1. Chapter 08 Lecture Outline
See separate PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes. 1

2. A Glimpse of History Barbara McClintock (1902–1992)
Observed that kernel colors in corn not inherited in a predictable manner
Concluded segments of DNA moved in and out of genes involved in color
Destroyed function of genes
Published results in 1950
Met with skepticism
DNA believed stable
Idea established by 1970s
Awarded Nobel Prize in 1983
Termed transposons
“Jumping genes”

3. Antibiotic Resistance
Staphylococcus aureus
Gram-positive coccus; commonly called Staph
Frequent cause of skin and wound infections
Since 1970s, treated with penicillin-like antibiotics
For example, methicillin
In 2004, over 60% of S. aureus strains from hospitalized patients were resistant to methicillin
~2.3 million healthy people in U.S. harbor methicillin-resistant S. aureus (MRSA)
Healthcare-associated MRSA (HA-MRSA) resistant to other antibiotics, including vancomycin
Vancomycin considered drug of last resort

4. 8.1. Genetic Change in Bacteria
Organisms adapt to changing environments
Natural selection favors those with greater fitness
Bacteria adjust to new circumstances
Regulation of gene expression (Chapter 7)
Genetic change (Chapter 8)
Bacteria excellent system for genetic studies
Rapid growth, large numbers
More known about E. coli genetics than any other
Change in organism’s DNA alters genotype
Sequence of nucleotides in DNA
Bacteria are haploid, so only one copy, no backup
May change observable characteristics, or phenotype
Also influenced by environmental conditions

5. 8.1. Genetic Change in Bacteria
Mutation and horizontal gene transfer

6. 8.1. Genetic Change in Bacteria
Mutations can change organism’s phenotype
Deletion of gene for tryptophan biosynthesis yields mutant that only grows if tryptophan supplied
Growth factor required; mutant termed auxotroph
Auxo = “increase”; troph = “nourishment”
Prototroph does not require growth factors
Proto = “first”
Geneticists compare mutants to wild type
Typical phenotype of strains isolated from nature
For example, wild-type E. coli strain is prototroph
Strains designated by three-letter abbreviations
for example, Trp– cannot make tryptophan
Streptomycin resistance designated StrR

7. 8.2. Spontaneous Mutations
Spontaneous mutations caused by normal processes
Occur randomly at infrequent characteristic rates
Mutation rate: probability of mutation each cell division
Typically between 10–4 and 10–12 for a given gene
Mutations passed to progeny
Occasionally change back to original state: reversion
Large populations contain mutants (for example, cells in colony)
Environment selects cells that grow under its conditions
For example, antibiotics select for resistant bacteria if present
Environment does not cause mutations
Single mutation rare; two even rarer
Physicians may use two antibiotics to reduce resistance
Chance is product of mutation rate for each

8. 8.2. Spontaneous Mutations
Base substitution most common
Incorrect nucleotide
incorporated during
DNA synthesis
Point mutation is change
of a single base pair

9. 8.2. Spontaneous Mutations
Base substitution leads to three possible outcomes
Silent mutation: wild-type amino acid
Missense mutation: different amino acid
Resulting protein may only partially function
Termed leaky
Nonsense mutation:
Specifies stop codon
Yields shorter protein

10. 8.2. Spontaneous Mutations
Base substitutions
A mutation that inactivates gene is termed a null or knockout mutation
“Silent mutation” sometimes used to indicate mutation that does not alter protein function
Base substitutions more common in aerobic environments
Reactive oxygen species (ROS) produced from O2
Can oxidize nucleobase guanine
DNA polymerase often mispairs with adenine

11. 8.2. Spontaneous Mutations
Deletion or addition of nucleotides
Impact depends on number of nucleotides
Three pairs changes one codon
One amino acid more or less
Impact depends on location
within protein
One or two pairs yields
frameshift mutation
Different set of codons translated
Often results in premature
stop codon
Shortened, nonfunctional protein
Knockout mutation

12. 8.2. Spontaneous Mutations
Transposons (jumping genes)
Can move from one location to another
Process is transposition
Gene insertionally inactivated
Function destroyed
Most transposons have transcriptional terminators
Blocks expression of downstream genes in operon

13. 8.2. Spontaneous Mutations
Transposons (jumping genes) (continued…)
Classic studies carried out by Barbara McClintock
Observed color variation in corn kernels resulting from transposons moving into and out of genes controlling pigment synthesis

14. 8.3. Induced Mutations Induced mutations result from outside influence
Agent that induces change is mutagen
Geneticists may use mutagens to increase mutation rate
Two general types: chemical, radiation

15. 8.3. Induced Mutations
Chemical mutagens may cause base substitutions or frameshift mutations
Some chemicals modify nucleobases
Change base-pairing properties
Increase chance of incorrect nucleotide incorporation
Nitrous acid (HNO2) converts cytosine to uracil
Base-pairs with adenine instead of guanine

16. 8.3. Induced Mutations Some chemicals modify nucleobases (cont…)
Alkylating agents add alkyl groups onto nucleobases
Nitrosoguanidine adds methyl group to guanine
Base-pairs with thymine

