Brooker Chapter 6

Report
Bacterial Genetics
Bacterial Genetics

Bacteria are haploid

identify loss-of-function mutations easier

recessive mutations not masked
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6-2
Bacterial Genetics

Bacteria reproduce asexually


Crosses not used
genetic transfer

bacterial DNA segments transferred
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6-3
Genetic Transfer


Enhances genetic diversity
Types of transfer

Conjugation


Transduction


direct physical contact & exchange
phage
Transformation

uptake from environment
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6-4
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
F-factor Plasmid

Genes for conjugation
Conjugation
Figure 6.4
Conjugation
Figure 6.4
Conjugation

Results of conjugation



F factor plasmid may carry additional genes


recipient cell acquires F factor
converted from F– to F+ cell
called F’ factors
F’ factor transfer can introduce genes & alter
recipients genotype
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
Hfr Conjugation

Conjugation of Hfr & F– transfers portion of Hfr chromosome

origin of transfer of integrated F factor


takes 1.5-2 hrs for entire Hfr chromosome to be transfered



starting point & direction of the transfer
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
Hfr Conjugation
F– now lac+ pro–
order of transfer is lac+ –
pro+
F– now lac+ pro+
Figure 6.5b
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

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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6-21
Figure 6.6
Interpreting the Data
Minutes that
Bacterial
Cells were
Allowed to
Mate Before
Blender
Treatment
After 10 minutes,
the thr+ leu+
genotype was
obtained
There were no surviving colonies
after 5 minutes of mating
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
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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6-26

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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6-27
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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6-28
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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6-29

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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6-30
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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6-31
It will undergo
the lytic cycle
Prophage can
exist in a dormant
state for a long
time
Virulent phages only
undergo a lytic cycle
Figure 6.9
Temperate phages can
follow both cycles
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6-32
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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Transduction
Any piece of bacterial
DNA can be incorporated
into the phage
This type of transduction is
termed generalized transduction
Figure 6.10
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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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
A region of mismatch
By DNA repair enzymes
Figure 6.12
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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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6-48
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
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
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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6-51
Contains the
genetic material
Figure 6.13
Used for attachment to
the bacterial surface
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6-52

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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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
6-54

Some mutations in the phage’s genetic material can
alter the ability of the phage to produce plaques


Plaques are visible with the naked eye


Thus, plaques can be viewed as traits of bacteriophages
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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6-55


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


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)


The bacterium lyses so quickly that it does not have time to produce many new
phages
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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6-56
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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6-57

Figure 6.16 shows the possible outcomes of
complementation experiments involving plaque
formation mutants
Figure 6.16
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6-58

Benzer carefully considered the pattern of
complementation & noncomplementation


Benzer coined the term cistron to refer to the
smallest genetic unit that gives a negative
complementation test


He determined that the rII mutations occurred in two
different genes, which were termed rIIA & rIIB
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
rII mutations
Viruses cannot
form plaques in
E. coli K12(l)
rII mutations
Viruses cannot
form plaques in
E. coli K12(l)
Figure 6.17
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
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6-60

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(l)
Both rII mutants &
wild-type phages
can infect this
strain
Total number
of phages
66 plaques
rII mutants cannot
infect this strain
Number of wild-type
phages produced by
intragenic recombination
11 plaques
6-62

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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
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 wildtype phages multiplied by two
Frequency of recombinants =
Frequency of recombinants =
2 [wild-type plaques
obtained in E. coli k12(l)]
Total number of plaques
obtained in E. coli B
2(11 X 106)
6.6 X 109
= 3.3 X 10–3 = 0.0033
In this example, there was approximately 3.3 recombinants per 1,000 phages
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6-64

As in eukaryotic mapping, the frequency of recombinants can
provide a measure of map distance along the bacteriophage
chromosome


The frequency of intragenic recombinants is correlated with
the distance between the two mutations


In this case the map distance is between two mutations in the same
gene
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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6-65
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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6-66

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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6-67
Figure 6.19
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6-68

As described in Figure 6.19, the first step in the deletion
mapping strategy localized rII mutations to seven regions


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


Six in rIIA & one in rIIB
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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6-69
Contain many mutations
at exactly the same site
within the gene
Figure 6.20
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6-70



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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6-71

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