1. Gene transmission in bacteria Transformation
Fahareen-Binta-Mosharraf
MNS

2. Bacteria reproduce asexually
Some sexual process-Parasexual process
Genetic material is transferred from one bacteria to another through three distinct processes

3. Mechanisms of Genetic Exchange in Bacteria
Bacteria transfer (or receive) genetic material 3 different ways:
Conjugation
Transformation
Transduction

4. Mechanisms of Genetic Exchange in Bacteria

5. Mutant phenotypes in bacteria
Bacteria contain genes that mutate (genetic trasformation)to produce altered phenotypes. Gene transfer in bacteria is unidirectional—from donor cells to recipient cells.

6. 1. Mutants Blocked in Their Ability to Utilize Specific Energy Sources:
Wild-type E. coli can use almost any sugar as an energy source.
Some mutants are unable to grow on the milk sugar lactose.
Grow well on other sugars but cannot grow on medium containing lactose as the sole energy source
Nomenclature
For phenotypes, the first letter is capitalized; for genotypes, all three letters are lowercase and italicized.
Therefore, wild-type E. coli is phenotypically Lac+ (able to use lactose as an energy source) and genotypically lac+.
Mutants that are unable to utilize lactose as an energy source are phenotypically Lac- and genotypically lac-

7. 2. Mutants Unable to Synthesize an Essential Metabolite
Prototrophs
cells can synthesize all of the metabolites—amino acids, vitamins, purines, pyrimidines, and so on—they need from these substances. These wild-type bacteria are called prototrophs
Auxotrophs
the bacterium carrying that mutation will have a new growth requirement. It will grow if the metabolite is added to the medium, but it will not grow in the absence of the metabolite. Such mutants are called auxotrophs; they require auxiliary nutrients for growth
Example, wild-type E. coli can synthesize tryptophan de novo; these cells are phenotypically Trp and genotypically trp. Tryptophan auxotrophs are Trp and trp

8. 3. Mutants Resistant to Drugs and Antibiotics
Wild-type E. coli cells are killed by antibiotics such as ampicillin and tetracycline.
Phenotypically, they are Amps and Tets. The mutant alleles that make E. coli resistant to these antibiotics are designated ampr and tet r

9. General Features of Gene Transfer in Bacteria
gene transfer is unidirectional rather than bidirectional. Recombination events in bacteria usually occur between a fragment of one chromosome(from a donor cell) and a complete chromosome (in a recipient cell)
crossovers must occur in pairs and must insert a segment of the donor chromosome into the recipient chromosome .If a single crossover (or any odd number of crossovers) occurs, it will destroy the integrity of the recipient chromosome, producing a nonviable linear DNA molecule

11. Even number crossing over

12. Basic test for transformation, conjugation and transduction
(1) Does the process require cell contact?
(2) Is the process sensitive to deoxyribonuclease (DNase),an enzyme that degrades DNA in free condition ?

13. bacteria with different genotypes are placed in opposite arms of a U-shaped culture tube
The two arms are separated by a glass filter that has pores large enough to allow DNA molecules and viruses, but not bacteria, to pass through it.

14. If gene transfer occurs between the bacteria growing in opposite arms of the U-tube,
the process cannot be conjugation, which requires direct contact between donor and recipient cells.
if the observed gene transfer occurs in the presence of DNase and in the absence of cell contact, it must involve transduction

17. Transformation

18. „ Transformation is the process by which a bacterium will take up extracellular DNA released by a dead bacterium
„ It was discovered by Frederick Griffith in 1928 while working with strains of Streptococcus pneumoniae
„ There are two types
1. Natural transformation
„ DNA uptake occurs without outside help
2. Artificial transformation
„ DNA uptake occurs with the help of special techniques

19. Factors affecting transformation
Transformation works best when donar and recipient cells are closely related
DNA size and state as sensitive to nucleases
Recipient cells are in a specific physiological state to take the donor DNA fragment

20. „ Natural transformation occurs in a wide variety of bacteria
„Artificial transformation Bacterial cells able to take up DNA are termed competent cells
They carry genes that encode proteins called competence factors
These proteins facilitate the binding, uptake and subsequent corporation of the DNA into the bacterial chromosome

21. Transformation discovery
Griffith’s discovery of transformation in Streptococcus pneumoniae.

23. Transformation process
The mechanism of transformation has been studied in considerable detail in S. pneumoniae, Bacillus subtilis, Haemophilus influenzae, and Neisseria gonorrhoeae
S. pneumoniae and B. subtilis will take up DNA from any source
whereas H. influenzae and N. gonorrhoeae will only take up their own DNA or DNA from closely related species.

