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Bacterial and
Phage Genetics
Chapter 15
meiosis
Why study Bacteria and Viruses
 Many cause human diseases, or affect agriculture.
 Many are beneficial to human health and mankind in
general.
 Because they can be manipulated so easily, they are
fantastic research tools. Used in nearly all labs.
 They can be genetically engineered to produce drugs,
plastics, or other useful products.
Bacterial and Phage Genetics
•  Very short doubling time.
Human: 25 years; Mice: 3-4 months; Drosophila: 12-20 days;
Bacteria: 20 min-24 hrs; Phage: 100x-1000x per infection cycle
•  Number of organisms examined is several orders of
magnitude larger than with plants or flies
•  Genetically pure cultures - asexual reproduction yields
clones (binary fission).
•  Haploid, genotype = phenotype.
•  Small Genome Size
Ecoli: 4 X 106 bp; Drosophila: 1.65 X 108 bp; Human: 2.9 X 109 bp
Methods of
growing
bacteria in
the
laboratory
Selectable genetic markers in bacteria
•  Nutritional markers
Prototrophic Bacteria can grow on minimal medium,
which only contains inorganic salts,
a carbon source and water (wild type) .
Auxotrophic Bacteria will not grow on minimal medium, but
can grow on minimal medium plus one or more
specific nutrients or supplements, or on rich
media (mutant).
Bacterial nomenclature
Genes are indicated using three letters (not
capitalized, and italic), followed by a + for wild type
or a - for mutant.
phe+
Wild type phe gene.
phe-
Mutant phe gene.
Phenotypes are indicated the same way, but not
italic and first letter capitalized.
Phe+
Does not require phenylalanine added as a supplement
to minimal medium
Phe-
Requires phenylalanine added as a supplement to
minimal medium
• Other nutritional markers
Anything the bacteria needs to synthesize
for its growth, such as Adenine, Biotin, Thiamine,.....
AdeAde+
Requires adenine added as a supplement to minimal medium
Does not require adenine added as a supplement to minimal
medium
• Carbon Source
Lactose
For energy, such as:
galactose, glucose, lactose,
maltose, .......
lacZ enzyme
Lac-
Lac+
Cannot grow on
lactose as its sole
carbon source.
Can can grow on
lactose as its sole
carbon source.
•  Antibiotic Selections
Bacteria will not grow colonies on the medium
containing certain antibiotics
Sensitive
Str
s
Resistant
Str
- streptomycin is an antibiotic that interferes with
the correct initiation of protein synthesis by
binding the 30S subunit of the ribosomal RNA.
Bacteria will grow colonies on the medium
containing certain antibiotics
r
- A mutation in a protein subunit of the 30S unit
confers resistance by preventing binding by the
antibiotic. The antibiotic is unable to exert its action
upon the substrate, and the bacteria continues to
grow.
Selection vs screen.
Selection: Establish conditions in which
only the desired mutant will grow.
Example: Select for StrR mutants on
streptomycin. Genetic screen: an examination of each
colony in a population for its
phenotype. Example: screening for
auxotrophs.
Ways to enrich for auxotrophs in a genetic screen.
1. Add a mutagen to increase mutation rate.
UV light
Proflavin
EMS
etc…………..
2. Replica plating to quickly look at lots of colonies.
3. Penicillin enrichment scheme.
Replica Plating
A method to quickly
transfer colonies from
one plate to another.
The original plate is called
the Master Plate. It is
pressed on a piece of
velvet, transferring about
50% of the cells from
each colony. A second
plate, replica 1, is then
pressed on the velvet, to
leave an imprint.
Identification of Arg- auxotroph using replica plating.
Penicillin enrichment scheme.
• Penicillin only kills bacteria cells that are actively dividing,
since it affects cell wall synthesis.
• Auxotrophs get selected since they stop dividing.
Three ways to get exchange of
genetic information in bacteria
•  Transformation
A process in which recipient cells acquire genes
from free DNA molecules in the surrounding medium.
• Conjugation
A process in which DNA is transferred from a bacteria
donor cell to a recipient cell by cell-to-cell contact.
• Transduction
A process in which a bacterial DNA fragment is
transferred from one bacterial cell to another by a
phage particlecontaining the bacterial DNA.
Do bacteria undergo sexual
mating and recombine their genes?
Joshua Lederberg and Edward Tatum,
1946
Strain A
met-bio-thr+leu+thi+
Strains
A+B
Strain B
met+bio+thr-leu-thi-
A
wash cells
AB
wash cells
Plate 108 cells Plate 108 cells
min. media
min. media
No colonies
B
wash cells
Plate 108 cells
min. media
met+bio+thr+leu+thi+
prototrophic colonies
No colonies
U- tube experiment
(Bernard Davis) Do bacteria have to touch each other to exchange?
Now there was no
growth on minimal
media. Bacteria have
to touch each other to
have genetic
exchange.
Genes are transferred in one direction
William Hayes, 1953
Strain A (met-thr+leu+thi+)
streptomycin (antibiotic)
kills cells slowly, but will die.
wash out strep.
MIX
Strain B
(met+thr-leu-thi-)
Plate on minimal
media
(colonies grew)
Strain B (met+thrleu-thi-)
streptomycin
(kills slowly)
wash out strep.
Strain A
(met-thr+leu+thi+)
MIX
Plate on minimal
media
(no colonies)
Conclusions
•  Genetic transfer is not reciprocal
•  In this experiment, transfer occurs from
Strain A to Strain B only, not B to A.
They came up with some terminology
Donor cells (male):
• Hayes experiment: A strain
• Cells that can donate DNA to other cells
• F+
Recipient cells (female):
• Hayes Experiment: B strain
• Cells that can receive DNA from other cells
• F-
Years later it was found that
there are two types of bacteria:
F+
carry the F factor in the cytoplasm
contain Pili on the surface
can transfer the F factor to F- Bacteria
Cannot receive DNA from other strains.
F-
do not carry the F factor in the cytoplasm
do not contain pili on the surface
cannot transfer DNA by conjugation
can receive DNA transferred from F+
bacteria
Overview of Conjugation
F factor is a small plasmid.
Explains unidirectionality.
F+ x FF+
F+
F-
F+