Genetics of Bacteria

E. coli (bacteria)
single cell (no nucleus)
circular chromosome (~4500 genes), 1 copy
In the laboratory growth is in liquid media or on agar plates
1Æ2Æ4Æ8Æ16Æ32Æ64
each step takes approximately 20 minutes in rich media

Topics:
Conjugation
Transformation
Transduction
Bacteriophage
Genetics
Conjugation
Marker genes are met bio thr
leu thi.
‘+’ means good/wild type
No ‘+’ means defective
Two strains used that differ in
assortment of markers
Neither can grow on minimal
media
But, after coincubation some
cells are able to grow on
minimal media
It looks like something caused
conversion of one strain to all
‘+’ markers. How does this
happen?
Bacterial
Conjugation
If strain A and strain
B are kept
physically apart by a
filter, then cells that
can grow on
minimal media are
no longer observed.
This suggests
contact is required
to generate cells
that are + for each
marker.
Bacterial Conjugation

Due to an independent genetic unit called the fertility factor (F).

This is a piece of DNA that encodes genes to make a pili.

1. cells that do not harbor the F factor are F-

2. Cells that have an extrachromosomal F factor are F+

3. Cells that have an F factor integrated into their genome are Hfr (high
frequency of recombination)

If an F factor integrates into genome the F+ cell is converted into an Hfr

Matings
F+ X F- makes F+ and F+
Hfr X F- results in gene transfer, usually no conversion to F+ or Hfr
F+ X F+ shouldn’t occur
Hfr X F+ shouldn’t occur
(being Hfr or F+ immunizes the cell against F transfer from F+ or Hfr)
If the F factor is integrated into
the genome, it can bring
cellular genes along with it
when it is transferred.
Only a single strand is
transferred: The sequences
on this strand can convert
genomic DNA to the donor
DNA sequence through
recombination.
Genes are transferred
depending starting at where
the F factor is integrated and
depends on the direction of F
factor.
The time that it takes a gene to
be transferred can be
measured in minutes.
This lets genes around the
circular E coli chromosome to
be measured in minutes (map
distance in minutes).
By this measure the E. coli
chromosome is about 90 to
100 minutes long
 aziR

tonR

Efficiency of
Transfer of
Markers lac+

gal+

Minutes
Graph shows appearance of donor markers in recipient bacteria
1. genes near integration site are transferred first with high efficiency
2. genes farther away from integration site are transferred later and have
lower efficiency due to interruptions of matings
Donor is aziR, tonR, lac+, gal+
Recipient is aziS, tonS, lac-, gal-
At the end of the experiment you want to only allow growth (count) colonies
that are due to the recipient bacteria having obtained donor markers. This
means that you need to have additional selectable markers that will allow you
to separate donor and recipient bacteria.
 Transformation
If you take naked DNA (not packaged with any proteins) and incubate it
with bacterial cells, the DNA will enter the bacteria with very low efficiency
In the case of plasmid DNAs which are used as cloning vectors, the cells
that have taken up the DNA are usually selected by some antibiotic
resistance marker. In this case the plasmid (vector) is able to replicate
itself autonomously once inside the cell

selected by
transformation
antiobiotic
resistance
marker on
plasmid

If the cell were to take up piece of E. coli DNA that carried a normal cellular
gene, it is possible that that sequence could recombine with the endogenous
cellular genome to convert it to the new genotype
a+
a+
a- a- a+
transformation
 c+ c- a+
a+ a- b+
b+ a+ b- c+
b+ a+ b+
c+
a+ b+

DNA from bacterial cells of genotype a+, b+, c+ is isolated (pieces).

The DNA is incubated with bacteria of strain a-, b-, and c-
In this process there is a chance to convert a- to a+, b- to b+, or c- to c+
This chance should be equal for each gene. But, there is also a chance to
convert both a- and b- to a+ and b+ at the same time. This happens if the a
and b genes are close enough that a DNA fragment might contain both genes.

Transformation efficiency is generally low. Organisms doesn’t want to

promiscuously take up DNA (programs) from the environment without
knowing what they are.
Then what happens to all the DNA you eat? Does it transform you into a carrot,
or a chicken, maybe just a little little bit? It gets degraded in the acidic
environment of the stomach by nucleolytic enzymes. Coding content is lost.
Through these and other types of protective mechanisms, cells in your mouth,
throat, stomach, don’t get ‘transformed’ by DNA from what you eat.
Transduction

