Ch. 7.

Microbial Genetics
Learning Objectives: You should be able to explain and
describe the following:
DNA structure and composition
Plasmids
DNA replication
How DNA codes for proteins (transcription and translation)
RNA structure and composition
Types of mutations (point and frameshift)
Mutagens
Methods for selecting mutants
The Ames Test
Genetic recombination and gene transfer (transformation,
conjugation, transduction, and transposition)



DNA structure and composition
The basic unit of DNA is the nucleotide that
contains a phosphate group, deoxyribose sugar,
and a nitrogenous base

In the helix the strands are oriented
antiparallel, base-pairing occurs in the center
(hydrophobic region), with a hydrophilic
phosphate-sugar backbone on the outside
Plasmids

Extrachromosomal, closed circular
small molecules of DNA that replicate
independently of the chromosome

The bidirectionality of DNA replication
Origin
Replication
proceeds in both
directions
Replication forks
Termination
of replication
How genes code for proteins
Transcription: process by which DNA is
copied into a messenger RNA (mRNA)
molecule

Translation: process by which mRNA is
read by ribosomes to assemble a protein (or
a polypeptide)
Transcription
Translation
by ribosomes
5
5
5 3
3
3
DNA
(genotype)
mRNA
Polypeptide
(made of
amino acids)
Phenotype
NH
2
Methionine Arginine Tyrosine Leucine
reading frame
for the
ribosomes
Note: differences between DNA and RNA
DNA RNA
Thymine (T) Uracil (U)
Guanine (G) Guanine (G)
Adenine (A) Adenine (A)
Cytosine (C) Cytosine (C)
Deoxyribose Ribose
Phosphate Phosphate
Double-stranded Single-stranded
Bases pair: (complementary base pairing)
In double-stranded (ds) DNA:DNA
G and C (3 hydrogen bonds)
A and T (2 hydrogen bonds)

In ds RNA:RNA or hybrid RNA:DNA
G and C (3 hydrogen bonds)
A and U (2 hydrogen bonds)
Genetic Change in Bacteria
A genetic change alters an organisms genotype

Bacteria are haploid

- have only 1 copy of their DNA, therefore a
change in DNA can easily alter the
phenotype of the bacteria

Mutations of Genes
Mutation change in the nucleotide base sequence

Almost always harmful to the bacterial cell

Types of Mutations

Point mutations one base pair is affected

E.g.: insertions, deletions, and substitutions


Frameshift mutations nucleotide triplets after
the mutation are displaced

E.g.: insertions and deletions
Point mutations
Changes in one base pair can lead to one of 3 possibilities:

1. silent mutation no change in amino acid sequence

2. missense mutation change in amino acid sequence

3. nonsense mutation premature termination of the
protein synthesis usually defective protein
Is the mutated proteins function likely to be altered?
Is the mutated proteins function likely to be altered?
Is the mutated proteins function likely to be altered?
Frameshift mutations
Ribosomes read the mRNA in codons
(groups of 3 nucleotides); this is called a
reading frame

When frameshift mutations occur, these lead to
a shift in the reading frame used by the
ribosomes, often leading to changes in the
amino acid sequence of the protein
Is the mutated proteins function likely to be altered?
Ultraviolet light
Thymine dimer
Mutagens

Ionizing radiation
induces breaks
in chromosomes

Nonionizing
radiation
induces
pyrimidine
dimers
(e.g. thymine
dimers)
What are the effects of thymine dimers on DNA?
Mutagens

One example of a chemical mutagen

Nucleotide analog (e.g. base analog)
structurally similar to a normal nucleotide, may be
incorporated into DNA in place of a normal
nucleotide; it disrupts DNA and RNA replication
and causes point mutations

An example of a base analog is 5-bromouracil
which incorporates in place of thymine and binds with
guanine

This will change the complementary strand being
synthesized during DNA replication
Wild types nonmutated cells

