Recombination in bacteria

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Recombination in bacteria

• Bacterial Review
• Conjugation
• Bacteriophage genetics
• Phage cycle
• Plaque assay
• Phage cross
• Transduction
• Generalized transduction
• Cotransduction

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I. Bacterial Review

• Can be grown both in liquid and agar medium,
  colony visible cluster of cells
• Antibiotic resistant mutants able to grow in
  the presence of an antibiotic
• Nutritional mutants
• Prototrophs wildtype cells can synthesize
  nutrients from simple molecules present in the
  growth media
• Auxotrophs cant synthesize an essential
  nutrient and cant grow unless that nutrient is
  supplied in the medium
• Minimal medium contains only inorganic salts,
  energy source and carbon atoms
• Nonselective medium all wild type cells form
  colonies
• Selective medium medium that allows growth of
  only one type of cell

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Selective plating

• Allows the desired mutant to reproduce but not
  wild-type genotypes
• antibiotic resistance (mutants able to grow in
  presence of antibiotic)
• Strr (mutants that are resistant to streptomycin)
• minimal medium supplemented with specific
  nutrient
• Met- auxotroph is able to grow if minimal medium
  has methionine

CNA (Columbia Naladixic Acid) Agarselective for
Gram-positive bacteria
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II. Conjugation A. Lederberg Tatums experiment
illustrated that DNA was transferred from one
bacterium to another. Strain 1
B-M-PT Strain 2 B M P- T-
These colonies are due to the transfer of genetic
material between two strains by conjugation.
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Bacterial sex Sex Pili required for a good
time!!!
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B. F plasmid (F factor)

• Ability to transfer based on presence of
  Fertility factor, now known as F plasmid
• 12 the size of the bacterial chromosome
• sex pili genes
• surface exclusion protein genes, preventing F
  conjugating with F
• transfer origin
• Episome F plasmid can remain as a free plasmid
  or be integrated into the bacterial chromosome

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1). Properties of F plasmid

• Can be replicated inside the cell
• Cells with F plasmid (F) have sex pili
• F and F- cells can conjugate
• Transfer of information is one-way from donor
  (F) to recipient (F-)
• Strain A Strain B
• F x F-
• (donor) (recipient)
• F cannot conjugate with other F cells
• F can become integrated into the host chromosome
  (rare event) F carries one or more insertion
  sequence elements (IS)
• F may leave genome, taking copies of some genes
  (F)

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host chromosome
F F-
F factor
F-
F
F pili promote cell to cell contact
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• F plasmid replication F replicates through
  rolling circle replication and it is transferred
  to a recipient cell via temporary cytoplasmic
  bridge between two cells. A copy remains in the
  donor cell.

F
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• 3. Intigration of F (Hfr)
• On rare occasions, the F plasmid is integrated
  into the circular chromosome, and there is
  genetic recombination this can then be
  transferred to a recipient cell and incorporated
  into the recipient's genome.

Hfr high frequency of recombination.
Still only partial transfer, not 100
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i.e. prior to mating, the recipient was lac- and
pro-, however after a short time period of
mating, the recipient is now lac, but still
pro- after a longer mating, the cell is lac and
pro (lac is always transferred first, pro later)
Chromosome transferred in a linear manner
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C. Mapping the E. coli chromosome using
interrupted mating

• A. Interrupted mating used a blender to
  separate bacterial cells that were in the act of
  conjugation, without killing them