Bacteria get infected by bacteriophage. The bacteriophage contain a

small genome that wants to do one thing….
MAKE MORE OF ITSELF

Phages typically
show two possible
life cycles. In the
lytic life cycle the
phage infects the cell,
makes more copies
of itself, and then
lyses the cell. The
progeny that emerge
go on to find other
bacteria to infect.
In the lysogenic life
cycle, the phage
enter the cell, then
integrate into the
bacterial
chromosomal DNA
(prophage). Some
phages integrate at
one location while
others integrate at
random positions.
While integrated,
the phage genome
replicates as part of
the bacterial
chromosome, as
though it were just
another gene.
Under certain
conditions, the
integrated phage
DNA can excise
itself and enter the
lytic cycle.
 Picture shows plaques where a circle region of
bacteria have been eaten away through lysis by a
bacteriophage
Lederberg-
Zinder
experiment

Consider two
different
auxotrophic
strains of
Salmonella
LA-2 and LA-22,
each with
distinct growth
requirements.
Incubating them
as shown,
without contact,
allowed
production of
fully wild type
(all ‘+’) strain.
Gene transfer in
this case is due
to transducing
phage P22.
Part of the
Salmonella
chromosome is
being packaged
as a phage
particle,
resulting in
infection and
delivery of phe+,
trp+ DNA to
recipient cells
beyond the filter.
Note
directionality of
transduction
from one cell to
another.
Generalized transduction mapping experiment

Cell with genotype aziR, thr+, leu+

infect with bacteriophage P1

use phage that are produced to then infect cells with genotype aziS,
thr, leu (sensitive to Na azide, cannot make thr, leu)

quantitate number of transductants

if leu+ 50% were also aziR (suggests close proximity)

2% were also thr+ (suggest far apart)

if thr+ 3% were also leu+

0% were also aziR (thr, azi very far apart)

From result 1: leu azi

From result 2: thr leu azi
Specialized Transduction
Involves lysogenic bacteriophage like lambda which integrate into
ONE specific location along the circular bacterial chromosome.
Integrated lambda sometimes wants to get out and start producing
more copies of itself through the lytic cycle. This might happen if
lambda can detect that its cellular host is in trouble (no food,
exposure to mutagens, etc). If excision is not precise, then the phage
particles may carry cellular genes located near the site of lambda
integration. In the case of lambda, this typically involves the lac and
bio genes.

What might transduction be similar to as far as humans are

concerned? Imagine a virus integrates into the genome next to a
gene that controls cell growth. When the virus leaves the host DNA it
may carry all or part of an adjacent gene with it. Viruses tend to
undergo rapid mutation, so the gene in the virus will probably mutate
more rapidly than when it was part of the genome. Sometimes viral
forms of the cellular gene will help the virus by stimulating cell growth
when the virus reinfects another cell, making it a ‘transforming’ virus.
Scientists were puzzled why viruses carry genes similar to normal
cellular genes, and why they stimulated cell growth (thus, indirectly,
viral growth). Investigation of this phenomena revealed that in
many cases viral versions of these genes were oncogenic (cancer
causing) forms of normal cellular genes. Later it was found that
mutations in the corresponding cellular gene was also associated
with carcinogenesis and unregulated cell growth. Thus, the
discovery of oncogenes.
BACTERIOPHAGE GENETICS
Bacteriophage T2 will infect and lyse E. coli cells grown on a petri dish.

The phage T2 h gene controls host range (susceptible E.coli strains)

h+ will infect and lyse E. coli B but not E. coli B/2
h will infect and lyse both E. coli B and E. coli B/2

The phage T2 r gene controls plaque morphology (efficiency of lysis)

r+ makes small fuzzy plaques
r makes large clear plaques

Normally one phage infects one cell. However, if you add a lot of phage to
few cells (high multiplicity of infection, MOI) multiple phage will enter
each single cell. Here we consider a high MOI infection with h+r phage
with hr+ phage in E. coli B.

If there is NO recombination between the phage genomes that have

entered the cell you would get parental h+r and hr+ phage back out
If recombination DOES occur, you should also expect to also obtain h+r+
and hr recombinant phage
The phage output is revealed by infecting a mixture of E. coli B and B/2

hr+ small clear fuzzy border

h+r large cloudy distinct border
h+r+ small cloudy fuzzy border
hr large clear distinct border

Since nonparental phenotypes ARE observed, we conclude that

recombination between phage strains (DNA) is taking place in the cell.

The relative frequency of recombinants can be used as a measure of how far

apart the phage genes are…..

#(h+r+) + # (hr) plaques X 100

total plaques

Gives you a percentage measure of how close the genes are.

Benzers fine structure mapping experiments is famous for showing that

recombination is possible within a gene, not just between genes. This
allowed him to map the position of mutations within a gene. We are not
going to cover fine structure mapping in the detail the book does.
Complementation is important and works like this…
Here phage 1 is defective for the rIIA gene, phage 2 is defective for rIIB.
However, phage 1 can provide rIIB, and phage 2 can provide rIIA. As a result
both phage, though defective individually, are produced with high efficiency.