1. Bacterial Genetics

2. Bacterial Genetics Bacteria are haploid recessive mutations not masked
identify loss-of-function mutations easier
recessive mutations not masked
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3. Bacterial Genetics Bacteria reproduce asexually genetic transfer
Crosses not used
genetic transfer
bacterial DNA segments transferred
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4. Genetic Transfer Enhances genetic diversity Types of transfer
Conjugation
direct physical contact & exchange
Transduction
phage
Transformation
uptake from environment
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5. Conjugation Many, but not all, species can conjugate
Only certain strains can be donors
Donor strain cells contain plasmid called F factor
F+ strains
Plasmid circular, extra-chromosomal DNA molecule

6. F-factor Plasmid
Genes for conjugation

7. Conjugation
Figure 6.4

8. Conjugation
Figure 6.4

9. Conjugation Results of conjugation
recipient cell acquires F factor
converted from F– to F+ cell
F factor plasmid may carry additional genes
called F’ factors
F’ factor transfer can introduce genes & alter recipients genotype

10. Hfr Strains
1950s, Luca Cavalli-Sforza discovered E. coli strain very efficient at transferring chromosomal genes
designated strain Hfr (high frequency of recombination)
Hfr strains result from integration of F' factor into chromosome
Figure 6.5a

11. Hfr Conjugation
Conjugation of Hfr & F– transfers portion of Hfr chromosome
origin of transfer of integrated F factor
starting point & direction of the transfer
takes hrs for entire Hfr chromosome to be transfered
Only a portion of the Hfr chromosome gets into the F– cell
F– cells does not become F+ or Hfr
F– cell does acquire donor DNA
recombines with homologous region on recipient chromosome

12. Hfr Conjugation Figure 6.5b F– now lac+ pro–
order of transfer is lac+ – pro+
F– now lac+ pro+
Figure 6.5b

13. Interrupted Mating Technique
Elie Wollman & François Jacob
The rationale
Hfr chromosome transferred linearly
interruptions at different times  various lengths transferred
order of genes on chromosome deduced by interrupting transfer at various time

14. Wollman & Jacob started the experiment with two E. coli strains
Hfr strain (donor) genotype
thr+ : Can synthesize threonine
leu+ : Can synthesize leucine
azis : Killed by azide
tons : Can be infected by T1 phage
lac+ : Can metabolize lactose
gal+ : Can metabolize galactose
strs : Killed by streptomycin
F– strain (recipient) genotype
thr– leu– azir tonr lac – gal – strr
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15. Figure 6.6

16. Percent of Surviving Bacterial Colonies with the Following Genotypes
Interpreting the Data
After 10 minutes, the thr+ leu+ genotype was obtained
Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment
Percent of Surviving Bacterial Colonies with the Following Genotypes
thr+ leu azis tons lac gal+ 5 –– 10
100 12 3 15 70 31 20 88 71 25 92 80 28
0.6 30 90 75 36 40 38 50 91 78 42 27 60
There were no surviving colonies after 5 minutes of mating
The azis gene is transferred first
It is followed by the tons gene
The lac+ gene enters between 15 & 20 minutes
The gal+ gene enters between 20 & 25 minutes
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17. From these data, Wollman & Jacob constructed the following genetic map:
They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome
Comparison of the order of genes among these strains, demonstrated that the E. coli chromosome is circular
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18. The E. coli Chromosome
Conjugation experiments have been used to map genes on the E. coli chromosome
The E. coli genetic map is 100 minutes long
Approximately the time it takes to transfer the complete chromosome in an Hfr mating
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19. Arbitrarily assigned the starting point
Units are minutes
Refer to the relative time it takes for genes to first enter an F– recipient during a conjugation experiment
Figure 6.7
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20. The distance between genes is determined by comparing their times of entry during an interrupted mating experiment
The approximate time of entry is computed by extrapolating the time back to the origin
Figure 6.7
Therefore these two genes are approximately 9 minutes apart along the E. coli chromosome
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21. Transduction
Transduction is the transfer of DNA from one bacterium to another via a bacteriophage
A bacteriophage is a virus that specifically attacks bacterial cells
It is composed of genetic material surrounded by a protein coat
It can undergo two types of cycles
Lytic
Lysogenic
Refer to Figure 6.9
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22. 6-32 Figure 6.9 It will undergo the lytic cycle
Prophage can exist in a dormant state for a long time
Virulent phages only undergo a lytic cycle
Temperate phages can follow both cycles
Figure 6.9
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23. Plaques
A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish
It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages
Figure 6.14
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24. Transduction
Any piece of bacterial DNA can be incorporated into the phage
This type of transduction is termed generalized transduction
Figure 6.10

