The Genetics of Bacteria and Their Viruses

Problems assigned the week of Jan 27,

for presentation during the week of Feb 3,
as model for quiz during the week of Feb 10

Chapter 5: 2, 6, 9, 14, 15, 20, 24, 26, 34, 40
Gene linkage Gene mapping in Eukaryotes

Physically linked alleles are inherited together except when there is a
cross-over in the region of the chromosomes between the genes.
A B
a B
a b
A b
All the events that we have been observing are reciprocal;
information can be exchanged, but the number of copies of a given
allele remains the same during the process
Meiosis is a carefully controlled process to insure the proper
segregation of the genetic information
Chromosomes segregate independently, and even genes on
the same chromosome get segregated by recombination
A B
a B
A b
a b
A B
a B
a b
A b
Bacteria are single-celled, prokaryotic organisms
Their cells do not have a nucleus or other
organelles.
They typically have circular chromosomes.
They are typically haploid.
They have genes; they have phenotypes.
They have cell division (binary fission), but not meiosis.

Escherichia coli or E. coli is the most commonly
used bacterial organism for genetic studies.
Bacteria and other single celled organisms offer the
advantage of large populations.

Rare mutations can be selected from large populations
when there is a way to select for or identify the mutant.

Bacteria have fast generation times. Cell division
can be as short as 20 minutes.

In haploid organisms the genotype and phenotype
are the same since recessive alleles are not masked
by a second copy of the gene.
Bacteria used in experiments are most commonly seen
as liquid cultures or as colonies on agar plates.
Bacteria can be
streaked,
spread,
or arrayed
on semi-solid
agar with
growth media.
Dr. Axel Oberbremer
E. coli can be grown
in liquid media.

It grows best (fastest)
at 37C and with
aeration that comes
with shaking.
Bacteria can easily be counted on agar plates
after serial dilution.
Fresh media
1 ml
1/10 1/10 1/10 1/10
1/10
th
ml
6 colonies,
inoculum was
60 bacteria/ml
The original culture was 6 x 10
5
bacteria /ml
The phenotype of a bacteria is usually seen by
whether it grows or not on a selective media.
There are three major classes of genes that are often used
for bacterial genetics

I. Resistance to antibiotics

II. Requirement for a particular nutrient in order to grow.

III. Ability to grow on a particular compound
as a sole carbon source.
I. Resistance to antibiotics

ampicillin (amp
r
) kanamycin (kan
r
)
streptomycin (str
r
) tetracycline (tet
r
)
neomycin (neo
r
)
Rich media, Rich media + ampicillin
no antibiotics
I. Resistance to antibiotics

ampicillin (amp
r
) kanamycin (kan
r
)
streptomycin (str
r
) tetracycline (tet
r
)
neomycin (neo
r
)
Rich media, Rich media + ampicillin
no antibiotics
I. Resistance to antibiotics

ampicillin (amp
r
) kanamycin (kan
r
)
streptomycin (str
r
) tetracycline (tet
r
)
neomycin (neo
r
)
Rich media, Rich media + ampicillin
no antibiotics
I. Resistance to antibiotics

ampicillin (amp
r
) kanamycin (kan
r
)
streptomycin (str
r
) tetracycline (tet
r
)
neomycin (neo
r
)
Rich media, Rich media + ampicillin
no antibiotics
amp
r
amp
s
Gene symbol systems - E. coli
Escherichia coli and other bacterial genes

In class we will use three letter symbols with + for wild type,
normal and for the mutant type.

leu
+
leu

, dcm
+
dcm


Bacterial geneticists usually shorten leu

to leu
II. Requirement for a particular nutrient in order to grow.
Many of these gene mutations are related to a requirement for
particular amino acids; eg.
arginine arg- cysteine cys-
glycine gly- histidine his-
leucine leu- lysine lys-
methionine met- proline pro-
III. Cannot grow on a particular compound as a sole carbon
source. Many of these gene mutations prevent E. coli from
growing with a particular sugar as a sole carbon source.

