequency Mapping

Answer 33

-in general, homologous recombination occurs at random points all along the a chromosome -but there is greater recombination frequency between loci that are further apart Map Distance = No.ofRecombinants/TotalNo.of Progeny x 100%