24. Only cells that are expressing the genes that encode proteins required for the process are capable of taking up DNA. These bacteria are said to be competent, and the proteins that mediate the transformation process are called competence (Com) proteins.
Bacteria develop competence during the late phase of their growth cycle—when cell density is high but before cell division stops

27. Conjugation

28. Conjugation Definition:
Donor
Definition:
Gene transfer from a donor to a recipient by direct physical contact between cells
Unidirectional transfer
Genetic transfer is mediated by sex factor F
Mating types in bacteria
Donor
F factor (Fertility factor)
F (sex) pilus
Recipient
Recipient
Lacks an F factor

29. Conjugation requires direct contact between cells for unidirectional transfer of genetic material.
a. A segment of donor chromosome is transferred to the recipient, and may integrate into the recipient’s chromosome by homologous recombination.
b. The recipient is called a transconjugant.

30. Discovery of conjugation
Genetic transfer in bacteria was discovered in 1946 by Joshua Lederberg and Edward Tatum
„ They were studying strains of Escherichia coli that had different nutritional growth requirements
„ Auxotrophs cannot synthesize a needed nutrient
„ Prototrophs make all their nutrients from basic components

31. Lederberg and Tatum discovered conjugation (1946) using two E
Lederberg and Tatum discovered conjugation (1946) using two E. coli auxotrophic mutant strains:
Strain A’s genotype was met bio thr+ leu+ thi+. It grows on minimal medium supplemented with methionine and biotin.
Strain B’s genotype was met+ bio+ thr leu thi. It grows on minimal medium supplemented with threonine, leucine and thiamine.
Strains A and B were mixed, and plated onto minimal medium. About 1/106 cells produced colonies with the phenotype met+ bio+ thr+ leu+ thi+
d. Neither strain produced colonies when plated alone onto minimal medium, so the new phenotype resulted from recombination.

32. Discovery of conjugation
Lederberg and Tatum experiment showing that sexual recombination occurs between cells of E. coli

33. The genotype of the bacterial cells that grew on the plates has to be bio+ met+ leu+ thr+ thi+
„ Lederberg and Tatum reasoned that some genetic material was transferred between the two strains
Either the bio– met– leu+ thr+ thi+ strain got the ability to synthesize biotin and methionine (bio+ met +)
„ Or the bio+ met+ leu– thr–thi-strain got the ability to synthesize phenylalanine and threonine (leu+ thr+ thi+)

34. 2. Davis (U-tube) tested whether cell-to-cell contact was required:
Strain A cells were placed on one side of a filter, and strain B on the other. Cells could not move through the filter but molecules moved freely, encouraged by alternating suction and pressure.
b. No prototrophic colonies appeared when the cells were plated on minimal medium. This indicates that cell-to-cell contact is required, and the genetic recombination results from conjugation.

35. the sex factor F:
William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction.
F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).
F factor can integrate bacterial chromosome
F plasmid contains an origin sequence (O), which initiates DNA transfer. It also contains genes for hair-like cell surface (F-pili or sex-pili), which aid in contact between cells.
No conjugation can occur between cells of the same mating type.

36. Conjugation and F+ factor
F+ factor (fertility factor)- a sophisticated plasmid
OriT, Origin of transfer
insertion sequences,
genes for own replication,
genes for own transfer
genes for own hairlike structure

37. F factor existance The F factor can exist in either of two states:
the autonomous state, in which it replicates independently of the bacterial chromosome, and
(2) the integrated state, in which it is covalently inserted into the bacterial chromosome and replicates like any other segment of that chromosome- called Hfr (for High frequency of recombination)

39. Hfr (for High frequency of recombination)
In the 1950s, Luca Cavalli-Sforza discovered a strain of E. coli that was very efficient at transferring chromosomal genes
„ He designated this strain as Hfr (for High frequency of recombination)
„ Hfr strains are derived from F+strains
Hfr cells can conjugate with F- cells. The F DNA is nicked at its origin, and transfer moves some F sequences, and then chromosomal DNA. Double crossover inserts the donor DNA into the recipient chromosome, and allelic recombination occurs

40. Conjugation process
The first step in conjugation is the contact between donor and recipient cells
„ This is mediated by sex pili (or F pili) which are made only by F+strains
These pili act as attachment sites for the F– bacteria
„ Once contact is made, the pili shorten. Donor and recipient cell are drawn closer together .A conjugation bridge is formed between the two cells .The successful contact stimulates the donor cells to begin the transfer process
Transfer of F factor between F+ x F- Crosses
Transfer of F factor from Hfr i.e. Hfr x F- Crosses