Hypothesis the time it takes for genes to enter
a recipient cell is directly related to their
order along the bacterial chromosome Interrupted
matings would lead to various lengths of the Hfr
chromosome being transferred to the recipient.
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F-
Whether or not bacteria could grow depended upon
their genotypes i.e. a cell that is Azs cant
grow on azide platesa cell that is Azr can
To determine gene order, colonies were picked
from previous plates and restreaked on plates
that had azide or bacteriophage T1, or on minimal
plates
Lac
T1
A2
T
Gal
?
s
Rate of transfer
T L A2 T1 Lac Gal ? S
100 100 90 70 40 25 15 Time 8 8 9 11 18 25 26
90
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Gradient of transfer Frequency of inheritance
corresponds to the order of transfer Genes are
mapped according to time of appearance of
recombinants Circular, low resolution map is made
by combining maps from different Hfr donors
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• Conclusions
• there was a point of origin (O), because transfer
  occurs from a fixed point on the donor chromosome
• O first, F last
• determined the gene order based on the gradient
  of transfer
• chromosome was circular
• the F is integrated at different points and
  different directions

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Last first
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ORDER OF TRANSFER
Random reshuffling of genes??? Pattern L next
to G, G next to E, L next to B
ORDER OF GENES ON ORIGINAL F XTJFPYLGEBDNA
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gene order CRUISE Using the E. coli map (that
is based on 100 minutes), identify the location
of the origin of both Hfr 1 Hfr 3. The Hfr2
origin has already been identified. MAP ORDER
USING Hfr 2 FIRST, then you can map the Hfr 1 3
relative to that.
1
R
U
C
3
I
E
S
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If leu str-r recombinants are desired from the
cross Hfr leu str-s x F- leu- str-r, on what
kind of medium should the matings pairs be plated?
Plate on minimal medium that lacks leucine
(select for leu) but contains streptomycin
(selects for Str-r)
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D. The F state and Merozygotes
Formation of a Partial diploid The F pops
out often taking a piece of the chromosome with
itbecoming an F F prime, in the process
creating a stable partial diploid MEROZYGOTE
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recombination occur in asexual prokaryotes via

• Conjugation, Transduction, Transformation

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IV. Bacteriophage genetics

• A. Phage Cycle
• Virus binds to host cell
• Tail sheath causes core penetration of cell wall
• DNA in head is extruded
• Once inside cell
• All processes halted
• Degradation of host DNA initiated
• Phage DNA replicated, transcribed translated
  using host machinery
• Virus parts assembled
• e) Host cell ruptures

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B. Plaque assay the Phage cross Plaques are
clear zones formed in a lawn of cells due to
lysis by phage. Different phages produce distinct
plaque morphologies

• Two parental strains
• h- r X h r -
• h only infects strain1
• h- infects both strains
• r small plaque
• r- large plaque
• Double infection
• Phage lysate analyzed, looked at different plaque
  types
• RF (hr) (h-r-)
• total plaques

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Double Infections
Plaque phenotypes produced by progeny of the
cross h-r x hr- Four plaque phenotypes can be
differentiated 2 parental types, (h-r and
hr-) and 2 recombinant types (hr and
h-r-) Thus, phage genomes can be mapped by
analysis of RF
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V. Transduction

• Generalized Transduction Small fraction of DNA
  from the donor bacterial strain are carried by
  the phage
• Only a few genes are carried
• Any host marker can be transduced
• Phage infects the recipient, transferring the DNA
  fragment, creating a merozygote
• Remains in cytoplasm (abortive transduction)
• Transduced bacterial genes may be incorporated
  into the bacterial chromosome (complete
  transduction)

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B. cotransduction

• If 2 genes are close together along the
  chromosome, a bacteriophage may package a single
  piece of the chromosome that carries both genes
  and transfer that piece to another bacterium
• The likelihood of this occurring depends upon how
  close together they are
• Can map genes using cotransduction

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Mapping genes using cotransduction

• Select for the transduction of one gene and then
  monitor whether or not another gene is
  cotransduced along with it
• Donor arg met strs (infected w/P1 and lysate
  mixed w/recipient)
• Recipient arg- met- strr
• Plated on minimal media w/arg and strep but no
  met,
• Pick each of the colonies and re-streak on plates
  without met and without arg.
• Calculate cotransduction frequency
• growing on media (no arg)
• total colonies

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