1. Chapter 10-Mutations and Gene Transfer
General Microbiology
Chapter 10-Mutations and Gene Transfer

2. Mutations
All organisms contain specific sequence of nucleotide bases in the genome
Mutation is a heritable change in the base sequence of that genome
Lead to changes
Good or bad, mostly neutral
Genetic alterations can be made
By physical exchange of DNA between genetic elements
Recombination creates new combinations of genes even in the absence of mutation

3. Unlike most eukaryotes, prokaryotes do not reproduce sexually.
To detect genetic exchange between two prokaryotes it is necessary to employ genetic markers
Markers-any gene whose presence is monitored during a genetics experiment
chosen that are relatively easy to detect

4. Mutations
=heritable change in the base sequence of the nucleic acid in the genome
A strain of any cell or virus carrying a change - mutant
differs from its parental strain in its genotype, the nucleotide sequence of the genome
Aditionally, its phenotype may also be altered relative to its parent
strain isolated from nature as a wild-type (WT) strain.
wild-type may refer to a whole organism or just to the status of a particular gene that is under investigation

5. Genotype vs phenotype
a mutant strain may or may not differ in phenotype from its parent
Genotype: designated by three lowercase letters followed by a capital letter (all in italics) indicating a particular gene
Example: hisC (encodes a protein called HisC that functions in biosynthesis of the amino acid histidine)
Mutations in the hisC gene would be designated as hisC1, hisC2 etc.

6. Phenotype
designated by a capital letter followed by two lowercase letters, with either a plus or minus superscript to indicate the presence or absence of that property
Example: His+ strain of E. coli is capable of making its own histidine, whereas a His- strain is not

7. Isolation of mutants Screening vs selection
some mutations are selectable
confers a clear advantage on the mutant strain under certain environmental conditions
Example: drug resistance
An antibiotic-resistant mutant can grow in the presence of antibiotic concentrations that inhibit or kill the parent
It is relatively easy to detect and isolate selectable mutants by choosing the appropriate environmental conditions
Selection: extremely powerful genetic tool, allowing the isolation of a single mutant from a population containing millions or even billions of parental organisms

8. Screening nonselectable mutation Example: pigmented organisms
Nonpigmented cells usually have neither an advantage nor a disadvantage over the pigmented parent cells when grown on agar plates
although pigmented organisms may have a selective advantage in nature
Screening: detect such mutations only by examining large numbers of colonies and looking for the “different” ones

9. Molecular basis of mutation
Spontaneous or induced
Induced: due to agents in the environment and include mutations made deliberately by humans
result from exposure to natural radiation or chemicals
Spontaneous: occur without external intervention
result from occasional errors in the pairing of bases during DNA replication.

10. Point mutations: Mutations that change only one base pair
caused by base-pair substitutions in the DNA or by the loss or gain of a single base pair
do not actually cause any phenotypic change

11. Base pair substitution
If a point mutation is within the coding region
change in the phenotype of the cell is the result of a change in the amino acid sequence of the polypeptide gene that encodes a polypeptide

12. Frameshifts and other Insertions or Deletions
genetic code is read from one end of the nucleic acid in consecutive blocks of three bases (as codons)
any deletion or insertion of a single base pair results in a shift in the reading frame
Frameshift mutations have serious consequences
Single base insertions or deletions change the primary sequence of the encoded polypeptide
can result from replication errors

13. Insertions or deletions - result in the gain or loss of hundreds or even thousands of base pairs
result in complete loss of gene function
If deleted genes are essential-mutation will be lethal
Larger insertions and deletions may arise as a result of errors during genetic recombination

14. Sickle-cell hemoglobin
Hemoglobin structure and sickle-cell disease
Primary structure
Secondary and tertiary structures
Quaternary structure
Function
Red blood cell shape
Hemoglobin A
Molecules do not associate with one another, each carries oxygen.
Normal cells are full of individual hemoglobin molecules, each carrying oxygen  
10 m
Hemoglobin S
Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced.
 subunit 1 2 3 4 5 6 7
Normal hemoglobin
Sickle-cell hemoglobin
. . .
Exposed hydrophobic region
Val
Thr
His
Leu
Pro
Glul
Glu
Fibers of abnormal hemoglobin deform cell into sickle shape.

15. Site directed mutagenesis and Transposons
not directed at any particular gene
induce specific mutations in specific genes
generating mutations at specific sites is called site-directed mutagenesis
Mutations can also be deliberately introduced by transposon mutagenesis
If a transposable element inserts within a gene, loss of gene function generally results
transposons are widely used to generate mutations

16. Reversions
Point mutations are typically reversible, a process known as reversion
revertant is a strain in which the original phenotype that was changed in the mutant is restored
same-site revertants: mutation that restores activity is at the same site as the original mutation.
true revertant: back mutation is not only at the same site but also restores the original sequence

17. Mutation rates
rates at which different kinds of mutations occur vary widely
Some mutation occur frequently some of then rarely
all organisms possess a variety of systems for DNA repair

