bacterial genetics. bacteria are haploid identify loss-of-function mutations easier recessive...
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Bacterial Genetics
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Bacterial Genetics Bacteria are haploid
identify loss-of-function mutations easier recessive mutations not masked
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Bacterial Genetics Bacteria reproduce asexually
Crosses not used genetic transfer
bacterial DNA segments transferred
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Enhances genetic diversity Types of transfer
Conjugation direct physical contact & exchange
Transduction phage
Transformation uptake from environment
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Genetic Transfer
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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
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Genes for conjugation
F-factor Plasmid
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Figure 6.4
Conjugation
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Figure 6.4
Conjugation
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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
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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
Hfr Strains
Figure 6.5a
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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 1.5-2 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
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Figure 6.5b
order of transfer is lac+ – pro+
F– now lac+ pro–
F– now lac+ pro+
Hfr Conjugation
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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
Interrupted Mating Technique
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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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Figure 6.6
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Interpreting the Data
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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 0 0
15 100 70 31 0 0
20 100 88 71 12 0
25 100 92 80 28 0.6
30 100 90 75 36 5
40 100 90 75 38 20
50 100 91 78 42 27
60 100 91 78 42 27
After 10 minutes, the thr+ leu+ genotype was obtained
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
There were no surviving colonies after 5 minutes of mating
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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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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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The E. coli Chromosome
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6-29Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 6.7
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
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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
Therefore these two genes are approximately 9 minutes apart along the E. coli chromosome
Figure 6.7
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Transduction is the transfer of DNA from one bacterium to another via a bacteriophage
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Transduction
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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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 6.9
Virulent phages only undergo a lytic cycle
Temperate phages can follow both cycles
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Prophage can exist in a dormant
state for a long time
It will undergo the lytic cycle
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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
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Plaques
Figure 6.14
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Figure 6.10
Any piece of bacterial DNA can be incorporated into the phage
This type of transduction is termed generalized transduction
Transduction
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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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Transformation
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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
Transformation
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6-47Figure 6.12
A region of mismatch
By DNA repair enzymes
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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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Transformation
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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
Horizontal Gene Transfer
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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
Horizontal Gene Transfer
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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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6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES
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Figure 6.13
Contains the genetic material
Used for attachment to the bacterial
surface
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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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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
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Plaques
Figure 6.14
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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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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() (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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Benzer collected many rII mutant strains that can form large plaques in E. coli B & none in E. coli K12()
But, are the mutations in the same gene or in different genes?
To answer this question, he conducted complementation experiments
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Complementation Tests
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Figure 6.16 shows the possible outcomes of complementation experiments involving plaque formation mutants
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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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At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred
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Coinfection
rII mutations
rII mutations
Viruses cannot form plaques in E. coli K12()
Viruses cannot form plaques in E. coli K12()
Function of protein A will be restored
Therefore new phages can be made in E. coli K12()
Viral plaques will now be formed
Figure 6.17
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Figure 6.18 describes the general strategy for intragenic mapping of rII phage mutations
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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()
66 plaques 11 plaques
Total number of phages
Number of wild-type phages produced by intragenic recombination
Both rII mutants & wild-type phages
can infect this strain
rII mutants cannot infect this strain
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The data from Figure 6.18 can be used to estimate the distance between the two mutations in the same gene
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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() 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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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() Therefore, the total number of recombinants is the number of wild-
type phages multiplied by two
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2 [wild-type plaques obtained in E. coli
k12()]Frequency of recombinants =Total number of plaques
obtained in E. coli B
2(11 X 106)
6.6 X 109Frequency of recombinants = = 3.3 X 10–3 = 0.0033
In this example, there was approximately 3.3 recombinants per 1,000 phages
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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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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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Deletion Mapping
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Let’s suppose that the goal is to know the approximate location of an rII mutation, such as r103
E. coli k12() 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
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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-68Figure 6.19
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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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Contain many mutations at exactly the same site within
the gene
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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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