Download Chapter 7 – Recombination in Bacteria and

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Chapter 7 – Recombination in Bacteria and Their Viruses
Questions to be addressed:
1) By what mechanisms is genetic information recombined in
bacteria?
2) How are the distances between genes determined in bacteria?
Terminology
conjugation: the union of bacterial cells during which genetic
information is transferred from donor (F+) to recipient(F-)
plasmid: extrachromosomal, circular DNA
F (Fertility) factor: bacterial episome (present on plasmid or
chromosome) which allows a bacterial cell to be the donor during
conjugation
Hfr (high frequency of recombination): a bacterial cell in which the F
factor is integrated into the chromosome; during conjugation, the F
factor acts as the origin of chromosomal transfer
exconjugate: a bacterium that has undergone conjugation
endogenote: the endogenous recipient chromosome
exogenote: the exogenous donor chromosome
merozygote: a cell which is a partial diploid containing both an
endogenote and an exogenote
transformation: introduction of foreign DNA material through
external application
bacteriophage: "bacteria eater" - a virus that infects bacteria
lytic cycle: mode of infection in which the bacteriophage genome
enters the bacterium, replicates, lyses the cell and progeny are
released
virulent bacteriophage - undergo the lytic cycle
prophage: bacteriophage genome which is integrated into the bacterial
chromosome
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lysogenic: "lysis causing" - prophage is replicated with bacterial
chromosome; if prophage is excised from the chromosome, lytic cycle is
initiated
temperate bacteriophage - phage can enter the lysogenic cycle
transduction: movement of genetic material from donor to recipient
through a bacteriophage vector
Characteristics of Bacteria:
• single-celled
• prokaryotic (i.e. no nucleus or membrane-bound organelles)
• DO NOT exhibit meiosis
Typical phenotypes in bacteria (see Table 7-1)
-often alternative phenotypes of protrophy (self-feeding) and
auxotrophy (outside-feeding) are used as markers for genetic analysis
CLASS 1: pairs of alleles that confer ability to synthesize amino
acids, nucleotides, or other essential macromolecules
ad+ - wild type allele, able to synthesize amino acid adenine prototrophic
ad- - mutant allele, unable to synthesize amino acid adenine auxootrophic
CLASS 2: pairs of alleles that confer ability to utilize energy sources
gal
+
- wild type allele, able to utilize galactose as a carbon source,
prototrophic
gal - - mutant allele, unable to utilize galactose as a carbon source,
auxotrophic
CLASS 3: pairs of alleles that confer resistance to compounds that
normally inhibit bacterial growth (e.g. antibiotics)
Strs - wild type allele, sensitive to streptomycin, unable to form
colonies in presence of streptomycin
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Strr - mutant allele, resistant to streptomycin, able to form colonies in
presence of streptomycin
Scoring phenotypes (Figure 7-4):
-
a selection system is used
-
e.g. to select antibiotic resistant bacteria, cells are grown on
medium containing antibiotic
-
wild type cells die, resistant cells form colonies which can be
scored and selected
-
e.g. to select revertants, auxotrophic bacteria are grown on minimal
medium
-
cells of original (auxotrophic) phenotype die, cells which have
reverted to wild type allele form colonies
-
e.g to select for auxotrophic mutants, bacteria are grown in a low
concentration of penicillin
-
rapidly dividing cells (prototrophs) die, auxotrophs which cannot
divide survive
-
if cells are washed of penicillin and replated on various
supplemented media, auxotrophs can be selected
Question 1: How are new combinations of genetic information
achieved?
Genetic material enters the bacterial cell by one of three mechanisms,
and then undergoes RECOMBINATION with homologous regions of the
original bacterial chromosome.