17. 8.3. Induced Mutations Base analogs resemble nucleobases
Have different hydrogen-bonding properties
Can be mistakenly incorporated by DNA polymerase
5-bromouracil resembles
thymine, often base-pairs
with guanine
2-amino purine resembles
adenine, often pairs with
cytosine

18. 8.3. Induced Mutations Intercalating agents cause frameshift mutations
Flat molecules that intercalate (insert) between adjacent base pairs in DNA strand
Pushes nucleotides apart, produces space
Causes errors during replication
If in template strand, a base pair added to synthesized strand
If in strand being synthesized, a base pair deleted
Often results in premature stop codon
Ethidium bromide is common intercalating agent
Likely carcinogen
Chloroquine, used to treat malaria, is another example

19. 8.3. Induced Mutations Transposition
Transposons can be used to generate mutations
Transposon inserts into cell’s genome
Generally inactivates gene into which it inserts

20. 8.3. Induced Mutations Radiation: two types
Ultraviolet irradiation forms thymine dimers
Covalent bonds between adjacent thymines
Cannot fit into double helix; distorts molecule
Replication and transcription stall at distortion
Cell will die if damage not repaired
Mutations result from cell’s SOS repair mechanism
X rays cause single- and
double-strand breaks in DNA
Double-strand breaks
often produce lethal
deletions
X rays can alter nucleobases

21. 8.4. Repair of Damaged DNA
Enormous amount of spontaneous and mutagen-induced damage to DNA
If not repaired, can lead to cell death; cancer in animals
For example, in humans, two genes associated with breast cancer code for DNA repair enzymes; mutations in either result in 80% probability of breast cancer
Mutations are rare; alterations in DNA generally repaired before being passed to progeny
Several different DNA repair mechanisms

22. 8.4. Repair of Damaged DNA

23. 8.4. Repair of Errors in Nucleotide Incorporation
During replication, DNA polymerase sometimes incorporates wrong nucleotide
Mispairing slightly distorts DNA helix
Recognized by enzymes
Mutation prevented by repairing before DNA replication
Two mechanisms: proofreading, mismatch repair
Proofreading by DNA polymerase
Verifies accuracy
Can back up, excise nucleotide
Incorporate correct nucleotide
Very efficient but not flawless

24. 8.4. Repair of Errors in Nucleotide Incorporation
Mismatch Repair
Fixes errors missed by DNA polymerase
Enzyme cuts sugar-phosphate backbone
Another enzyme degrades short region of DNA strand
Methylation of DNA indicates template strand
Methylation takes time, so newly synthesized strand is unmethylated
DNA polymerase, DNA ligase make repairs

25. 8.4. Repair of Modified Nucleobases in DNA
Modified nucleobases lead to base substitutions
Glycosylase removes
oxidized nucleobase
Another enzyme cuts
DNA at this site
DNA polymerase
removes short section;
synthesizes replacement
DNA ligase seals gap

26. 8.4. Repair of Thymine Dimers
Several methods to repair damage from UV light
Photoreactivation: light repair
Enzyme uses energy from light
Breaks covalent bonds of thymine dimer
Only found in bacteria
Excision repair: dark repair
Enzyme removes damage
DNA polymerase, DNA ligase repair

27. Repair of Thymine Dimers
Several methods to repair damage from UV light (continued…)
SOS repair: last-ditch repair mechanism
Induced following extensive DNA damage
Photoreactivation, excision repair unable to correct
DNA and RNA polymerases stall at unrepaired sites
Several dozen genes in SOS system activated
Includes a DNA polymerase that synthesizes even in extensively damaged regions
Has no proofreading ability, so errors made
Result is SOS mutagenesis

28. 8.5. Mutant Selection Mutants rare, difficult to isolate
Two main approaches
Direct selection: cells inoculated onto medium that supports growth of mutant but not parent
for example, antibiotic-resistant mutants exposed to antibiotic
Indirect selection: isolates
auxotroph from prototrophic
parent strain
More difficult since parents will
grow on any media on which
auxotroph can grow
Replica plating

29. 8.5. Mutant Selection

30. 8.5. Mutant Selection Penicillin enrichment of mutants sometimes used
Increases proportion of auxotrophs in broth culture
Penicillin kills growing cells
Prototrophs
Auxotrophs survive
Penicillinase then added
Cells plated on rich medium

31. 8.5. Mutant Selection Screening for Possible Carcinogens
Carcinogens cause many cancers; most are mutagens
Animal tests expensive, time-consuming
Mutagens increase low frequency of spontaneous reversions
Ames test measures effect of chemical on reversion rate of histidine-requiring Salmonella auxotroph
Uses direct selection
If mutagenic, reversion rate increases relative to control
Rat liver extract added since non-carcinogenic chemicals often converted to carcinogens by animal enzymes
Additional tests on mutagenic chemicals to determine if carcinogenic

32. 8.5. Mutant Selection

33. Horizontal Gene Transfer as a Mechanism of Genetic Change
Microorganisms commonly acquire genes from other cells: horizontal gene transfer
Can demonstrate recombinants
with auxotrophs
Combine two strains
for example, His–, Trp– with Leu–, Thr–
Spontaneous mutants unlikely
Colonies that can grow on
glucose-salts medium most likely
acquired genes from other strain

