Transcript F factor and Conjugation F

Chapter 7
The Genetics of Bacteria and
Their Viruses
Plasmids
• Many DNA sequences in bacteria are mobile and can
be transferred between individuals and among
species.
• Plasmids are circular DNA molecules that replicate
independently of the bacterial chromosome.
• Plasmids often carry antibiotic resistance genes
• Plasmids are used in genetic engineering as gene
transfer vectors
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F factor and Conjugation
• F (fertility) factor is a conjugative plasmid
transferred from cell to cell by conjugation
• F factor is an episome–a genetic element that can
insert into chromosome or replicate as circular
plasmid
• The F plasmid is a low-copy-number plasmid ~100
kb in length and is present in 1–2 copies per cell
• It replicates once per cell cycle and segregates to
both daughter cells in cell division
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F factor and Conjugation
• Conjugation is a process in which DNA is transferred
from bacterial donor cell to a recipient cell by cell-tocell contact
• Cells that contain the F plasmid are donors and are
designated the F+
• Cells lacking F are recipients and are designated the F–
• The transfer is mediated by a tube-like structure called
a pilus, formed between the cells, through which the
plasmid DNA passes
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Figure 7.3: Transfer of F from an F+ to an F- cell
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Transposable Elements
• Transposable elements are DNA sequences that can
jump from one position to another or from one DNA
molecule to another
• Bacteria contain a wide variety of transposable
elements
• The smallest and simplest are insertion sequences,
or IS elements, which are 1–3 kb in length and
encode the transposase protein required for
transposition and one or more additional proteins
that regulate the rate of transposition
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Transposable Elements
• Other transposable elements in bacteria contain one
or more genes unrelated to transposition that can be
mobilized along with the transposable element; this
type of element is called a transposon
• Transposons can insert into plasmids that can be
transferred to recipient cells by conjugation
• Transposable elements are flanked by inverted
repeats and often contain multiple antibiotic
resistance genes
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Figure 7.4: Transposable elements in bacteria
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Figure 7.5: Cointergrate formed between
two plasmids by recombination between
homologous sequences present in both
plasmids
Figure 05: Cointegrate
Transposable Elements
• Integron is a DNA element that encodes a site-specific
recombinase as well as a recognition region that
allows other sequences with similar recognition
regions to be incorporated into the integron by
recombination.
• The elements that integrons acquire are known as
cassettes
• Integrons may acquire multiple-antibiotic-resistance
cassettes, which results in the plasmid resistant to a
large number of completely unrelated antibiotics
• Bacteria with resistance to multiple antibiotics are an
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increasing problem in public health
Figure 06: Site-specific recombinase
Figure 7.6: : Site-specific recombinase
Figure 07: Mechanism by which an integron sequentially captures
Figure 7.7: Mechanism by which an integron sequentially captures
cassettes by site-specific recombination
cassettes by site-specific recombination
Figure 7.8: Mechanism of cassette excision
Bacterial Genetics
• Three principal types of bacterial mutants use in
bacterial genetics:
• Antibiotic-resistant mutants are able to grow in
the presence of an antibiotic.
• Nutritional mutants are unable to synthesize an
essential nutrient and thus cannot grow unless
the required nutrient is supplied in the medium.
Such a mutant bacterium is said to be an
auxotroph.
• Carbon-source mutants cannot utilize particular
substances as sources of energy or carbon
atoms.
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Figure 09: Bacterial colonies on petri dish
Figure 7.9: Bacterial colonies on petri dish
Courtesy of Dr. Jim Feeley/CDC
Bacterial Transformation
• The process of genetic alteration by pure DNA is
transformation.
• Recipient cells acquire genes from DNA outside
the cell.
• DNA is taken up by the cell and often recombines
with genes on bacterial chromosome.
• Transformation may alter phenotype of recipient
cells.
• Bacterial transformation showed that DNA is the
genetic material.
