1. Bact 303
Bacterial Genetics II
Mutation

2. Mutation is a change in the sequence of bases in DNA
If a gene that encodes for a specific protein is mutated, it
may result in a change in the sequence of amino acids
comprising the protein. The activity of the protein may be
altered.

3. Frequency of naturally occurring (spontaneous) mutation
varies from 10-6
to 10-9
(avg = 10-8
)
This means that if a bacterial population increases from 108
to 2 x 108
, on the average, one mutant will be produced for
the gene in question.

4. Classes of Mutations
Mutations result from from two underlying causes
1. substitutions - One base is substituted in the DNA for
another base. Incorrect base pairing results from the
change of a single nucleotide base. Substitutions usually
result in “point mutations”
2. insertions or deletions of one or more bases - may result
in the the addition or deletion of one or more amino acids to
the growing protein, or more likely result in a “frame-shift”
mutation

6. A segment of the genetic code
DNA mRNA codon tRNA anticodon Amino acid
ACG UGC ACG CYSTEINE
CCT GGA CCU GLYCINE
AAA UUU AAA PHENYLALANINE
CGA GCU CGA ALANINE
TCG AGC UCG SERINE
CCG GGC CCG GLYCINE
TAA AUU UAA ISOLEUCINE
ACT UGA None None
A C G C C T A A A C G A DNA
Transcription
U G C G G A U U U G C U mRNA
Translation
_____ _____ _____ _____
cys gly phe ala Protein

7. A C G C C T A A A C G A DNA
U G C G G A U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein
TEMPLATE
A C G C C T A A A C G A DNA
U G C G G A U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein

8. A C G C C T A A A C G A DNA
U G C G G A U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein
T C G C C T A A A C G A DNA
A G C G G A U U U G C U mRNA
_____ _____ _____ _____
ser gly phe ala Protein
Substitution mutation T--->A at position #1 in DNA
changes the mRNA codon to code for the amino acid
serine in place of cysteine. This is a missense
mutation

9. A C G C C T A A A C G A DNA
U G C G G A U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein
Substitution mutation in the third base of DNA G--->T
generates terminator “stop” codon which terminates the
translation of the protein. A shortened or “truncated”
protein may be produced. This is called a nonsense
mutation.
A C T C C T A A A C G A DNA
U G A G G A U U U G C U mRNA
_____ ------------------------->
stop no translation

10. A C G C C T A A A C G A DNA
U G C G G A U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein
Substitution mutation in the sixth base of DNA T--->C
causes degenerate codon for glycine so there is no
change in the primary structure of the protein. This is
called a silent mutation.
A C G C C C A A A C G A DNA
U G C G G G U U U G C U mRNA
_____ _____ _____ _____
cys gly phe ala Protein

12. Consequences of substitution mutations
• none - altered protein retains same enzymatic or
structural activity
• change in enzymatic activity - could be beneficial or
harmful to the organism
• termination - incomplete (truncated) protein produced

13. A C G C C G T A A A C G A DNA
<---- insert into DNA
U G C G G C A U U U G C U mRNA
_____ _______ _____ _____ <---- old reading frame
cys gly phe ala Protein
Mutation caused by the insertion of a new base (G) between bases 5 and 6 in the DNA
(gene). When 1 or 2 bases are inserted, this causes a “frame shift” mutation because the
translation reading frame becomes shifted by one or two bases generating a whole new
set of triplets beyond the mutation. The same is true for mutations that result in the
deletion of bases.
A C G C C G T A A A C G A DNA
U G C G G C A U U U G C U mRNA
_____ _____ _____ _____ <---- new reading frame
cys gly isoleu cys Protein

15. Consequences of a frame shift mutation
• usually a more severe type of mutation that will lead to
translation of a totally different protein or it will generate a
stop codon that will terminate protein synthesis.

16. Mutagenic agents or mutagens
• Mutagens are physical, chemical or biological agents that
increase the rate of spontaneous mutation.
• Some mutagens cause point mutations and some cause
frame-shift mutation and some cause both.

