L12 - genetics of bacteria 2

Question 1

how was conjugation discovered?

Answer 1

1. mixed two strains of E.coli of different requirements
   (met- bio- leu+) (met+ bio+ leu-) and grew on minimal media
2. colony growth seen meaning bacteria had to be (met+ bio+ leu+)
3. bacteria had to be in contact with each other

Question 2

briefly describe process of conjugation

Answer 2

donor cell transfers genetic info to recipient cell via hollow tube called conjugation pilus (or sex pilus)

Question 3

what cell forms the conjugation pilus

Answer 3

donor cell

Question 4

what is the F plasmid

Answer 4

first plasmid to be discovered

carries genes that enable conjugation

Question 5

features of F plasmid

Answer 5

1. origin of replication (oriV)
2. origin of transfer (oriT)
3. 3x insertion sequences (IS)
4. tra genes

Question 6

what is meant by F+ and F-

Answer 6

F+ = has F plasmid (donor cell) 
F- = doesn't have F plasmid (recipient cell)

Question 7

describe process of F plasmid transfer

Answer 7

1. donor cell forms conjugation pilus
2. relaxosome complex cleaves the F plasmid DNA at oriT then binds to the 5’ end
3. relaxosome moves through pilus with single strand of DNA into recipient cell (peels single strand away)
4. new complimentary strands synthesised (in donor and recipient)
5. ends joined up, there is now F+ in both cells

Question 8

what forms conjugation pilus?

Answer 8

donor cell - from pilin proteins encoded by tra genes

Question 9

how long is the process of F plasmid transfer

Answer 9

2 mins

Question 10

role of relaxosome complex in conjugation of F plasmid

Answer 10

1. cleaves F plasmid DNA at oriT and binds to 5’ end

2. moves single strand of DNA through pilus to recipient cell

Question 11

name of proteins that form the conjugation pilus

Answer 11

pilin proteins

Question 12

what is the single strand of F plasmid DNA that is transferred called?

Answer 12

T strand

Question 13

what is Hfr strain of E.coli?

Answer 13

E.coli that contain F plasmid integrated into genome

high frequency of recombination

Question 14

what is the importance of the IS sequence?

Answer 14

it is where homologous recombination occurs to integrate the F plasmid into the bacterial genome

Question 15

describe conjugation of Hfr strains

Answer 15

1. Hfr strain can conjugate with an F- recipient cell
2. when the integrated F plasmid transfers (is peeled) from the donor genome it may take small regions of bacterial DNA with it (it is peeled, the whole F plasmid doesn’t excise)
3. these regions of bacterial DNA could be transferred and integrate into the recipient genome with the F plasmid

Question 16

how long would it take to transfer the entire E.coli genome to recipient cell? does this happen often? why/why not

Answer 16

100 mins
no
conj pilus usually breaks down before then

Question 17

after Hfr conjugation, is the recipient cell F+?

Answer 17

no , the conjugation pilus breaks down before the entire transfer of the F plasmid

Question 18

use of interrupted conjugation (mating)

Answer 18

map E.coli genome

Question 19

how does interrupted mating produce a genetic map

Answer 19

the earlier a gene is transferred to the F plasmid, the closer it is to the integrated F plasmid

Question 20

what E.coli strains are used in interrupted mating

Answer 20

Hfr :

F-

Question 21

how can you see if a gene has transferred to the F- plasmid?

Answer 21

grow it on selective media for the gene

Question 22

describe process of interrupted mating

Answer 22

Hfr strain will be sensitive to an antibiotic but + for all other genes - that are trying to be transferred

1. mix Hfr and F-
2. at 5, 10 , 15 , 20 , 25 mins take sample, vortex and plate on different selective media for all of the genes of interest
3. count colonies and plot graph

Question 23

what does the plateu of an interrupted mating graph show

Answer 23

the breakage of the conj pilus - no. of colonies wont just keep increasing because eventually it breaks

Question 24

what genes are first to transfer if the F plasmid integrates into IS1

Answer 24

leu / val

depending on orientation of ori of transfer

Question 25

what genes are first to transfer if the F plasmid integrates into IS2

Answer 25

leu / thr

depending on orientation of ori of transfer

Question 26

features to note about naturally occurring plasmids

Answer 26

• not all can transfer between bacteria

- not all can integrate into bacterial genome

Question 27

describe features of R plasmids

Answer 27

• have tra genes so can conjugate

- carry genes that confer antibiotic resistance

Question 28

describe features of ColE 1

Answer 28

• found in E.coli
• no tra genes (cant conjugate)
• produces the protein colicin which is toxic to other bacteria

Question 29

describe features of Ti plasmids

Answer 29

• in soil bacteria
• has tra genes
• can also transfer their T region into plant cells - integration into plant chromosome causes crown gall disease

Question 30

what causes crown gall disease in plants?

Answer 30

integration of T region of Ti plasmid of soil bacteria into the plant chromosome