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Cotransformation of Linked Genes
• Cotransformation: genes located close together are
often transferred as a unit to recipient cell.
• Cotransformation of two genes at a frequency
substantially greater than the product of the singlegene transformation frequencies implies that the
two genes are close together in the bacterial
chromosome.
• Genes that are far apart are less likely to be
transferred together
• Cotransformation is used to map gene order
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Figure 7.10: Cotransformation of linked markers
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Conjugation
• In bacterial mating, conjugation, DNA transfer is
unidirectional
• F factor can integrate into chromosome via genetic
exchange between IS elements present in F and
homologous copy located anywhere in bacterial
chromosome
• Cells with the F plasmid integrated into the bacterial
chromosome are known as Hfr cells
• Hfr: High Frequency of Recombination
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Hfr
• In an Hfr cell the bacterial chromosome remains
circular, though enlarged ~ 2 percent by the
integrated F-factor DNA
• When an Hfr cell undergoes conjugation, the
process of transfer of the F factor is initiated in
the same manner as in an F+ cell
• However, because the F factor is part of the
bacterial chromosome, transfer from an Hfr cell
also includes DNA from the chromosome
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Hfr and Conjugation
• Transfer begins within an integrated F factor and
proceeds in one direction
• A part of F is the first DNA transferred, chromosomal
genes are transferred next, and the remaining part of F
is the last
• The conjugating cells usually break apart long before
the entire bacterial chromosome is transferred, and the
final segment of F is almost never transferred
• The recipient cell remains F–
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Figure 11: Integration of F
Figure 7.11: Integration of F
Figure 7.12: Stages in the transfer and production of recombinants
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Chromosome Mapping
• It takes 100 minutes for an entire bacterial
chromosome to be transferred and about 2 minutes
for the transfer of F
• The difference reflects the relative sizes of F and the
chromosome (100 kb versus 4600 kb)
• Regions in the transferred DNA may incorporate into
the recipient chromosome and replace homologous
regions
• This results in recombinant F– cells containing one or
more genes from the Hfr donor cell
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Table T01: Data showing the production of recombinants when mating
is interrupted at various times
Chromosome Mapping
• Genes in the bacterial
chromosome can be mapped
by Hfr x F– mating
Figure 7.13a-e: Time-of-entry mapping
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Chromosome Mapping
Circular genetic map of E. coli shows map
distances of genes in minutes
Figure 7.13f: Time-of-entry mapping
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Figure 7.14: Circular genetic map of E. coli
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Figure 15: Formation of an F’ lac plasmid
Figure 15: Formation of an F’ lac plasmid by aberrant excision of F
from an Hfr chromosome
Transduction
• In the process of transduction, bacterial DNA is
transferred from one bacterial cell to another by a
phage
• A generalized transducing phage transfers DNA
derived from any part of the bacterial chromosome
• A specialized transducing phage transfers genes
from a particular region of the bacterial
chromosome.