41. Schematic Mechanism of F+ x F- Crosses
Pair formation
Conjugation bridge F+ F-
DNA transfer
Origin of transfer
Rolling circle replication

42. Transfer of the F factor

45. Conjugation of high-frequency recombinant strains:
Hfr strains replicate F factor as part of their main chromosome.
Conjugation in Hfr strains begins when F+ is nicked at the origin, and F+ and bacteria chromosomal DNA are transferred using the rolling circle mechanism.
Recombinants are produced by crossover of the recipient chromosome and donor in Hfr strains DNA containing F+.
In Hfr X F- the recipient virtually never acquires the Hfr phenotype, because this would require transfer of the entire chromosome, taking about 100 minutes at 37°C

46. Mechanism of Hfr x F- Crosses
Pair formation
Conjugation bridge
Hfr F-
DNA transfer
Origin of transfer
Rolling circle replication
Homologous recombination

49. Transfer of the Hfr F+ factor

50. In Hfr X F- the recipient virtually never acquires the Hfr phenotype
It generally takes about hours for the entire Hfr chromosome to be passed into the F– cell
„ Most matings do not last that long
„ Only a portion of the Hfr chromosome gets into the F– cell, only part of the plasmid is transferred and the F– cells does not become F+
„ The F–cell does pick up chromosomal DNA ,this DNA can recombine with the homologous region on the chromosome of the recipient cell
„ This may provide the recipient cell with new combination of alleles

51. Excision of the F+ factor also occurs spontaneously at low frequency.
Begin with Hfr cell containing F+.
Plasmids that “leave” the genome carrying chromosomal DNA are known as prime factors.
Small section of host chromosome also may be excised, creating an F’ plasmid.
F’ plasmid is named for the gene it carries, e.g., F’ (lac)

52. Exceptional physiological states of F Factor
Autonomous with donor genes (F’)
Characteristics of F’ x F- crosses
Error in excision of F factor fron Hfr
F factor containing segment of bacterial gene
F- becomes F’ while F’ remains F’
Hfr F’

53. Mechanism of F’ x F- Crosses
Pair formation
Conjugation bridge F’ F-
DNA transfer
Origin of transfer
Rolling circle replication

54. Hfr bacteria possess F factor integrated into the bacterial genome.
F+ bacteria possess F factor as a plasmid independent of the bacterial genome. The F plasmid contains only F factor DNA and no DNA from the bacterial genome.
F' (F-prime) bacteria possess an F plasmid that also includes some DNA taken from the bacterial genome. Sometimes it is formed by incorrect excision from the chromosome.
F- bacteria do not contain F factor. Will always act as the recipient and will never be a donor.

55. Transduction
Transduction: DNA is transferred from cell to cell via viruses.
A variety of prokaryotes can undergo transduction and a variety of phages can transduce.
Two types of transduction  virus ends up defective and homologous recombination. can occur in either case:

56. Transduction (cont’d)
There are two types of transduction:
generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.
specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

57. TRANSDUCTION Lederberg & Zinder
Transduction was first discovered in 1952 by Joshua Lederberg and Norton Zinder
Mixed two strains of bacteria
phe- trp- met+ his+ & phe+ trp+ met- his-
Plated mixture on minimal media
Media lacked these four amino acids
1 cell in 100,000 grew
Genotype phe+ trp+ met+ his+
Genetic material had been transferred

58. TRANSDUCTION Lederberg & Zinder
These two strains were placed in a U-tube
Separated by a filter
Presence of Dnase
Some cells became phe+ trp+ met+ his+
Genetic material was transferred from the second strain to the first
Transfer was unidirectional
Transfer was via a “filterable agent”

59. TRANSDUCTION Lederberg & Zinder
Pore sizes of the U-tube filter were altered
Filterable agent was less than 0.1 mm in diameter
Much smaller than a bacterium
Concluded (correctly) that the agent was a bacteriophage

60. Some Considerations Capsid size is finite
Limit to the amount of DNA that can be contained
Generally ~ 1 – 2.5% of genome
Two genes can be cotransduced only if they are very close together
Likelihood of cotransduction depends on how close together they are

61. Transfection
Transfection: Bacteria transformed by bacteriophage DNA instead of DNA from another bacterium.
Transfection by a lytic bacteriophage leads to normal virus production.

