Lecture 12 - bacterial genes & genetics: chromosomes, plasmids and gene transfer

Question 1

How can DNA from a donor cell be introduced into a recipient cell?

Answer 1

1. Transformation
2. Transduction
3. Conjugation

Question 2

What is transformation?

Answer 2

(uptake of naked DNA) - the DNA may of may not be degraded

Question 3

What is transduction?

Answer 3

(phage-mediated) - requires a bacteriophage that goes through an infective cycle

Question 4

What is conjugation?

Answer 4

(plasmid mediated) - the donor & recipient cell are different (transfer always from donor to recipient - and the recipient is always missing this plasmid)

Question 5

Describe Griffith’s classic experiments with Pneumococcus (1928)

Answer 5

• smooth & rough cells
• R stains lack carbohydrate capsule to protect bacteria from immune system
• Transformations changed R cells into S cells
• people weren’t sure whether this transformation principle causing heredity was either protein or DNA
• Oswald Avery showed that DNA was responsible for the genetic changes observed by Griffith, as he used proteases to break down protein, therefore leaving DNA responsible

Question 6

Describe the mechanism of transformation

Answer 6

1. DNA binds via proteins
2. ONE strand enters using the DNA translocate, the second strand is degraded
3. The internalised single strand is bound by RecA protein
4. As RecA has more than one DNA binding site, it can hold the ssDNA and dsDNA together
5. The RecA-ssDNA complex stretches the dsDNA to increase complementarity recognition (conformational proofreading)
6. Branch migration and homologous recombination follows
7. NOTE - the transforming DNA doesn’t need to be a plasmid (but often is)

Question 7

What is the mechanism of generalised transduction?

Answer 7

‘normal’ bacteriophage contains phage genes; transduction genes particles contain host genes as well

Question 8

What is T2 page-induced lysis of E.coli?

Answer 8

when phages lysed E.coli cells, regions of bacteria grow

Question 9

What is the mechanism of conjugation?

Answer 9

in conjugation there is a conjugation pair that connect via pillus

Bacterial colony growth was only seen through a mixture of 2 different types of mutants - through conjugation

double & triple mutants prevent spontaneous reversion

Question 10

What does conjugation require?

Answer 10

physical constant
- the fertility plasmid occurs only in dined cells

Question 11

What does mating require?

Answer 11

pili (s. pilus) responsible for cell pairing
- Pilus contracts by dis-assembling subunits
- the pilus is attached with phage virions

Question 12

How can transfer of the F plasmid by conjugation occur?

Answer 12

can happen via the pilus

Question 13

How can chromosomal genes be transferred?

Answer 13

due to integrated F-plasmid - Hfr (high frequency of recombination) strains were isolated - much more efficient genetic donors

Question 14

Describe how the transfer of chromosomal genes by an Hfr strain occurs

Answer 14

A bacterium with the ‘free’ F plasmid is called F+, when the plasmid is integrated, the bacterium is Hfr

Question 15

What occurs after integration and transfer of part of the plasmid?

Answer 15

chromosomal genes are transferred
- the Hfr strain transfers the F plasmid to a recipient as efficiently as an F+ would

Question 16

Why does different Hfr strains transfer genes in different order and form different regions?

Answer 16

The availability of many Hfr strains allowed microbiologists to identify the arrangement and orientation of almost all genes before the genome sequence of E.coli became known

Question 17

How is transformation used in the genetics laboratory?

Answer 17

widely used to get plasmids or fragments of linearlised DNA into numerous bacterial cell types.

• some bacteria are naturally competent to be transformed, others need chemical treatment or electroporation