25. Transformation Bacteria take up extracellular DNA
Discovered by Frederick Griffith,1928, while working with strains of Streptococcus pneumoniae
There are two types
Natural transformation
DNA uptake occurs without outside help
Artificial transformation
DNA uptake occurs with the help of special techniques
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26. Transformation
Natural transformation occurs in a wide variety of bacteria
Bacteria able to take up DNA = competent
carry genes encoding competence factors
proteins that uptake DNA into bacterium & incorporate it into the chromosome

27. A region of mismatch
By DNA repair enzymes
Figure 6.12
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28. Transformation
Sometimes, the DNA that enters the cell is not homologous to any genes on the chromosome
It may be incorporated at a random site on the chromosome
This process is termed nonhomologous recombination
Like cotransduction, transformation mapping is used for genes that are relatively close together
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29. Horizontal Gene Transfer
Transfer of genes between different species vs
Vertical gene transfer - transfer of genes from mother to daughter cell or from parents to offspring
Sizable fraction of bacterial genes have moved by horizontal gene transfer
Over 100 million years ~ 17% of E. coli & S. typhimurium genes have been shared by horizontal transfer

30. Horizontal Gene Transfer
Genes acquired by horizontal transfer
Genes that confer the ability to cause disease
Genes that confer antibiotic resistance
Horizontal transfer has contributed to acquired antibiotic resistance