Three mechanisms by which genetic information enters a bacterial cell:
1) CONJUGATION - exchange of genetic material between bacteria
involving cell to cell contact
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2) TRANSFORMATION - exchange of genetic material between bacteria
and their environment
3) TRANSDUCTION - bacteriophage (virus) mediated transfer of DNA
between bacteria
CONJUGATION
- one member of the conjugating pair carries a fertility factor (F) within
the F PLASMID
F plasmid - a small, circular, extrachromosomal piece of DNA
F+ strains - bacteria contain the F factor
F- strains - bacteria lack the F factor
Properties of the F Factor (See figure 7-5):
1) enables the production of PILI (proteinaceous attachment tube
between bacteria facilitating cell to cell contact)
2) replication of F plasmid permits F to be maintained (F factor moves
into F- cells during conjugation)
3) prevents conjugation between two F+ cells
Lederberg and Tatum (1952) studied bacterial gene transfer and
conjugation in Escherichia coli (see box 7-1)
STRAIN A = -bio- cys leu+ phe thi thr+
STRAIN B = bio+ cys+ leu phe+ thi thr
- both strains are AUXOTROPHIC
- if strains A and B are combined, a low frequency (1 x 10-7) of the
resulting bacteria are PROTOTROPHIC
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Question: How does recombination of genetic information
occur?
Hypothesis 1: physical contact between the cells is required.
Experiment: place bacterial strains on either side of a filter, and
determine if gene transfer occur
Results:
• unable to transfer genes through filter
Hypothesis 2: the direction of gene transfer is directed, with one
strain acting as donor, the other as recipient.
Experiment: expose one bacterial strain to streptomycin (an
antibiotic; allows transfer of DNA by bacterium, but inability to produce
progeny)
STUDY
Strain A
1) treat with streptomycin
2) incubate with Strain B
RESULT = prototrophs
Strain B
1) treat with streptomycin
2) incubate with Strain A
RESULT = no prototrophs
CONCLUSION: transfer is directed; only Strain A may act as the donor
and only Strain B acts as the recipient
Observation: Some F+ strains spontaneously become strains with a
high frequency of recombination (Hfr) (1000x more frequent
recombination) of chromosomal genes
Question: Are some genes more likely to be recombined than others?
Experiment (see figure 7-9):
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s tonr lac+ gal+ azir
Hfr strain: str
r tons lac- gal- azis
F- strain: str
NOTE: To control the experiment, the time of contact between the two
cells must be constant.
•
combine the two strains
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interrupt matings (disrupt conjugation) at specific times and sample
cells resulting from mating
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plate the samples on medium containing streptomycin
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streptomycin kills the original Hfr cells (since strs) but permits the
-
growth of F cells (strr)
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determine the genotype (test for tonr lac+ gal+ azir genetic markers)
of the surviving bacteria by evaluating their ability to carry out
metabolic pathway (e.g. a lac+ bacterium can use lactose as a
carbon source)
Results: the genetic markers are present with different frequencies at
Frequency of Hfr
geneticcharacters among strr exconjugates
the different sampling (mating) times (see figure 7-9)
100
azir
tonr
80
60
lac+
40
gal+
20
0
0
10 20 30 40 50 60 70
Mating Time (minutes)
azir - high frequency at short matings
gal+ - greatest frequency only in long matings
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Conclusions:
1) In Hfr strains, F factor is integrated into the bacterial chromosome
promoting transfer of chromosomal genes (Figure 7-6)
2) there is a fixed point at which transfer begins (origin) and a linear
order to the transfer process of the genes (Figure 7-8)
3) the time taken to transfer a gene is related to the distance from the
origin to that gene (Figure 7-9)
Additional observation: if the mating is permitted to continue (i.e. it
is not interrupted), the recipient cells become Hfr
Conclusions (Figure 7-7):
F-factor consists of two parts
• the first part to enter = origin
• the last part to enter = terminus
- a cell must receive both the origin and the terminus to become Hfr