18. Mutagenesis
Mutagens: agents that can increase the mutation rate and are therefore said to induce mutations
Chemical
Physical

19. Chemical mutagenesis By chemical mutagens
Induce chemical modifications in one base or another
Result: faulting base pairing
Diverse chemical mutagens:
Alkylating agents
Acridines (function as intercalating agent)

20. Physical mutagenesis Radiation Highly mutagenic
2 main categories: nonionizing and ionizing
Nonionizing (like UV) has the widest use
Purine and pyrimidine bases absorb UV strongly
Absorbtion maximum for DNA and RNA is 260 nm
Consequence: production of pyrimidine dimers
Two pyrimidine bases on the same DNA strand are covalently bounded to each other
Leads to wrong reading of DNA polymerase

21. Benefits of UV radiation
Most commonly used for mutagenesis
UV lamp emits radiation in 260 nm range
Dose of UV radiation is used to kill 50-90% of the cell population
Mutants are then screend among survivors
When used at correct dose, very convinient tool
Avoids the need to use toxic chemicals

22. Ionizing radiation More powerful form of radiation
Uses short-wavelength rays like X-rays, cosmic and gamma rays
Consequence: radicals are formed (mostly OH radicals)
React and damage macromolecules in the cell
Causing ds and ss breaks
Leads to rearrangement of large deletions
Higher dose can lead to fragmentations of DNA
Sometimes cannot be repaired
Goes through the glass and other materials
Frequently used to introduce mutations in plants and animals
Less usage in microbial genetics

23. DNA repair system
If damaged DNA can be repaired before cell division, mutation will not occur
Cells have variety of repairing mechanisms
Virtually are error-free
Some of them are error-prone (repair process can introduce mutation)
Can be grouped into 3 categories:
Direct reversal
Repair of single-strand damage
Repair of double-strand damage

24. Direct reversal
applies to bases that have been chemically altered but whose identity is still recognizable
No base pairing (no template strand) is needed
Example: alkylated bases are repaired by direct chemical removal of the alkyl group
Example 2: photoreactivation (cleaves pyrimidine dimers generated by UV radiation)

25. Repair of single-strand damage
Several systems
damaged DNA is removed from only one strand
opposite (undamaged) strand is used as a template for replacing the missing nucleotides
In base excision repair, a single damaged base is removed and replaced
In nucleotide excision repair and mismatch repair, a short stretch of single-stranded DNA containing the damage is removed and replaced.

26. Repair of double-strand damage
Double-strand damage is very dangerous!
repaired by recombinational mechanisms and may require error-prone repair

27. The SOS system
Lesions on the template DNA may lead to stalling of DNA replication
lethal event
Stalled replication activates SOS repair system
initiates a number of DNA repair processes, some of which are error-free
also allows DNA repair to occur without a template, that is, without base pairing
this results in many errors and hence many mutations.
This permits cell survival under conditions that are otherwise lethal.

28. Gene transfer
For genetic analyses, the microbial geneticist must cross strains of an organism that have different genotypes (and phenotypes) and look for recombinants…
Three mechanisms of genetic exchange are known:
Transformation (free DNA released from one cell is taken up by another)
Transduction (which DNA transfer is mediated by a virus)
Conjugation (DNA transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell)

29. Incoming DNA faces 3 different „fates“:
It may be degraded by restriction enzymes
It may replicate by itself (only if it possesses its own origin of replication such as a plasmid or phage genome)
It may recombine with the host chromosome

30. Genetic recombination
Recombination: physical exchange of DNA between genetic elements
Major form is homologous recombination
results in genetic exchange between homologous DNA sequences from two different sources
homologous DNA sequences have nearly the same sequence
Involved in the process referred to as “crossing over” in classical genetics

31. Transformation Genetic transfer process
free DNA is incorporated into a recipient cell and brings about genetic change
Several prokaryotes are naturally transformable (Gram+ and Gram- Bacteria and also some Archaea)
A single cell usually incorporates only one or a few DNA fragments
so only a small proportion of the genes of one cell can be transferred to another by a single transformation event

32. Competence in Transformation
only certain strains or species are transformable
Competent cell: one which is able to take up DNA and be transformed
his capacity is genetically determined
Competence in most naturally transformable bacteria is regulated
special proteins play a role in the uptake and processing of DNA
Specific proteins include:
membrane-associated DNA-binding protein
cell wall autolysin
various nucleases

33. Types of competence High efficiency among bacteria is very rare
Treatment of bacterial cells (E.coli) with high concentrations of calcium ions and then chilled for several minutes, they become adequately competent.
Chemical competence
Cells of E. coli treated in this manner take up double-stranded DNA, and therefore transformation of this organism by plasmid DNA is relatively efficient
getting DNA into E. coli—the workhorse of genetic engineering—is critical for biotechnology

34. Types of competence Electroporation
physical technique that is used to get DNA into organisms that are difficult to transform
cells are mixed with DNA and then exposed to brief high-voltage electrical pulses
This makes the cell envelope permeable and allows entry of the DNA
quick process and works for most types of cells, including E. coli
Also for some yeast, Archaea and plant cells