31. 6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES
Viruses are not living
However, they have unique biological structures & functions, & therefore have traits
We will focus our attention on bacteriophage T4
Its genetic material contains several dozen genes
These genes encode a variety of proteins needed for the viral cycle
Refer to Figure 6.13 for the T4 structure
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32. 6-52 Figure 6.13 Contains the genetic material
Used for attachment to the bacterial surface
Figure 6.13
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33. In the 1950s, Seymour Benzer embarked on a ten-year study focusing on the function of the T4 genes
He conducted a detailed type of genetic mapping known as intragenic or fine structure mapping
The difference between intragenic & intergenic mapping is:
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34. Plaques
A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish
It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages
Figure 6.14
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35. Plaques are visible with the naked eye
Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaques
Thus, plaques can be viewed as traits of bacteriophages
Plaques are visible with the naked eye
So mutations affecting them lend themselves to easier genetic analysis
An example is a rapid-lysis mutant of bacteriophage T4, which forms unusually large plaques
Refer to Figure 6.15
This mutant lyses bacterial cells more rapidly than do the wild-type phages
Rapid-lysis mutant forms large, clearly defined plaques
Wild-type phages produce smaller, fuzzy-edged plaques
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36. Benzer studied one category of T4 phage mutant, designated rII (r stands for rapid lysis)
It behaved differently in three different strains of E. coli
In E. coli B
rII phages produced unusually large plaques that had poor yields of bacteriophages
The bacterium lyses so quickly that it does not have time to produce many new phages
In E. coli K12S
rII phages produced normal plaques that gave good yields of phages
In E. coli K12(l) (has phage lambda DNA integrated into its chromosome)
rII phages were not able to produce plaques at all
As expected, the wild-type phage could infect all three strains
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37. Complementation Tests
Benzer collected many rII mutant strains that can form large plaques in E. coli B & none in E. coli K12(l)
But, are the mutations in the same gene or in different genes?
To answer this question, he conducted complementation experiments
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38. Figure 6.16 shows the possible outcomes of complementation experiments involving plaque formation mutants
Figure 6.16
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39. A cistron is equivalent to a gene
Benzer carefully considered the pattern of complementation & noncomplementation
He determined that the rII mutations occurred in two different genes, which were termed rIIA & rIIB
Benzer coined the term cistron to refer to the smallest genetic unit that gives a negative complementation test
So, if two mutations occur in the same cistron, they cannot complement each other
A cistron is equivalent to a gene
However, it is not as commonly used
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40. Function of protein A will be restored
At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred
rII mutations
Viruses cannot form plaques in E. coli K12(l)
Coinfection
Function of protein A will be restored
Therefore new phages can be made in E. coli K12(l)
Viral plaques will now be formed
Figure 6.17
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41. Figure 6.18 describes the general strategy for intragenic mapping of rII phage mutations
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42. r103
r104
Take some of the phage preparation, dilute it greatly (10-8) & infect E. coli B
Take some of the phage preparation, dilute it somewhat (10-6) & infect E. coli K12(l)
Both rII mutants & wild-type phages can infect this strain
11 plaques
66 plaques
Number of wild-type phages produced by intragenic recombination
rII mutants cannot infect this strain
Total number of phages
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43. The data from Figure 6.18 can be used to estimate the distance between the two mutations in the same gene
The phage preparation used to infect E. coli B was diluted by 108 (1:100,000,000)
1 ml of this dilution was used & 66 plaques were produced
Therefore, the total number of phages in the original preparation is
66 X 108 = 6.6 X 109
or 6.6 billion phages per milliliter
The phage preparation used to infect E. coli k12(l) was diluted by 106 (1:1,000,000)
1 ml of this dilution was used & 11 plaques were produced
Therefore, the total number of wild-type phages is
11 X 106
or 11 million phages per milliliter
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44. In this experiment, the intragenic recombination produces an equal number of recombinants
Wild-type phages & double mutant phages
However, only the wild-type phages are detected in the infection of E. coli k12(l)
Therefore, the total number of recombinants is the number of wild-type phages multiplied by two
2 [wild-type plaques obtained in E. coli k12(l)]
Frequency of recombinants =
Total number of plaques obtained in E. coli B
2(11 X 106)
6.6 X 109
Frequency of recombinants =
= 3.3 X 10–3 =
In this example, there was approximately 3.3 recombinants per 1,000 phages
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45. Homoallelic mutations
As in eukaryotic mapping, the frequency of recombinants can provide a measure of map distance along the bacteriophage chromosome
In this case the map distance is between two mutations in the same gene
The frequency of intragenic recombinants is correlated with the distance between the two mutations
The farther apart they are the higher the frequency of recombinants
Homoallelic mutations
Mutations that happen to be located at exactly the same site in a gene
They are not able to produce any wild-type recombinants
So the map distance would be zero
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46. Deletion Mapping
Benzer used deletion mapping to localize many rII mutations to a fairly short region in gene A or gene B
He utilized deletion strains of phage T4
Each is missing a known segment of the rIIA and/or rIIB genes
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47. Let’s suppose that the goal is to know the approximate location of an rII mutation, such as r103
E. coli k12(l) is coinfected with r103 & a deletion strain
If the deleted region includes the same region that contains the r103 mutation
No intragenic wild-type recombinants are produced
Therefore, plaques will not be formed
If the deleted region does not overlap with the r103 mutation
Intragenic wild-type recombinants can be produced
And plaques will be formed
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48. Figure 6.19
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49. As described in Figure 6.19, the first step in the deletion mapping strategy localized rII mutations to seven regions
Six in rIIA & one in rIIB
Other strains were used to eventually localize each rII mutation to one of 47 regions
36 in rIIA & 11 in rIIB
At this point, pairwise coinfections were made between mutant strains that had been localized to the same region
This would precisely map their location relative to each other
This resulted in a fine structure map with depicting the locations of hundreds of different rII mutations
Refer to Figure 6.20
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50. Contain many mutations at exactly the same site within the gene
Figure 6.20
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51. Intragenic mapping studies were a pivotal achievement in our early understanding of gene structure
Some scientists had envisioned a gene as being a particle-like entity that could not be further subdivided
However, intragenic mapping revealed convincingly that this is not the case
It showed that
Mutations can occur at different parts within a single gene
Intragenic crossing over can recombine these mutations, resulting in wild-type genes
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