1
Q
minimal media
A
contains a carbon source, essential elements such as nitrogen and phosphorus, vitamins and other nutrients
2
Q
complete media
A
contains all of the substances required by bacteria for growth and reproduction
3
Q
prototroph
A
- can grow on minimal media that contains an energy source and salts
- can synthesize everything they need for growth and reproduction with just minimal media
4
Q
auxotroph
A
- requires additional metabolites in order to grow because they lack one or more enzymes necessary for synthesizing essential molecules
- they will grow only on media supplemented with these essential molecules
5
Q
bacterial genomes
A
most are a circular chromosome of one DNA molecule several million bp in length
6
Q
plasmids
A
- small, replicating circular DNA molecules that may be present in addition to the chromosome
- in general, carry genes that are NOT essential to bacterial function but may an important role in the life cycle and growth of bacterial hosts
7
Q
episomes
A
class of plasmids that are capable of free replication and able to integrate into the bacterial chromosome.
8
Q
Lederberg and Tatum
A
- demonstrated that bacteria transfer and recombine genetic information
- Y10 and Y24 strain with opposite genotypes could not grow on minimal media. When the two strains were mixed, a few colonies grew on minimal media.
- genetic exchange and recombination took place between the two mutant strains
9
Q
U-shaped tube
A
- two sides separated by a sintered glass filters. the pores in the filter are too small to allow bacteria to pass through
- the two stains were placed on opposite sides of the filter, and a slight suction was applied alternatively to either side to allow media to flow back and forth across the filter
- after hours of incubation in the U-tube, the bacteria from neither side could grow on minimal media
- these experiments demonstrated that the exchange of bacterial genes requires direct contact between the cells
10
Q
F factor
A
- fertility factor
- contains a number of genes required for conjugation and the formation of a sex pili
11
Q
F+
A
- cells that contain the F plasmid
- donor cell
12
Q
F-
A
- cells that lack the F plasmid
- recipient cell
13
Q
sex pilus
A
- a slender extension of the cell membrane
- a sex pilus from an F+ cell makes contact with a receptor on an F- cell and pulls the two cells together
- DNA is then transferred from the F+ cell to the F- cell
14
Q
conjugation
A
1) F+ donor and F- recipient
2) F pili of F+ make contact with F- and cells pulled together. F factor codes for conjugation bridge to form.
3) one strand of DNA cleaved at oriT of F factor
4) rolling circle replication, one strand of F factor transfers to recipient (literally the circle rolls over and reforms)
5) Replication of F factor in both cells. The F- cell is now F+.
15
Q
Hfr
A
- the F factor is integrated into the bacterial chromosome
- Hfr cells behave as F+ cells and can mate with F- cells
- chromosomal genes are transferred from an Hfr strain to an F- strain with much higher frequency than from F+ strain
- The F- cell rarely becomes F+ or Hfr because the F factor is nicked in the middle to begin conjugation
16
Q
F’
A
- F factor present as separate circular DNA carrying some bacterial genes that it donates
- producing a partial diploid with two copies of the gene
17
Q
F+* F-
A
Two F+ cells (F- becomes F+)
18
Q
Hfr * F-
A
One Hfr cell and one F- (no change)
19
Q
F’ * F-
A
Two F’ cells (F- cell becomes F’)
20
Q
F’lac
A
- the F factor may pick up lac genes when it excises.
21
Q
Interrupted conjugation between Hfr and F-
A
only a portion of the chromosome will pass into the F- cell and have a chance to recombine with the recipient chromosome.
22
Q
Chromosome transfers always begin
A
within the integrated F factor at oriT and proceeds in a continuous direction
23
Q
genes are transferred according to
A
their arrangement on the chromosome
24
Q
the time for individual genes to be transferred
A
indicates their relative positions on the chromosome
25
bacterial maps with interrupted conjugation
the basic unit of distance is a minute
26
Different Hfr strains of a given species of bacteria have
the F factor integrated into the bacterial chromosome at different sites and in different orientations.
27
The order of gene transfer in a series of Hfr strains shows
the bacterial chromosome is circular
28
Transformation was discovered by
Frederick Griffith in 1928.
29
Type S
- smooth polysaccharide capsules
- virulent
- large colonies on blood agar
30
Type R
- rough and not encapsulated
- avirulent
- small colonies on blood agar
31
Experiment of Avery, MacLeod, and MacCarty
- used enzymes to destroy various biological molecules in a heat-killed lysate
- proteins in S lysate were treated with protein digesting enzymes, trypsin and chymotrypsin, yet still it transformed
- both DNA and RNA precipitated from the lysate using alcohol. RNA was destroyed using RNase, but solution could still transform. DNA was destroyed using DNase and the solution was unable to transform.
- concluded that DNA is the transforming principle.