34. Horizontal Gene Transfer as a Mechanism of Genetic Change
Genes naturally transferred by three mechanisms
Transformation: naked DNA uptake by bacteria
Transduction: bacterial DNA transfer by viruses
Conjugation: DNA transfer during cell-to-cell contact

35. Horizontal Gene Transfer as a Mechanism of Genetic Change
DNA replicated only if replicon
Has origin of replication
Plasmids, chromosomes
DNA fragments added to chromosome via homologous recombination
Only if sequence similar to region of recipient’s genome

36. 8.6. DNA-Mediated Transformation
Naked DNA is not within cell or virus
Cells release when lysed
Addition of DNase prevents transformation
Demonstration of transformation in pneumococci
Only encapsulated cells pathogenic

37. 8.6. DNA-Mediated Transformation
Recipient cell must be competent
Most take up regardless of origin
Some accept only from closely related bacteria (DNA sequence)
Process tightly regulated
Bacillus subtilis has two-component regulatory system
Recognizes low nitrogen or carbon
High concentration of bacteria (quorum sensing)
Only a fraction of population becomes competent

38. 8.7. Transduction Transduction: transfer of genes by bacteriophages
Specialized transduction: specific genes (Chapter 13)
Generalized transduction: any genes of donor cell
Rare error
during phage
assembly
Transfer of
DNA to new
bacterial host

39. 8.8. Conjugation Conjugation: DNA transfer between bacterial cells
Requires contact between donor, recipient cells
Conjugative plasmids direct their own transfer
Replicons
F plasmid (fertility)
of E. coli most
studied
Other plasmids encode resistance to some antibiotics
Spread resistance easily

40. 8.8. Conjugation Conjugation (continued…) F plasmid of E. coli
F+ cells have, F– do not
Encodes proteins including F pilus
Sex pilus
Brings cells into contact
Enzyme cuts plasmid
Single strand transferred
Complementary strands synthesized
Both cells are now F+

41. 8.8. Conjugation Chromosomal DNA transfer less common
Involves Hfr cells (high frequency of recombination)
F plasmid is integrated into chromosome via homologous recombination
Process is reversible
F′ plasmid results when small piece of chromosome is removed with F plasmid DNA
F′ is replicon

42. 8.8. Conjugation Chromosomal DNA transfer less common (continued…)
Hfr cell produces F pilus
Transfer begins with genes on one side of origin of transfer of plasmid (in chromosome)
Part of chromosome transferred to recipient cell
Chromosome usually breaks before complete transfer (full transfer would take ~100 minutes)
Recipient cell remains F– since incomplete F plasmid transferred

43. 8.9. The Mobile Gene Pool
Genomics reveals surprising variation in gene pool of even a single species
Perhaps 75% of E. coli genes found in all strains
Termed core genome of species
Remaining make up mobile gene pool
Plasmids, transposons, genomic islands, phage DNA

44. 8.9. The Mobile Gene Pool Plasmids found in many bacteria and archaea
Some eukarya
Usually dsDNA with origin of replication
Generally nonessential; cells survive loss
Few to many genes
Low-copy-number
One or a few per cell
High-copy-number
Many up to 500
Most have narrow host range
Single species
Some broad host range
Includes Gram– and Gram+

45. 8.9. The Mobile Gene Pool Resistance plasmids (R plasmids)
Resistance to antimicrobial medications, heavy metals (mercury, arsenic)
Compounds found in hospital environments
Often two parts
R genes
RTF (resistance
transfer factor)
Codes for conjugation
Often broad host range
Normal microbiota can
transfer to pathogens
Copyright © 2016 McGraw-Hill Education. Permission required for reproduction or display.
Use fig in 8th edition

46. 8.9. The Mobile Gene Pool Transposons provide mechanism for moving DNA
Can move into other replicons in same cell
Simplest is insertion sequence (IS)
Encodes only transposase enzyme, inverted repeats
Composite transposons include one or more genes
Integrate via non-homologous recombination

47. 8.9. The Mobile Gene Pool
Transposons yielded vancomycin resistant Staphylococcus aureus strain
Patient infected with S. aureus
Susceptible to vancomycin
Also had vancomycin resistant
strain of Enterococcus faecalis
Transferred transposon-
containing plasmid to S. aureus
Transposon jumped to
plasmid in S. aureus

48. 8.9. The Mobile Gene Pool Bacteria can conjugate with plants
Natural genetic engineering
Agrobacterium tumefaciens causes crown gall
Different properties, produces opine, plant hormones
Piece of tumor-inducing (Ti) plasmid called T-DNA (transferred DNA) is transferred to plant
Incorporated into plant chromosome via non-homologous recombination

49. 8.9. The Mobile Gene Pool
Genomic islands: large DNA segments in genome
Originated in other species
Nucleobase composition very different from genome
G-C base pair ratio characteristic for each species
May provide different characteristics
Utilization of energy sources
Acid tolerance
Development of symbiosis
Ability to cause disease
Pathogenicity islands