17. Examples of chemical mutagens
CHEMICAL ACTION
HNO2 React chemically with one or more bases so
Nitrogen mustard that they pair improperly
NTG
Intercalating agents Insert into DNA and cause frame-shift
(acridine dyes) mutations by inducing an addition or the
subtraction of a base
Base analogs Incorporate into DNA and cause mispairing
e.g.
5-bromouracil Analog of T which can pair with C
2-amino purine Analog of A which can pair with C

18. Examples of physical mutagens
PHYSICAL AGENT ACTION
UV irradiation Causes formation of adjacent T-T dimers that
distorts the DNA backbone, altering the
binding properties of bases near the dimer
X-ray Alters bases chemically, causes deletions and
induces breaks in DNA chain

19. Examples of biological mutagens
BIOLOGICAL AGENT ACTION
Insertion sequences (IS) Pieces of DNA about a thousand nucleotide
bases in length which can insert into a genetic
sequence
Transposons genetic elements goverened by IS which can
insert into the chromosome within a gene
Viruses Some bacteriophage (e.g. phage mu) can
integrate their DNA into random positions in
the bacterial chromosome

20. Designations for Bacterial Mutants
CLASS DESIGNATION EXAMPLE
NUTRITIONAL MUTANTS
1. Lack ability to utilize a carbon source Sub-
Lac-
2. Have ability to utilize a carbon source Sub+
Lac+
STRUCTURAL MUTANTS
1. Lack fimbriae Fim-
Fim-
2. Lost motility Mot-
Mot-
3. Lost flagella Fla-
Fla-
RESISTANCE TO ANTIBIOTIC ABr
Ampr
SENSITIVITY TO ANTIBIOTIC ABs
Amps
AUXOTROPHIC MUTANT
1. Blocked in metabolic pathway
for synthesis of an amino acid AA-
Trp-
Wild type AA+
Trp+
2. Blocked in metabolic pathway
for the synthesis of a vitamin Vit-
Thi-
Wild type Vit+
Thi+

21. Detection of Mutations in Bacteria
DIRECT SELECTION OR ENRICHMENT (for Selective Mutants)
1. Plate the bacteria on a medium on which a mutant but not the parent will grow.
2. Antibiotic or virus senstive parents are killed or do not grow. Dru resistant or virus
resistant mutants will grow.
3. Pick and purify colony. Confirm resistance.

22. Direct Selection of Antibiotic Resistance Mutant

23. Direct Selection of mutants able to use a specific carbon source

24. Isolation of a motility mutant by direct selection

25. Isolation of an auxotrophic revertant by direct selection

26. The Ames Test

27. Modification of the Ames Test

28. Detection of Mutations in Bacteria
PENICILLIN SELECTION OR ENRICHMENT (for Nutritional or Auxotrophic Mutants -
mutants that require a growth factor not needed by the parent)
1. Add penicillin to culture growing in a medium without the growth factor:
a. growing bacteria (can make their own growth factor) are killed in the presence of
penicillin
b. nongrowing bacteria (because they require the missing growth factor) are not killed
(Note: penicillin kills ONLY growing cells)
2. Remove Penicillin, add growth factor and auxotrophs (GF-
) will grow. This step selects or
enriches for the growth of the survivors.
3. Select a survivor colony, and pick, purify and confirm growth factor requirement
(auxotrophy).

29. Detection of Mutations in Bacteria
REPLICATE PLATING (Also for Nutritional or Auxotrophic Mutants)
1. Transfer part of every colony growing on a complete medium (contains growth factor) to
a minimal medium (lacks the growth factor).
2. Identify colonies growing on the complete medium which do not grow on the minimal
medium. These are auxotrophic colonies - they grow with, but not without, the added
growth factor.
3. Pick and purify auxotrophic colonies from the complete medium and confirm auxotrophy.

31. Replicate Plating Technique