Question: Is the position of genes, the position of the origin, and the
direction of transfer constant in different Hfr strains? (see figure 7-11)
experiment: Consider 5 different Hfr strains (all derived from the
same F+ strain) and determine the sequence of gene transfer
= origin
Results:
a - e = genetic marker
F = F factor
strain 1 =
a
b
c
d
e
F
strain 2 =
c
d
e
a
b
F
strain 3 =
b
c
d
e
a
F
strain 4 =
c
b
a
e
d
F
strain 5 =
e
d
c
b
a
F
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1) in each strain, the gene which enters first is different
2) the relative position of each gene is constant, (e.g. gene a in each
strain is always flanked by genes e and b
conclusion: the difference between Hfr strains is the position and
orientation of the origin within the circular chromosome
Useful Terminology:
EXCONJUGATE: a bacterium that has undergone conjugation
- select for recipient cells
ENDOGENOTE: the endogenous recipient chromosome
EXOGENOTE: the chromosome from the donor
MEROZYGOTE: partial diploid formed through conjugation
Hypothesis: using the sequence and time of transfer, a map of the
chromosome can be obtained (Figure 7-10)
1) based on the order of transfer it is possible to deduce the linear order
of genes
2) based on the first gene transferred it is possible to determine the
position and orientation of the F-factor
3) the time it takes to transfer the genes from the donor to the
recipient is equal to the distance between the genes
EXAMPLE
Five Hfr strains derived from the same E. coli original strain are
allowed to conjugate with an F- strain. The following table shows entry
times (minutes) of the first four genes.
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Strain 1
a (10)
Strain 2
c (5)
Strain 3
b (3)
Strain 4
c (6)
Strain 5
e (25)
b (18)
d (20)
c (13)
b (16)
d (45)
c (28)
e (40)
d (28)
a (24)
c (60)
d (43)
a (70)
3 (48)
e (54)
b (70)
question: derive a map of the original E. coli strain
MAPPING BASED ON RECOMBINATION FREQUENCY (Figure 7-12)
hypothesis: once genes are transferred into the F- strain, they are
able to recombine with genes in the host chromosome and the
frequency of recombination between two genes will depend upon the
distance between those genes
gal+
arg+
met+
gal
gal- -
arg--
met
met- -
transferred DNA
host chromosome
Solving the problem of gradient of transfer - gene transfer is based
on the distance from the origin, with genes nearest the origin being
transferred at the highest frequency, and genes farthest from the origin
transferred at the lowest frequency = gradient of transfer
INTEGRATION = transfer and recombination
•
a gene must be transferred before it can be integrated into the
chromosome.
•
gradient of transfer means that genes closer to the origin will be
transferred and therefore integrated more frequently than genes
further from the origin
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•
distorts the recombination frequency as a measure of distance
How to remove the gradient of transfer distortion?
EXAMPLE
Hfr strain gal+ arg+ met+
X
-Fstrain gal- arg- met-
RESULT
gene
met+
arg+
gal+
frequency of
inheritance
100%
70%
30%
implication
first gene to be transferred
last gene to be transferred
• since met+ is the most frequently integrated gene, it is the first gene
transferred
• since gal+ is the least frequently integrated gene, it is the last gene
transferred
recall that:
integration = transfer and recombination
•
if transfer is equal for all genes then integration = recombination
therefore, frequency of integration
= frequency of recombination
= distance between two genes
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TRANSFORMATION
- introduction of DNA fragments from the environment through the
bacterial cell wall
- recombination leads to integration
- transformation can also be induced in plant and animal cells
- the frequency of bacterial transformation can be increased by
manipulating [Ca+2] and electric shock
(a treated cell is said to be COMPETENT to take up DNA)
Linkage Information and Transformation
-DNA is introduced as fragments, no direction of transfer
- the closer together two genes are, the more likely that they will be on
the same fragment and integrate into the chromosome together
-
can deduce the relative distance between pairs of genes based on
COTRANSFORMATION frequency (the introduction of more than one
gene simultaneously)
-
