Bacteria as a Model System

1
Bacteria as a Model System

• Analysis of Drosophila has led to estimates of
  gene sizes which are comparable to those of the
  largest known moleculesgenes are nothing less
  than a particular kind of molecule except that
  their detailed structure is not yet known- Max
  Delbrück, On the Nature of Gene Mutation and
  Gene Structure, 1935

2
Bacteria as a Model System

• 1945 Erwin Schrödingers What is life?
  published
• How do living organisms order themselves? Do
  molecules govern heredity? What is the physical
  nature of molecules that govern heredity?

3
Bacteria as a Model System

• the obvious inability of present-day physics
  and chemistry to account for events taking place
  in a living organism is no reason at all for
  doubting they can be accounted for by these
  sciences
• ...likely to involve hitherto unknown other
  laws of physics, which, however, once they have
  been revealed will form just as integral a part
  of this science, as the former.

4
Birth of Bacterial Genetics and Molecular
Biology

• Since many.physical scientists were suffering
  in the immediate post-war period from a general
  professional malaise, they were eager to direct
  their efforts toward a new frontier.- Stent
• The influx of individuals trained in physics
  provided a basis for the revolution in molecular
  biology (e.g. Delbrück, Szilard, etc.)

5

• Selective media
• missing a nutrient
• containing an antibiotic
• viral exposure

• Phenotypes
• growth characteristics
• colony morphology

6
Bacterial Growth Characteristics

• prototroph an organism with wild-type growth
  requirements will be able to grow on minimal
  medium
• auxotroph an organism that will proliferate only
  when the media is supplemented with some specific
  substance not required by wild-type organisms

7
Tonr mutants resistant to T1 phage
8
Studies on Bacteria Are changes in growth
characteristics a result of random mutation or
physiological selection?

• 1943 The Delbrück-Luria Fluctuation test
• Designed to determine if variant phenotypes were
  due to physiological adaption of the bacterial
  cells or to random mutation
• test based on a statistical argument...

9
Innoculate with 103 cells/ml

Bulk Culture (20 ml)
20 Individual cultures (0.2 ml)
Grow cells to 108 cells/ml
withdraw 0.2 ml sample from bulk culture and
plate on dishes of T1 phage
plate individual 0.2 cultures on dishes
containing T1 phage
Score for resistant cells
10
Small variance among samples
11
Large variance among samples
12
Individual cultures great amount of variation in
number of tonr colonies
Bulk culture all samples show similar numbers of
tonr colonies
13
Replica Plating (1952) Joshua and Esther
Lederberg...
14
resistant cells are due to random mutation in
original population, NOT physiological selection
15
Recombination in BacteriaChapter 14

• How are genes in bacteria organized?
• Are there correlates to eukaryotic chromosomes?
  Is there linkage?
• How universal are information storage and
  retrieval systems in Nature?

16
Lederberg and Tatum Experiment (1946)

• Evidence supporting genetic exchange in bacteria

1/107
17
B. Davis (1950)
Genetic exchange required physical contact
between the two strains
18
W. Hayes (1953) Transfer of information is
unidirectional
Streptomycin treatment prevents cell division
and subsequently kills cells
Recombinants recovered
No recombinants
Donor/recipient relationship strain A Donor
strain B recipient
19
Physical Differences between Donor and Recipient
Strains
Donor cell contains a genetic factor that
converts recipient cell to donor status...
20
The F Factor

• F cells donor strain
• F- cells recipient strain
• Donor cells contain a genetic entity (F factor)
  that determines donor status
• F factor must replicated and transferred to
  recipient F- cells in culture become F
• F factor mediates directed transfer (e.g. genes
  for pili formation)

21
F factor and chromosome as circles? (to be
discussed...)

• F factor exists as independently replicating
  factor (distinct from bacterial chromosome)

• during replication it is transferred to recipient
  cell with high frequency

• F- (recipient) cell now converted to F (donor)
  cell

N.B. bacterial chromosome not involved in F- to
F conversion... conversion to prototroph (a- to
a) is a low frequency event
22
Discovery of Hfr Cells (1950-1953)

• Hayes discovered a strain of cells that could
  transfer chromosomal genes at much higher
  frequencies than seen in normal F cell
  populations Hfr (high frequency of
  recombination) strain
• Unlike F cells, this strain only transferred
  certain genes at high frequency (1000x)
• Hfr did not transfer donor status to recipient
  cells

23
Selection for Hfr Cells

• Wollman and Jacob (1957) different Hfr strains
  could be isolated from F populations
• Isolation of different Hfr strains could be
  accomplished by replica plating and selection...

24
Hfr Cells

• Unlike F cells, Hfrs donate specific genes to
  recipient cells with high frequency (Hfr high
  frequency of recombination)
• Unlike F cells, only an extremely small portion
  of the F- recipient cell population attains a
  donor phenotype.
• Wollman and Jacob developed technique to select
  for different Hfr strains...

25
replica plate
F
F-
Strain A (auxotroph e.g. lac- pro-)
Strain B (auxotroph e.g. met- bio-)
Isolate Hfr from master plate
F x F-
Minimal media plate (select for recombinants)
26
Reason for unidirectional transfer of chromosomal
genetic information...
1/1,000 cells an integration event will occur
27

• F factor exists as independently replicating
  factor
• during replication it is transferred to
  recipient cell with high frequency
• F- (recipient) cell now converted to F (donor)
  cell

28
Rarely, F factor crosses over into bacterial
chromosome
29
Hfr
What is the evidence that both F factor and
bacterial chromosome are circular?
Genetic studies predict that F factor will
contain an origin of replication, fertility genes
(to make pili) and region for pairing with
bacterial chromosome...
30
If both are circles, a single x-over integrates
the entire F factor
Hfr
What is the evidence that both F factor and
bacterial chromosome are circular?
Genetic studies predict that F factor will
contain an origin of replication, fertility genes
(to make pili) and region for pairing with
bacterial chromosome...
31
Interrupted Mating Experiments

• Start Hfr x F- mating at a precise time
• remove portion of culture...
• disrupt conjugation by shearing conjugation
  bridge in a Waring blender and plate on media to
  select for recombinants...

32
Interrupted Mating Experiments

• e.g. Selection Hfr strs a and F- strr a- cells
  are used
• exconjugants are plated on streptomycin and
  minimal media plate to select for particular
  auxotroph
• all donor cells will die and only recipients
  that are recombinant for wild-type gene will
  survive...

33
Use strep-resistant recipients to kill off donor
cells...

• Transfer of genes
• genes closest to origin transferred first will
  be present in greatest frequency in population

• genes farther away will be transferred to smaller
  fraction of cells...

34
(No Transcript)
35
Time-of-Entry Mapping
36
Different integration events determine location
and direction of transfer
37
Time of entry mapping experiment data was only
consistent with a circular map
38
F' factorswhat goes in can come out...

• F factors can excise from the genome either
  precisely or imprecisely
• Sometimes imprecise excision can produce F'
  factors that contain bacterial genes

• sexduction when the F' factor replicates, it
  passes the bacterial gene AND the F factor genes
  to recipient cell...

39
F' factorswhat goes in can come out...

• F factors can excise from the genome either
  precisely or imprecisely
• Sometimes imprecise excision can produce F
  factors that contain bacterial genes
• sexduction when the F' factor replicates, it
  passes the bacterial gene(s)AND the F factor
  genes to recipient cell...

Merodiploid (partial diploid)