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Transduction
• A generalized transducing phage P1 cuts bacterial
chromosome into pieces and can package
bacterial DNA into phage particles – transducing
particle
• Transducing particle will insert ‘transduced”
bacterial genes into recipient cell by infection
• Transduced genes may be inserted into recipient
chromosome by homologous recombination
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Figure 7.16: Transduction
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Transduction
• A typical P1 transducing particle contains from
100 to 115 kb of bacterial DNA or about 50 genes
• The probability of simultaneous transduction of
both markers (cotransduction) depends on how
close to each other the genes are. The closer they
are, the greater the frequency of cotransduction
• Cotransduction provides a valuable tool for
genetic linkage studies of short regions of the
bacterial genome
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Figure 7.17: Demonstration of linkage of the gal and bio genes
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Transduction
• Specialized transducing phages transduce
bacterial genes at the site of prophage insertion
into the bacterial chromosome
• Transduction of bacterial genes occurs by
aberrant excision of viral DNA, which results in
the incorporation of bacterial genes into phage
chromosome
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Temperate Bacteriophages
• Temperate bacteriophages have two life cycles:
 lytic cycle = infection that results in production
of progeny phage and bacterial cell lysis
 lysogeny = nonproductive viral infection
results in insertion of viral DNA into bacterial
chromosome
• Viral DNA integration = site-specific insertion into
bacterial chromosome
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Lytic Cycle
• The reproductive cycle of a phage is called the lytic
cycle
• In lytic cycle:
Phage DNA enters the cell and replicates repeatedly
Cell ribosomes produce phage proteins
• Phage DNA and proteins assemble into new phage
particles
• Bacterium is split open (lysis), releasing phage
progeny with parental genotypes
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Figure 7.18A: The absence of a phage
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Figure 7.18B: Large plaques in lawn of E.coli
Courtesy of CDC
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Lytic Cycle
• When two phage particles that have different
genotypes infect a single bacterial cell, new
genotypes can arise by genetic recombination
• This process differs from genetic recombination in
eukaryotes:
 the number of participating DNA molecules
varies from one cell to the next
 reciprocal recombinants are not always
recovered in equal frequencies from a single cell
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Figure 7.19: Progeny of a phage cross
Fine Structure of the Gene
• The mutation and mapping studies of rII locus of
phage T4 performed by S. Benzer provided an
experimental proof to important conclusions:
 Genetic exchange can take place within a gene
and probably between any pair of adjacent
nucleotides
 The unit of mutation is an individual pair of
nucleotides
 Mutations are not produced at equal frequencies
at all sites within a gene
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Figure 7.20: Array of deletion mutations
Adapted from S. Benzer, Proc. Natl. Acad. Sci. USA 47(1961): 403-426.
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Figure 7.21: Genetic map
Adapted from S. Benzer, Proc. Natl. Acad. Sci. USA 47(1961): 403-426
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Lysogenic Cycle
• All phage species can undergo a lytic cycle
• Phages capable of only the lytic cycle are called
virulent
• The alternative to the lytic cycle is called the
lysogenic cycle: no progeny particles are produced,
the infected bacterium survives, and a phage DNA
is transmitted to each bacterial progeny cell when
the cell divides
• Those phages that are also capable of the lysogenic
cycle are called temperate
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Lysogenic Cycle
• In the lysogenic cycle, a replica of the infecting
phage DNA becomes integrated into the bacterial
chromosome
• The inserted DNA is called a prophage, and the
surviving bacterial cell is called a lysogen
• Many bacterial generations, after a strain has
become lysogenic, the prophage can be activated,
excised from the chromosome, and the lytic cycle
can begin
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Figure 7.22: The general mode of lysogenization
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Bacteriophage 
• E. coli phage  is a temperate phage capable of
both lytic and lysogenic, cycles
• The DNA of  is a linear molecule with cohesive
ends (cos) that pairing yields a circular molecule
• In lysogen prophage  is linearly inserted between
the gal and bio genes in the bacterial DNA
• The sites of  integration in the bacterial and
phage DNA are called the bacterial attachment site
and the phage attachment site
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Figure 7.23: Molecular and genetic maps of bacteriophage 
Figure 7.25: Linear DNA molecule showing the cohesive ends
Figure 26: Geometry of integration and excision of phage
Figure 7.26: Geometry of integration and excision of phage 
Figure 7.24: The map order of genes in phage  as determined by phage
cycle) and
in the
Figure recombination
23: Molecular (lytic
and genetic
maps
ofprophage
bacteriophage
Bacteriophage 
• Prophage genetic map is a permutation of the
genetic map of the phage progeny obtained from
standard phage crosses.
• Upon induction, the prophage  is usually excised
from the chromosome precisely. However, once in
every 106 or 107 the excision error leads to
formation of aberrant phage particles that can carry
either the bio genes (cut at the right) or the gal
genes (cut at the left)
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Figure 7.27: Aberrant excision leading to the production of specialized 
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transducing phages