1. Chapter 9 Lecture Outline
Gene Transfer, Mutations, and Genome Evolution

2. Covered in this Lecture
9.1 The mosaic nature of genomes
9.2 Gene transfer: Transformation, conjugation, transduction
9.3 Recombination
9.4 Mutations
9.5 DNA repair
9.6 Mobile gene elements
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

3. The Mosaic Nature of Genomes
DNA sequence is not static
Over the millennia microbes have undergone extensive gene loss and gain
Horizontal gene transfer
Recombination events
Mutations
Single bases
Large deletions
Large insertions of sequence
Transferred from other species
New functions useful in particular situations
Pathogenicity islands
Fitness islands
Maintained via interaction with environment
Survival determined by having appropriate genes for the specific environment
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

4. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Gene Transfer
Transformation
Uptake of naked DNA
Conjugation
Uptake of DNA from cell to cell with direct contact
Transduction
Transfer of DNA via virus
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

5. Transformation: History
Griffith: demonstrated the phenomena of transformation in 1928
Avery, MacLeod, and MacCarty: proved that DNA is the transforming element in 1944
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

6. Gene Transfer: Transformation
Uptake of DNA directly from surrounding medium
DNA used as nutrient
For repair
Adjustment to the environment
Natural transformation is found in many species
Competent cells
Gram +: Streptococcus, Bacillus
Gram-: Haemophilus, Neisseria
Translocasome takes up DNA
Located in envelope
Competence induced in different species
Gram+ cells secrete signal
Species specific competence factor
Gram- cells do not produce competence factor
Always competent
Stress induces competence
Starvation
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

7. Artificial Transformation
In the laboratory
Alter membrane to allow DNA to pass
Chemical
CaCl2
Electroporation
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

8. Gene Transfer: Conjugation
Sex pilus connecting 2 E. coli cells.
Requires cell to cell contact
“Bacterial sex”
In gram- bacteria: sex pilus
In gram+ bacteria: non-pilus attachment proteins
Requires transferable plasmid
Contains all the genes for pilus/attachment information and DNA export (ex.: F factor)
If plasmid incorporates into host chromosome it may also transfer host genes
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

9. The Conjugation Process
1. Sex pilus from the F+ donor cell attaches to receptors on recipient cell.
Donor (F+) Cell
Recipient (F-) Cell
2. Contraction of the pilus draws the two cells together and forms a conjugation bridge.
3. The F factor is nicked at oriT and the 5’ end begins transfer through the bridge.
4. The strand remaining in the donor is replicated by rolling circle with Pol III.
5. Once in the recipient the transferred strand circularizes and replicates.
6. The recipient has been converted to a donor.
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
F+ Cell
F+ Cell

10. Gene Transfer: Conjugation
Episome
Plasmid that can exist in extrachromosomal and integrated form
Hfr
High frequency recombination
F factor integrates on bacterial chromosome
Bacterial strain is called Hfr strain
More cells are capable to transfer chromosomal DNA
Tries to transfer entire chromosome
Requires 100 minutes for E. coli (44 kbp/min)
Transfers genes in order
Can determine order of genes on chromosome by interrupted mating
F (F prime) factor
Integrated F factor is excised along with some chromosomal DNA
F plasmid contains extra genes
In addition to genes for pilus, transfer
Transfers extra genes to recipient
F factor
Bacterial
chromosome
lac genes
oriT
Flac
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

11. Conjugation across Species
Relatively frequently across species
Ex.: from E. coli to Salmonella
Some bacteria can cross biological domain
From Agrobacterium tumefaciens to plants
Tumor inducing plasmid
Crown gall disease
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

12. Gene Transfer: Transduction
Bacteriophage inject DNA into cell
After replication and capsid production package DNA into viral capsid
Viral DNA
Sometimes accidentially package bacterial host DNA
Transfer DNA to new host
Host DNA
Generalized transduction
Specialized transduction
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

13. Generalized Transduction
During lytic life cycle host DNA is degraded
Can be packaged into capsid
Can replace entire virus genome
Transducing particle is generated
Defective: can infect a new host cell but not be replicated
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

14. Specialized Transduction
During lysogenic life cycle host DNA is accidentally excised when prophage is reactivated
New virus genome contains host genes lying adjacent to the phage insertion site
Can be packaged into capsid along with viral DNA
Infective virion: can infect a new host cell and will be able to replicate
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

15. Defense against Transferred DNA
Bacteria cut entering DNA to pieces
Cut at specific restriction sites with restriction enzymes
Bacteria add methyl groups to DNA
Prevents restriction at those sites
Added as cell replicates chromosome
Entering DNA is destroyed
Unless it comes from a similar species
And has methyl groups protecting DNA
Prevention of promiscuous DNA transfer
EcoRI
restriction/ modification
site
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

16. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Recombination
New DNA enters via transformation, conjugation, transduction
Fate of DNA
Independent existence as plasmid
Degradation
Integration into host chromosome through recombination
Recombination is the process by which DNA sequences can be exchanged between DNA molecules
Replaces variable-sized section of DNA
DNA repair
Enhancement of fitness
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