32
Transformation
the uptake of DNA from the surrounding medium and its incorporation into a bacterial chromosome or plasmid
33
competent
cells that take up DNA
34
competence influenced by
growth stage and the concentration of DNA in the environment among other factors
35
Mechanism of transformation
1. Double stranded DNA reaches the cell membrane
2. As the DNA fragment enters the cell, one strand is hydrolyzed while the other traverses the membrane
3. The single-stranded fragment pairs with the bacterial chromosome and recombination takes place (two crossover events).
4. The remainder of the single-stranded DNA fragment is degraded by bacterial enzymes
5. When the cell replicates and divides, one of the resulting cells is transformed and the other is not.
36
Techniques for increasing transformation efficiencies
treatment with calcium chloride, heat shock, or an electrical field makes bacterial membranes more porous and permeable to DNA, and increases efficiency of transformation with high concentrations of DNA.
37
Transformation mapping
able to show that genes that are close to one another on the chromosome are more likely to be present on the same DNA fragment and be recombined together.
38
transformants
cells that receive genetic material through transformation
39
cotransformed
when two or more genes are transferred to the host chromosome
40
bacteriophage
viruses that infect bacteria
41
virulent phages
reproduce strictly through the lytic cycle and always kill the host cell
42
temperate phages
undergo either the lytic or lysogenic cycle
43
lytic cycle
1. phage binds to bacteriaum
2. phage DNA enters host cell
3. host DNA digested
4. phage DNA replicates
5. host cell transcribes, translates phage DNA, producing phage proteins
6. assembly complete. cells lyse
44
lysogenic cycle
1. phage binds to bacterium
2. phage DNA enters host cell
3. phage DNA incorporates itself into bacterial chromosome and becomes a prophage
4. prophage replicates
5. prophage my separate and enter lytic cell
45
Mapping bacterial genes by generalized transduction
- mixed two opposite strains
- when plated on minimal media, a few prototropic colonies appeared, suggesting conjugation
- when tested in a U-tube experiment, and placed on minimal medium, prototrophs were obtained from only one side of the tube
- later the agent was shown to be a bacteriophage
46
generalized transduction
- early 1950s
- trying to produce recombination between s. typhimurium by conjugation. stumbled upon transduction instead
- any bacterial gene can be transferred
47
transducing phages
- a piece of the bacterial chromosome instead of phage DNA becomes packaged into a phage coat
48
transductants
- the recombinant bacteria produced when the transducing phage infects a new cell, releasing the bacterial DNA which can become integrated into the bacterial chromosome.
49
Map genes using transduction
- when the phages have lysed the donor cells, a suspension the progeny phages is mixed with a recipient strain of bacteria, which is then plated on several different types of media to decide the phenotypes of the transducing phages.
- able to show if two genes are transduced together.
50
transduction
1. virus attaches to bacterial cell
2. injects DNA and replicates taking up bacterial DNA
3. Virus infects new bacterium carrying bacterial DNA with it
4. crossover in recipient leads to creation of recombinant chromosome
51
cotransduced
genes located close together that are transduced together.
52
Conjugation between F+ and F- cells
1. The F+ cell has an autonomous F plasmid. The F- cell lacks the F plasmid
2. the pili of the F+ donor cell makes contact with the F- recipient cell and the cells are pulled close together
3. One strand of DNA is cleaved at the oriT of the F factor. The F plasmid replicates by rolling circle replication. the new strand transfers to the recipient cell through conjugation channel
4. Replication of the F factor occurs in both cells during transfer. The new strand becomes double-stranded in the recipient cell, and the strand in the F+ cell also becomes double stranded
5. both cells are now F+
53
Conjugation between Hfr and F- cell
1. Hfr contains F plasmid incorporated into chromosome
2. Replication begins at oriT and a single-stranded product is made containing part of the F plasmid as well as part of the attached bacterial chromosome
3. The new strand crosses the conjugation tube and becomes double stranded
4. The chromosome genes on the new strand pair up with the homologous sequence on the chromosome of the recipient cell. Recombination occurs between the homologous sequences on the bacterial chromosome can be replaced by those on the transferred DAN.
5. After crossing over, the remainder of the transferred DNA is degraded. The recipient chromosome remains and can be replicated and passed on to daughter cells. The cell is still F- because it lacks an F plasmid. Original remains an Hfr cell
54
Conversion of Hfr into F' cell
1. integrated F factor in Hfr excises and takes with it a piece of the adjacent bacterial chromosome (lac)
2. The cell is now F'
3. F' can conjugate with F- during which the F factor is replicated
4. The donor cell contains the lac region only on the F factor while the recipient cell (now F') contains two copies of the lac region - one on the bacterial chromosome and one on the F factor. The recipient is known as partial diploid because it has two copies of the lac region.
55
rolling circle replication
the transfer of genetic material is unidirectional.
56
mapping with interrupted conjugation
- Donor Hfr cell and recipient F- cell
- strains mixed in nutrient medium and allowed to conjugate.
- at regular intervals, a sample of cells was removed and agitated vigorously to stop conjugation
- cells from each sample were placed on a selective medium
- only cells that underwent conjugation could grow on this medium.
GN 311
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