62. Types of Bacteriophage
Lytic or virulent – Phage that multiply within the host cell, lyse the cell and release progeny phage (e.g. T4)
Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell.

63. Generalized Transduction
Infection of Donor
Phage replication and degradation of host DNA
Assembly of phages particles
Release of phage
Infection of recipient
Legitimate recombination

64. Specialized Transduction Lysogenic Phage
Excision of the prophage
Replication and release of phage
Infection of the recipient
Lysogenization of the recipient
Legitimate recombination also possible

65. Seven steps in Generalised Transduction
1. A lytic bacteriophage adsorbs to a susceptible bacterium.
2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes
3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

66. 4. The bacteriophages are released.
5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

67. Seven steps in Generalised Transduction (contd)
6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .
7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

68. Six steps in Specialised Transduction
1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome .
2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

69. 3. Occasionally during spontaneous induction, a small
piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid.
4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

70. Six steps in Specialised Transduction (cont’d)
5. The bacteriophage adsorbs to a recipient bacterium and injects its genome.
6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

71. Specialized transduction

72. Three types of gene transfer

74. Genetic Mapping in Bacteria by Transformation
1. Transformation is used to map genes in situations where mapping by conjugation or transduction is not possible.
a. Donor DNA is extracted and purified, broken into fragments, and added to a recipient strain of bacteria. Donor and recipient will have detectable differences in phenotype, and therefore genotype.
If the DNA fragment undergoes homologous recombination with the recipient’s chromosome, a new phenotype may be produced. Transformants are detected by testing for phenotypic changes.
2. Some bacterial cells take up DNA naturally (e.g., Bacillus subtilis), while others require engineered transformation for efficient transfer (e.g., E. coli).

75. Genetic Mapping in Bacteria
Completed transformation occurs in a small proportion of the cells exposed to new DNA. Bacillus subtilis is an example
a. Donor is wild-type (a+). Recipient is mutant (a).
b. One of donor DNA strands is degraded, leaving ssDNA with the a+ allele.
c. The donor ssDNA pairs with homologous DNA in recipient’s chromosome, forming a triple-stranded region.
d. A double crossover event occurs, replacing one recipient DNA strand with the donor strand.
e. The recipient now has a region of heteroduplex DNA. One strand has the recipient’s original a allele and the other strand has the new a+ allele.
f. DNA replication will produce one chromosome with the original (a) genotype, and one with the recombinant (a+) genotype.
g. The cell with the recombinant genotype is then selected by its phenotypic change.

76. Transformation in Bacillus subtilis

77. Transformation experiments are used to determine gene sequence:
a. Whether genes are linked (physically close on the bacterial chromosome).
i. Transformation works best with small DNA fragments that hold only a few genes.
ii. Cotransformation is an indication that two genes are near each other. It is analyzed by
(1) Experimentally, if cotransformation is more frequent the genes must be close together.
(2) If the cotransformation rate is close to the transformation rate for each gene alone, the genes are linked.
b. The order of genes on the genetic map.
i. Suppose two genes (e.g., p and q) cotransform and are thus linked. One of them (e.g., q) often cotransformations with another gene (e.g., o).
ii. Determining the distance between p and o involves analyzing their cotransformation frequency.
(1) If p and o rarely cotransform, the gene order is p-q-o.
(2) If p and o frequently cotransform, the gene order is o-p-q.
c. The map distance between genes.

78. Fig. 14.11 Demonstration of determining gene order by cotransformation
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

79. Using Conjugation to Map Bacterial Genes
1. Conjugation experiments to map genes begin with appropriate Hfr strains selected from the progeny of F+ X F- crosses.
Jacob and Wollman (1950s) used Hfr donor strains with allelic differences from the F- recipient strains, in interrupted-mating experiments.
a. Donor: HfrH thr+ leu+ aziR tonR lac+ gal+ strR.
Recipient: F- thr leu aziS tonS lac gal strS.
c. The 2 cell types are mixed in liquid medium at 37°C. Samples are removed at time points and agitated to separate conjugating pairs.
d. Selective media are used to analyze the transconjugants. Results in this experiment:
i. The 1st donor genes to be transferred to the F- recipient are thr+ and leu+, and their entry time is set as 8 minutes.
ii. At 9 minutes, aziR is transferred, and tonR follows at 10 minutes.
iii. At about 16 minutes lac+ transfers, followed by gal+ at about 25 minutes.
e. Recombination frequency becomes less at later time points, because more pairs have already broken apart before the sample was taken.
3. A map may be constructed with the distance between genes measured in minutes. (The E. coli chromosome map spans about 100 minutes.)

