L12 Genetics Of Bacteria Ii

Front 1

What confirmed the physical union of bacteria in conjugation?

Back 1

Microscopic studies have confirmed the physical union of bacteria,called conjugation. During conjugation, the donor cell transfers someof its genetic information to a recipient cell.

Front 2

How is genetic information

transferred?

Back 2

Genetic information is transferred through a hollow tube made ofproteins, called a conjugation pilus or conjugation tube or sex pilus

Front 3

What is the F plasmid?

Back 3

Initially called the fertility factor the F plasmid of E. coli was the first plasmidto be discovered. F plasmid carries genes which enable genetic exchangeby conjugation.

Front 4

What are the features of the F plasmid?

Back 4

The F plasmid is 100 kb in size and has the following features

1. Approx. 30 transfer (tra) genes

2. An origin of replication (oriV)

3. An origin of transfer (oriT).

4. Three insertion sequences (IS).

Front 5

How does the transfer of the F plasmid by conjugation work?

Back 5

Front 6

What is the first step in the conjugation?

Back 6

1. The F+ cell has a conjugation pilus that attaches to the F- celland retracts drawing the cells closer together and forms a tube made ofpilin proteins through which the F plasmid can transfer.

Front 7

What is the second step in the conjugation?

Back 7

2. A single strand copy of the F plasmid is transferred from the F+ cell into theF- cell. Prior to transfer a single strand nick is produced at oriT in the F plasmidby the relaxosome (protein complex). A single strand of the F plasmid DNA calledthe T strand is transferred to the F- cell.

Front 8

What is the third step in the conjugation?

Back 8

3. The DNA replication process ensures that both copies of the F plasmidbecome intact circular double stranded DNA molecules. Each cell now hasa F plasmid and is referred to as F+.

Front 9

How long does the transfer of the F plasmid take?

Back 9

It takes 2 minutes for the F plasmid to transfer into the F- cell.

Front 10

How many cells in an E.coli cell culture will have the F plasmid integrated into their genome?

Back 10

1 in 100,000 cells in an E. coli cellculture of F+ cells will have the Fplasmid integrated into the genome.

Front 11

What are the E.coli cells that have a copy of the F plasmid in their genome known as?

Back 11

E. coli cells which have a copy of theF plasmid in their genome are knownas Hfr strains. Hfr for high frequencyof recombination

Front 12

When will the F plasmid integrate?

Back 12

The F plasmid integrates when ahomologous recombination eventoccurs between one of its ISsequences and one of the many ISsequences on the E. colichromosome.

Front 13

How can the bacterial chromosome be mobilised?

Back 13

The bacterial chromosome canbe mobilised by the integratedF plasmid.

A Hfr cell can conjugate with a F cell. Bacterial DNA flanking the integrated F plasmid is transferred across into the F- or recipient cell.

Front 14

How long would it take to transfer the entire E. coli chromosome?

Back 14

It would take 100 minutes to transfer the entire E. coli chromosome.

Front 15

What normally happens to interrupt the transfer?

Back 15

The mating pair of E. coli usually break apart before the wholechromosome is transferred.

Front 16

What does the recipienr (F-) cell become?

Back 16

The recipient (F-) cell remains F- (as it never receives all the Fplasmid).

Front 17

What must the transferred linear DNA do to survive?

Back 17

For the transferred linear DNA to survive it must integrate intobacterial chromosome by homologous recombination as it has nomechanism to replicate itself independently

Front 18

What is the procedure of mapping the E. coli chromosome by interrupted conjugation (mating) based on?

Back 18

The procedure is based on the artificial interruption of conjugationbetween Hfr and F- cells. In each mating a number of selectablemarkers are chosen for analysis at each time point

Front 19

What are these selectable markers?

Back 19

gal- unable to use galactose as a carbon sourcelac- unable to use lactose as a carbon sourcetonS sensitive to colicin and T1 phage

aziS sensitive to azide

strS sensitive to antibiotic streptomycin

Front 20

What are colicins?

Back 20

Colicins are proteins produced by E. coli containing ColE1 plasmids which bindreceptors in the cell envelope and cause cell lysis.

Front 21

In the example what strains are mated?

Back 21

The two stains mated in this example are

Hfr strain strS gal+ lac+ tonR aziR

F- strain strR gal- lac- tonS aziS

Front 22

What happens at time 0?

Back 22

At time 0 the Hfr and F- cells are mixed together

Front 23

What happens after 5 mins?

Back 23

After 5 minutes a sample of the cells is taken out, vortexed to breakthe cell-cell contact and plated on the selective media

Front 24

What selective media are used?

Back 24

Minimal media with streptomycin and lactose (lac+)

Minimal media with streptomycin and galactose (gal+)

Minimal media with streptomycin and azide (aziR)

Minimal media with streptomycin and colicin (tonR)

Samples of cells are removed at 10, 15, 20, 25 minutes etc. andplated on the same set of selective media plates.

Front 25

What is the aim of the process?

Back 25

The aim is to find the time the selectable marker gene is first transferred intothe strR F- cell. The first genetic maps of E. coli were constructed using thismethod.

Front 26

Where can the F plasmid integrate into the E.coli genome?

Back 26

The F plasmid can integrate into different sites (IS sites) in the E. coligenome creating different Hfr strains. They can also integrate in the sameIS site in the E.coli genome in two different orientations.

Front 27

How can a more detailed circular map of the positions of genes in the E.coli genome be created?

Back 27

Different Hfr strains of bacteria were the F plasmid has integrated intodifferent sites and in different orientations in the E. coli chromosome havebeen used to produce a genetic map of different parts of the E. coli genomeusing the interrupted mating technique. By overlapping these linear maps itis possible to create a more detailed circular map of the positions of genesin the E. coli genome.

Front 28

What do you need to be aware of when using naturally occurring plasmids?

Back 28

Since the discovery of the F plasmid in E. coli thousands of naturally occurringplasmids have been isolated from many different types of bacteria

Note

1. Only some naturally occurring plasmids can transfer between bacteria

2. Only some naturally occurring plasmids can integrate into the bacterialgenome

Front 29

What are R plasmids?

Back 29

R plasmids are a group of plasmids found in a range of bacterial speciesthat

a. have tra genes so can conjugate between different species of bacteria

b. carry genes that confer multiple antibiotic resistancec. Large ~ 100 kb

Front 30

What are CoIEI plasmids?

Back 30

ColEI plasmid found in E. coli

a. small (6.6 kb) and does not carry tra gene cluster so can NOT move byitself between different bacteria.

b. carries ColEI gene that produces a protein colicin which is excreted formthe cell and toxic to other bacteria (not the host).

Front 31

What are Ti plasmids?

Back 31

Found in soil bacteria Agrobacterium tumefaciens

a. Large (~ 200 kb) have tra genes so can conjugate between differentspecies of bacteriab. Also able to transfer the T-region of plasmid into plant cells.

c. Integration of T-region into plant chromosomes causes crown galldisease.

Front 32

What do transfomation, transduction and conjugation allow?

Back 32

Transformation, transduction and conjugation allow bacteria toexchange DNA between each other.Can be same species of bacteria or different species