Mutants cells with one or more mutations


We use several methods to recognize mutants

Positive selection
Negative (indirect) selection
Ames test

Positive selection
of mutants
In this example we
are looking for
cells that after
exposure to a
mutagen, have
mutated and
become resistant
to penicillin, and
can grow in
medium with
penicillin
Negative
(indirect)
selection
Mutagen
Incubation
Bacterial
suspension
Inoculate bacteria onto
complete medium
containing tryptophan.
Bacterial colonies
grow. A few may be
tryptophan auxotrophs.
Most are wild types.
Stamp sterile velvet onto
plate, picking up cells
from each colony.
Stamp replica plates
with velvet.
Sterile velvet
surface
Bacteria
In this
example we
are looking for
cells that after
exposure to a
mutagen, have
mutated and
cannot grow
without the
amino acid
tryptophan
Stamp replica plates
with velvet.
Bacteria
Identify auxotroph
as colony growing on
complete medium but
not on lacking medium.
Inoculate auxotroph
colony into complete
medium.
Complete medium
containing tryptophan
Medium lacking
tryptophan
Incubation
All colonies grow. Tryptophan auxotroph
cannot grow.
The Ames Test
Liver extract is used to mimic what happens in the human
liver (our liver helps us detoxify compounds, sometimes
leading to the production of carcinogenic compounds)

A particular type of Salmonella mutant is used. This mutant
is his
-
which means it cannot grow without the addition of
the amino acid histidine in the medium

The goal of this assay is to test whether a chosen chemical is
capable of causing a mutation (i.e. capable of being a
mutagen) and causes the Salmonella mutant to revert its his
-

mutation and become his
+
(his
+
means that the Salmonella
is able to grow in medium without the addition of the amino
acid histidine).
The
Ames
Test
Liver
extract
Liver
extract
Suspected
mutagen
Experimental
tube
Control
tube
Culture of
his

Salmonella
Medium
lacking
histidine
Incubation
Colony of revertant (his
+
) Salmonella No growth
Genetic Recombination and Transfer
Homologous recombination:
exchange of nucleotide sequences (homologous sequences)

Recombinants cells with DNA molecules that contain new
nucleotide sequences

Vertical gene transfer organisms replicate their
genomes and provide copies to descendants
Genetic
recombination
Homologous
sequences
Enzyme nicks one strand of
DNA at homologous sequence.
3
5
3
5
DNA A
DNA B
B
A
Recombination enzyme inserts the cut strand into
second molecule, which is nicked in the process.
Ligase anneals nicked ends
in new combinations.
Molecules resolve
into recombinants.
Recombinant A
Recombinant B
Horizontal Gene Transfer Among Prokaryotes


Three types of horizontal gene transfer:

Transformation
Transduction
Bacterial conjugation

Horizontal Gene Transfer Among Prokaryotes

Transformation

Cells that take up DNA are competent
Transformation in
Streptococcus pneumoniae
Why do we see living S
cells recovered from the
dead mouse? What has
happened? This is the
process of
transformation.
Horizontal Gene Transfer Among Prokaryotes

Transduction

A virus (phage) carries DNA segment from donor
cell to recipient cell

Phage injects
its DNA.
Phage enzymes
degrade host DNA.
Phage DNA
Bacterial
chromosome
Cell synthesizes new
phages that incorporate
phage DNA and, mistakenly,
some host DNA.
Transducing phage
Phage
Transducing phage
Transducing phage
injects donor DNA.
Recipient host cell
Donor DNA is incorporated
into recipients chromosome
by recombination.
Transduced cell
Inserted
DNA
Conjugation is another way to transfer DNA between
neighboring cells (horizontal transfer)
Bacterial conjugation
F plasmid
Origin of
transfer
Conjugation pilus
Chromosome
F
+
cell F

cell
Donor cell attaches
to a recipient cell
with its pilus.
Pilus may draw
cells together.
F
+
cell
Donor chromosome
Pilus
Pilus
F
+
cell
Hfr cell
F

recipient
F plasmid integrates
into chromosome by
recombination.
Cells join via a
conjugation pilus.
Formation of an Hfr (High frequency of recombination cell) and what
happens when it encounters a F- cell:
F
+
cell (Hfr)
F plasmid
Part of F plasmid
Donor DNA
Portion of F plasmid partially
moves into recipient cell.
Conjugation ends with pieces
of F plasmid and donor DNA
in recipient cell.
Donor DNA and recipient
DNA recombine, making a
recombinant F

cell.
Incomplete F plasmid;
cell remains F

Recombinant cell (still F

)
Does this process require cell cell contact? How does this affect
the size of the DNA that is transferred from the donor to the
recipient cell?
Transposons and Transposition

Transposons are segments of DNA that move from one
location to another in the same or different molecule

Result is a frameshift insertion
Is this an example of horizontal or vertical transfer?