17. Generalized Recombination
Considerable stretch of DNA homology
Involves RecA and intermediate ssDNA
Cross over sites appear as crosses
Holliday junction
Deinococcus radiodurans uses RecA homologue for radiation induced DNA repair
Has multiple copies of each of its 2 chromosomes
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

18. Site-Specific Recombination
Exganche between DNAs with little overall sequence homology
Does not require RecA
Requires insertion sequences
10 – 20 bp sequence recognizable by restriction enzymes is required
Intergrases mediate site-specific recombination
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

19. Click box to launch animation
Recombination
Animation: Recombination
Click box to launch animation
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

20. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Mutation
Heritable permanent change in DNA sequence
Harmful, beneficial, or neutral
Change in genotype
May result in a change in phenotype
Change is base sequence and failure to repair
Point mutations
Change in one base pair
Transition: purine  purine, pyrimidine  pyrimidine
Transversion: purine  pyrimidine
Larger mutations
Insertion
addition of 1 or > nucleotides
Deletion
subtraction of 1 or > nucleotides
Inversion
flipping a fragment of the DNA
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

21. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
(Missense)
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

22. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Silent Mutations
Normal
Most mutations are silent
No effect on organism
Mutations in regions between genes
Mutations that change 3rd base of a codon
Mutations that change 1 amino acid into a similar one
Protein still retains normal function
Mutations that change a protein that is not needed
In current environment of organism
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

23. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Types of Mutations
Silent
No effect on organism
Missense mutation
Change one codon to another
Nonsense mutation
Change a codon to Stop
Frameshift mutation
Insert or delete a single base
Changes bases read by ribosome
Alters all codons downstream of mutation
CGA  CGC
Arginine  Arginine
CGA  CAA
Arginine  Glutamine
CGA  TGA
Arginine  STOP
AAA CGA CCC  AAA CTG ACC C
Lysine Arginine Proline  Lysine Leucine Threonine
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

24. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Mutagens
Increase the rate of mutations
Number of mutations formed per cell doubling
Spontaneous mutation rate is 106 to 108 per generation
Increase of mutation frequency
Number of mutant cells per population (ratio of mutant cells/ total number of cells)
Most mutagens are carcinogens
Cancer results from multiple mutations
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

25. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

26. Sectored Colonies reflect Mutation
Mutant yeasts accumulate red compound
Normal
Lac- bacteria mutate and revert to Lac+.
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

27. Measurement of Mutagens: Ames Test
Reversion test
Uses bacterial strain auxotrophic for histidine
Has mutation in hisG gene
Cannot grow unless histidine is supplied
Expose bacteria to the mutagen
Mutagen causes reversion
Changes mutation to normal form
Normally rare mutation
More colonies = stronger mutagen
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

28. Classical and Modified Ames Test
Bacteria are plated on medium w/o his and a disk with mutagen is added
Modified
Bacteria are incubated with mutagen and liver extract and then plated onto medium w/o his
Mutagen on
paper disk
Revertant
colonies
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

29. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
DNA Repair
Methyl mismatch repair
Parent strand is methylated
Newer strand is less methylated and more likely to contain errors
Nucleotide excision repair
Clips out patches of ssDNA
Base excision repair
Excision of structurally altered bases without cleaving the phosphodiester backbone resulting in an apurinic and apyrimidic site (AP site)
AP nucleases follow up
Recombinational repair
Intact strand is used to replace a homologous damaged strand
SOS response
Coordinated response to extensive damage
Set of genes and repair mechanisms activated
Error-Proof
Error-Prone
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

30. Mobile Genetic Elements
Chromosomes sequence is not fixed
Transposable elements insert into chromosome
Found in all species
Retroviruses (HIV) insert DNA in mammals
Can jump from one site to another
Nonreplicative transposition
Can copy itself to a new site
Replicative transposition
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

31. Transposable Elements
DNA sequences
Are not autonomous
Cannot exist outside of a larger DNA molecule
Insertion sequences (IS)
500 – 1500 bp
Transposase gene
Flanked by short inverted repeat sequences
Transposons (Tn)
More complex than IS
Carry additional genes
Antibiotic resistance or catabolic genes
5’-AATCGAT……….ATCGATT-3’
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

32. Basic Transposition and Origin of Target Site Duplication
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

33. Non-Replicative and Replicative Transposition
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

34. Mobile Genetic Elements
Animation: Transposition
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Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

35. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Concept Quiz
Gene transfer by means of a bacteriophage is called:
Transformation
Conjugation
Transduction
Answer: C
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

36. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Concept Quiz
In an Ames Test, a very strong mutagen will cause the appearance of:
Many colonies over the plate, with a clear space close to the disk of mutagen.
A few colonies throughout the plate up to the edge of the disk.
Many colonies covering the whole plate, extending up to the disk.
Answer: A
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

37. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Concept Quiz
Even though transposons disable genes into which they jump, transposons aren’t immediately detrimental for a cell because:
Transposons speed evolution through lateral transfer.
Transposons are unmethylated, and are destroyed by the recipient cell.
Transposons bring antibiotic genes into the cell.
Answer: A
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.