lactose lac-
galactose gal-
Rich media Minimal MM+ arginine
media
Wild type (wt)
arg
-
arg
-

does not grow on Minimal Media (MM)
It does grow on MM + arginine
It cannot synthesize arginine.
The wild type strain that can grow on minimal media is
called a prototroph, the mutants that need
supplements are called auxotrophs
Rich media
(LB)
Minimal
media (MM)
MM + arginine MM + glycine
arg
-
gly
-
arg

mutants cannot synthesize the amino acid arginine. Arginine must be in the
media for them to grow
gly
-
mutants cannot synthesize the amino acid
glycine. It must be in the media for them to grow
Rich media
(LB)
III. Mutants that cannot grow on a particular compound as a sole
carbon source.
SM + lactose SM+ glucose
lac
lac
-
mutants cannot grow on lactose as a sole source of
carbon, i.e energy. They require some other energy source.
Rich media
(LB)
III. Mutants that cannot grow on a particular compound as a sole
carbon source
SM + lactose SM+ glucose
lac
-
lac
-
lac
-
SM + glycerol SM+ fructose
lac
-
mutants cannot grow on
lactose as a sole source of carbon,
i.e energy. They require some
other energy source.
lac
-
lac
-
II. Requirement for a particular nutrient in order to grow.
Many of these gene mutations are related to a requirement for
particular amino acids; eg.
arginine arg- cysteine cys-
glycine gly- histidine his-
leucine leu- lysine lys-
III. Cannot grow on a particular compound as a sole carbon
source.
Many of these gene mutations prevent E. coli from growing with
a particular sugar as a sole carbon source.

lactose lac-
galactose gal-
I. Resistance to antibiotics
ampicillin (amp
r
) kanamycin (kan
r
)
streptomycin (str
r
) tetracycline (tet
r
)
These mutants provide the tools to look for genetic
exchange among bacteria.

If we think about the complexity of the system for genetic
exchange in eukaryotes (the whole system of meiosis)
the idea that bacteria would undergo genetic exchange
seemed, to most researchers, very unlikely
These mutants provide the tools to look for genetic
exchange among bacteria.

If we think about the complexity of the system for genetic
exchange in eukaryotes (the whole system of meiosis)
the idea that bacteria would undergo genetic exchange
seemed, to most researchers, very unlikely

However, with collections of auxotrophic, drug resistant
and sugar-requiring mutants, researchers looked for
genetic exchange

They mixed together strains of E. coli with different
combinations of multiple auxotrophic mutants, and
looked for the appearance of prototrophs
Bacterial Mating
The F
+
fertility factor is about 100 kb in length, about
1/50 the size of the 4600 kb bacterial chromosome
www.bio.utexas.edu/faculty/
sjasper/bio212/microbial.html
In high frequency recombination (Hfr) bacterial strains
the F factor is integrated into the chromosome.
Hfr strains can transfer a copy of their chromosome to a
recipient (F
-
) strain.
New DNA introduced into the bacterial cell is incorporated
into the circular chromosome by a double crossover.
The linear
fragments
are lost during
cell replication.
Individual strains have a fixed starting point for the
transfer a copy of their chromosome.
It takes about 60 minutes to transfer the whole chromosome.
Gene order can be determined by the time it takes to
transfer different genes to the recipient strain.
Bacterial mating - can be interrupted by vigorous agitation
of the culture.
Donor Recipient
Media: no arginine no leucine no histidine lactose
after
30 min
disrupt
Repeat at 10, 20, 30 and 40 minutes
arg
+
leu
+
his
+
lac
+
Bacterial mating

Str
s
, arg
+
, leu
+
, his
+
, lac
+
(Hfr) X (F-) Str
r
, arg
-
, leu
-
, his
-
, lac
-

Selective media to determine the genotype.

