Transcript Ch. 8: Presentation Slides

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
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
F factor and Conjugation
• Conjugation is a process in which DNA is transferred
from bacterial donor cell to a recipient cell by cell-to-cell
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
Figure 07.03: Transfer of F from an F+ to an F- cell.
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
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
Figure 07.04: Transposable elements in bacteria.
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 and 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
• Bacteria with resistance to multiple antibiotics are an
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.
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.
• Bacterial transformation showed that DNA is the
genetic material.
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
Figure 07.10: Cotransformation of linked markers.
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
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
Figure 11: Integration of F
Figure 7.11: Integration of F
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–
Figure 07.12: Stages in the transfer and production of recombinants.
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
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 07.13AE: Time-of-entry mapping.
Chromosome Mapping
Circular genetic map of E. coli shows
map distances of genes in minutes
Figure 07.13F: Time-of-entry mapping.
Figure 07.14: Circular genetic map of E. coli.
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.
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
Figure 07.16: Transduction.
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
Figure 07.17: Demonstration of linkage of the gal and bio genes.
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
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
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
Figure 07.18A: The absence of a phage.
Figure 07.18B: Large plaques in lawn of E.coli.
Courtesy of CDC
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
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
Figure 07.20: Array of deletion mutations used to divide the rII locus of phage T4.
Adapted from S. Benzer,
Proc. Natl. Acad. Sci. USA 47(1961): 403-426.
Figure 07.21: Genetic map of part of the rII locus of phage T4.
Adapted from S. Benzer,
Proc. Natl. Acad. Sci. USA 47(1961): 403-426
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 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
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
Figure 07.22: The general mode of lysogenization.
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
Figure 7.23: Linear DNA molecule showing the cohesive ends
Figure 26: Geometry of integration and excision of phage
Figure 7.24: Geometry of integration and excision of phage 
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 07.25: Aberrant excision leading to the production of specialized l
transducing phages.