if two genes are unlinked, the frequency of cotransformation will be
equal to the product of their individual transformation frequencies
EXAMPLE
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BACTERIOPHAGES (refer to Figures 7-16, 7-17, 7-18)
- viruses that infect bacteria (e.g. T2, T4, l = lambda)
- consist of nucleic acid surrounded by a protein coat (= capsid)
Lytic Cycle (refer to Figure 7-17, 7-18)
1) phage recognizes and attaches to bacterium
2) viral genome enters bacterium
3) bacterium makes copies of the viral genome and capsid
4) new phages assemble within host bacterium
5) bacterial cell is lysed and viral particles are released
- if bacteria are infected and then grown on an agar plate, the lysis of
the bacterial cell results in the formation of a clear area of dead
bacteria known as a PLAQUE
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Mapping in Bacteriophages
-
requires that two genetically distinct phages come together and
have the opportunity to recombine
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-
the Phage Cross (Figure 7-20, 7-21, Table 7-2)
coinfection of a bacterium with two distinct phage genotypes by
incubating bacteria with a very high phage concentration (Figure 720)
-
e.g. phage 1 = h+ rphage 2 = h- r+
-
-
h = "host range" - can only affect one strain of E. coli
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h - can affect two strains of E. coli
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r - "rapid lysis" - rapidly lyses E.coli
-
r - slowly lyses E.coli
progeny of infection is incubated with a combination of both E.coli
strains, and 4 genotypes (2 parental , 2 recombinant) scored
-
recombination frequency = #recombinant plaques/total plaques
Lysogenic Cycle
•
the viral genome is integrated into the bacterial chromosome
(PROPHAGE)
•
integration of the prophage occurs at a particular site within the
bacterial chromosome
•
example:
•
lambda and E. coli (both have circular genomes)
•
integration is accomplished through a recombination event at
specific ATTACHMENT SITES (att sites) in the phage and
bacterial genomes (see figure 7-23)
•
the incorporated viral genome is replicated each time the
bacterial chromosome is duplicated
•
adverse conditions may trigger the viral genome to become
released from the bacterial chromosome and subsequently
induce lysis
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•
excision (entry into the lytic cycle) is through reversal of the
recombination event
•
if a lysogenic Hfr bacterium conjugates with a nonlysogenic Fbacterium, the transferred prophage immediately enters the
lytic cycle = zygotic induction (Figure 7-22)
•
repressors present in cytoplasm of lysogenic cell maintain
prophage in lysogenic state
•
nonlysogenic cell has no repressors, therefore lysis is induced
TRANSDUCTION: the bacteriophage mediated transfer of genetic
material
- information is transferred between two bacteria via a bacteriophage
Hypothesis: physical contact is necessary for transfer of genetic
material
experiment: assess if genetic material can be transferred between
bacteria when they are separated by a filter
results:
• the exclusion limit (point at which genetic material cannot be
transferred) is determined by the size of the phage particle
• the phage is the VECTOR of genetic material transfer
- there are two types of transduction
1) Generalized Transduction (Figure 7-24) - random sample of
genes are transduced
- the bacterial chromosome disintegrates when the cell is lysed
- genomic fragments may be incorporated into the phage particle
(FAULTY HEAD STUFFING)
- subsequent phage infection of a new bacterial cell introduces the old
disintegrated fragment into new bacterium
- recombination leads to integration of the fragment
- each gene is equally likely to be transduced
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- proximity of genes is implied by …? (figure 7-26)
2) Specialized Transduction (see Figure 7-25)
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excision of prophage initiates the lytic cycle
•
if recombination is not exact, bacterial genes close to the
attachment site may be incorporated
•
in lambda (l), the gal+ genes is adjacent to the attachment site
•
if the phage leaves it genes it may be incapable of integrating (i.e.
becomes defective)
•
•
(lambda d gal)
the gene most frequently transduced is nearest the attachment site