35. Transfection
Bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium
Transfection
useful for studying the mechanisms of transformation and recombination
If the DNA is from a lytic bacteriophage, transfection leads to virus production and can be measured by the standard phage plaque assay

36. Transduction
a bacterial virus (bacteriophage) transfers DNA from one cell to another
2 ways:
generalized transduction (DNA derived from any portion of the host genome is packaged inside the mature virion in place of the virus genome)
specialized transduction (DNA from a specific region of the host chromosome is integrated directly into the virus genome)

37. Generalized transduction
any gene on the donor chromosome can be transferred to the recipient
Example: Phage
during lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes package host DNA accidentally
result is called a transducing particle
cannot lead to a viral infection because they contain no viral DNA
said to be defective
On lysis of the cell, the transducing particles are released along with normal virions that contain the virus genome
Consequence: the lysate contains a mixture of normal virions and transducing particles

38. Specialized transduction
allows extremely efficient transfer
Is selective and transfers only a small region of the bacterial chromosome

39. Conjugation
mechanism of genetic transfer that involves cell- to-cell contact
Plasmid encoded mechanism
Conjugative plasmids use this mechanism to transfer copies of themselves to new host cells
involves a donor cell, which contains the conjugative plasmid, and a recipient cell, which does not
genetic elements that cannot transfer themselves can sometimes be mobilized during conjugation
can be other plasmids or the host chromosome itself

40. Gene transfer in Archaea
contain a single circular chromosome like most Bacteria
the development of gene transfer systems lags far behind that for Bacteria
problems include the need to grow many Archaea under extreme conditions
the temperatures necessary to culture some of them will melt agar
alternative materials are required to form solid media and obtain colonies

41. Another problem: antibiotics
most antibiotics do not affect Archaea
penicillins do not affect Archaea because their cell walls lack peptidoglycan
choice of selectable markers for genetic crosses is therefore often limited

42. No single species of Archaea has become a model organism for archaeal genetics
more genetic work on select species of extreme halophiles than on any other Archaea
individual mechanisms for gene transfer have been found scattered among Archaea
Examples of transformation, transduction, and conjugation are known
several plasmids have been isolated from Archaea
have been used to construct cloning vectors, allowing genetic analysis

43. Transformation procedures vary in detail from organism to organism.
Transposon mutagenesis has been well developed in certain methanogen species
Transformation procedures vary in detail from organism to organism.
One approach involves removal of divalent metal ions, which in turn results in the disassembly of the glycoprotein cell wall layer surrounding many archaeal cells and hence allows access by transforming DNA

44. Mobile DNA: Transposable Elements
Mobile DNA: discrete segments of DNA that move as units from one location to another within other DNA molecules
most mobile DNA consists of transposable elements
stretches of DNA that can move from one site to another
always found inserted into another DNA molecule such as a plasmid, a chromosome, or a viral genome
do not possess their own origin of replication
replicated when the host DNA molecule into which they are inserted is replicated

45. two major types of transposable elements in Bacteria:
insertion sequences (IS)
transposons
have two important features in common:
carry genes encoding transposase, the enzyme necessary for transposition
they have short inverted terminal repeats at their ends that are also needed for transposition

46. Insertion sequences (IS)
simplest type of transposable element
short DNA segments, about 1000 nucleotides
typically contain inverted repeats of 10–50 bp
The only gene they possess is for the transposase
IS elements are found in the chromosomes and plasmids of both Bacteria and Archaea
Also in certain bacteriophages

47. Transposons larger than IS elements Same two essential components:
inverted repeats at both ends
gene that encodes transposase
The transposase recognizes the inverted repeats and moves the segment of DNA flanked by them from one site to another
Genes included inside transposons vary widely
confer important new properties on the organism harboring the transposon (antibiotic resistance genes)
most highly investigated transposons have antibiotic resistance genes as selectable markers

48. Mechanism of Transposition
Two mechanisms of transposition are known: conservative and replicative
In conservative transposition: the transposon is excised from one location and is reinserted at a second location
The copy number of conservative transposon therefore remains at one
During replicative transposition, a new copy is produced and is inserted at the second location
After a replicative transposition, one copy of the transposon remains at the original site, and there is a second copy at the new site.

49. Mutagenesis with Transposons
When a transposon inserts itself within a gene, a mutation occurs in that particular gene
Mutations due to transposon insertion do occur naturally
use of transposons is a convenient way to create bacterial mutants in the laboratory
The transposon is introduced into the target cell on a phage or plasmid that cannot replicate in that particular host
Consequently, antibiotic-resistant colonies will mostly be due to insertion of the transposon into the bacterial genome.

50. most transposon insertions will occur in genes that encode proteins (bacterial genomes contain relatively little noncoding DNA)
If inserted into a gene encoding an essential protein, the mutation may be lethal under certain growth conditions and be suitable for genetic selection.
More recently, transposons have even been used to isolate mutations in animals, including mice

51. End of chapter 10

52. Quiz 2
Start: 15:00
Everything from the Chapter 6 (including Chapter 6)