80. WOLLMAN AND JACOB Elie Wollman and Francois Jacob (1950s)
Donor (Hfr) strain genetic composition
thr+ (Able to synthesize the amino acid threonine)
leu+ (Able to synthesize the amino acid leucine)
azir (Resistant to killing by the chemical azide)
tonr(Resistant to infection by bacteriophage T1)
lac+ (Able to metabolize the sugar lactose)
gal+ (Able to metabolize the sugar galactose)
strs (Sensitive to killing by the antibiotic streptomycin)

81. WOLLMAN AND JACOB Elie Wollman and Francois Jacob (1950s)
Recipient (F-) strain genetic composition
thr- (Unable to synthesize the amino acid threonine)
leu- (Unable to synthesize the amino acid leucine)
azis(Sensitive to killing by the chemical azide)
tons (Sensitive to infection by bacteriophage T1)
lac- (Unable to metabolize the sugar lactose)
gal- (Unable to metabolize the sugar galactose)
strr (Resistant to killing by the antibiotic streptomycin)

83. Fig. 14.7 Interrupted-mating experiment
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

84. Fig. 14.8 Genetic map of genes in the experiment in Fig. 14.7

85. Genetic Mapping in Bacteria by Transduction
Some bacteriophage carry genes from former host to new host, transferring genes between bacterial cells.
The DNA capacity of these phage vectors is limited to ≦ 1% of the host chromosome.
Once introduced into a new host, the recombinant viral DNA undergoes homologous recombination into the chromosome of the new host (transductant).

86. Gene mapping by generalized transduction
Closely linked genes are cotransduced at high frequency, allowing a detailed genetic map to be generated. For example:
P1 was used to map E. coli genes.
i. Donor strain is able to grow on minimal medium, and is also resistant to the metabolic poison sodium azide (leu+ thr+ aziR).
ii. Recipient strain can’t make leucine or threonine, and is poisoned by cndiiim azide (ieu thr aziS).
iii. P1 lysate grown on donor cells is used to infect recipient cells.
iv. Transductants can be selected for any of these traits (e.g., leu+, and then checked for the unselected markers (e.g., thr+ aziR)

88. For example:
(1) Of the leu+ selected transductants, 50% have aziR and 2% have thr+.
(2)Of the thr+ selected transductants, 3% have leu+, and 0% have aziR.
(3) This gives the map order: thr—leu--azi.
Map distances are calculated from the cotransduction frequency of gene pairs. It is effective only with genes located near each other on the chromosome.

89. Gene mapping of bacteriophages
1. Phage genes are mapped by 2-, 3- or 4-gene crosses, involving bacteria infected with phages of different genotypes.
a. Progeny phage are counted using a plaque assay in which each phage produces a cleared area in a bacterial lawn.
b. Distinguishable phage phenotypes include mutants with different plaque morphology. An example is strains of T2 differing in plaque morphology and/or host range.
i. One T2 strain has the genotype h+ r (h+ lyses E. coli strain B, but not strain B/2, and r is a mutant producing large distinct plaques).
ii. The other T2 strain has the genotype h r+ (h lyses both B and B/2 E. coli, and r+ produces the wild-type small plaque with fuzzy borders).
iii. When the E. coli lawn includes both B and B/2 strains, T2 with h produce clear plaques, while T2 with h+ produce cloudy plaques (due to uninfected strain B/2).

90. 2. The h and r genes are mapped by infecting E
2. The h and r genes are mapped by infecting E. coli strain B simultaneously with two phages, h+ r and h r+
i. Crossover can occur between the 2 types of T2 DNA, producing recombinant T2 chromosomes (h+ r+ and h r).
ii. Recombinant chromosomes are assembled into phage, as are parental-type chromosomes.
iii. The resulting lysate is plated on a mixed lawn E. coli strains B and B/2. The resulting plaques have 4 possible phenotypes (Figure 14.17):
(1) Parental type h r+ has small clear plaques with a fuzzy border.
(2) Parental type h+ r has large cloudy plaques with a distinct border.
(3) Recombinant type h+ r+ has small cloudy plaques with a fuzzy border.
(4) Recombinant type h r has large clear plaques with a distinct border.
iv. Recombination frequency reflects the relative genetic distance between the phage genes under study.

91. Fig. 14.16 The principles of performing a genetic cross with bacteriophages