Media Phenotype selected
MM + Str (control) Str
r
, arg
+
,leu
+
his
+


MM + Str + leu, his, glucose (no arg) Str
r
, arg
+

MM + Str + arg, his, glucose (no leu) Str
r
, leu
+

MM + Str + arg, leu, glucose (no his) Str
r
his
+


MM + Str + arg, leu, his, lactose Str
r
lac
+
Str
s
, arg+,leu+,his+,lac+ (Hfr) X Str
r
,arg-,leu-,his-,lac-

phenotype 10 min. 20 min. 30 min. 40 min.
arg+ 0 0 3 20
leu+ 6 30 60 61
his+ 0 8 41 56
lac+ 0 0 0 10

What is the gene order?
Str
s
, arg+,leu+,his+,lac+ (Hfr) x Str
r
,arg-,leu-,his-,lac-

phenotype 10 min. 20 min. 30 min. 40 min.
arg+ 0 0 3 20
leu+ 6 30 60 61
his+ 0 8 41 56
lac+ 0 0 0 10

The gene order is: leu, his, arg, lac
Different High Frequency Recombination (Hfr) strains
have different starting points and different orientations
for the transfer of a copy of their chromosome.

Mapping data from different Hfr strains can be
combined to produce the complete circular genetic
map of a bacteria.
Different Hfr strains have the fertility factor in different positions
and orientations in their genome
Genes are in the same order in different strains, but the first
genes
to be transferred will be different in different strains.
Bacterial
genetic maps
are circular.

Bacterial
chromosomes
are circular
There are three E. coli strain types to remember in reference to
bacterial conjugation.

1. F
+
strains have the fertility factor as a plasmid. F
+

can donate the fertility factor but cannot receive DNA.
They donate the F
+
factor readily. A very small proportion
of the population have the F
+
factor integrated into the
chromosome and can donate other genes.

2. F
-
strains can receive DNA but cannot donate DNA. If they
receive the F
+
factor, they become F
+
.

3. Hfr strains have the F
+
integrated into a specific place in their
chromosome. They transfer their chromosome into a F
-
strain,
starting at the place adjacent to the F factor, with the F factor
itself being the last DNA to transfer.
Masamichi Kohiyama, Sota Hiraga, Ivan Matic, Miroslav Radman
Science (2003) 301:802-803
2003 - 50th
Anniversary
of discovery of
Bacterial
Mating

Donor: C (spots are hemi-
methylated DNA)

Recipient: D

Mated cells are green
with yellow spots - which
mark the presence of
hemi-methylated DNA

A shows Hfr mating, B an F
+

transfer
Some background information

Bacteria have simple immune systems that result from
enzymes called restriction enzymes that cut foreign DNA
that enters the cell
Bacteria protect their own DNA from these systems by
modifying the DNA, typically by methylation of specific
bases these combined systems are restriction-
modification systems.
We will talk more about restrictions enzymes when we talk
about genetic engineering
The Dam methylase methylates the A of the sequence
GATC
Recipient F
-
bacteria are expressing GFP (green
fluorescent protein) and thus are green, and are dam
-
and
thus cannot methylate their DNA

Donor F
+
or Hfr bacteria have red labeled DNA because a
fluorescent antibody is binding the SeqA protein, which in
turn is binding to hemi-methylated DNA (this is DNA that
has a methyl group added to specific bases, but only on
one strand of the double helix). Hemi-methylated regions
occur transiently during replication before the newly
synthesized strand is itself methylated

The F
-
strains do not have visible DNA because they are
methylation defective dam
-
mutants, and fail to methylate
the dam sites, so SeqA does not bind
Because only one strand of the F+ or Hfr donated DNA
enters the recipient cell, it is always hemi-methylated. The
incoming strand is methylated because the donor is Dam
+
,
the recipient duplicates the DNA but does not add any
methyl groups, so the GATC sites of the transferred DNA
are completely hemi-methylated