Microbiology Chapter 9

Front 1

• Mutations can be divided into three main classes

Back 1

1) Point mutations
2) Frame-shift mutations
3) Inversions

Front 2

1) Point mutation is

Back 2

is a change in single nucleotide

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i) Transition = A mutation in which a
DNA level

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purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine

Front 4

ii) Transversion = A mutation in which a
DNA level

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purine is replaced by a pyrimidine or vice versa

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i) Silent mutation =
At the protein level

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i) Silent mutation = The resulting triplet codes for the same amino acid
- AAA (Lysine) → AAG (Lysine)

Front 6

ii) Missense mutation
At the protein level

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The resulting triplet codes for a different amino acid
- AAA (Lysine) → GAA (Glutamic acid)
- If the resulting triplet codes for a functionally equivalent amino acid, the mutation is said to be a neutral mutation
- If the missense change decreases or eliminates protein activity, the mutation is said to be a loss of function mutation
- If the missense change allows the protein to gain a new activity, the mutation is said to be a gain of function mutation

Front 7

iii) Nonsense mutation =
At the protein level

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The resulting triplet codes for a stop codon- AAA (Lysine) → TAA (Stop Codon)

Front 8

2) Frame-shift mutation is a

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change in the ORF that changes all the downstream amino acids

Front 9

3) Inversion occurs when _____

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a fragment of DNA is rotated 180o

Front 10

• The effect of mutations on proteins depends on

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type of mutation and position of mutation

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A mutation away from the wild-type

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forward mutation

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reverse mutation

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A second mutation that makes the mutant appear to be a wild-type organism again

Front 13

• Non-ionizing radiation

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- Includes UV light
- Characterized by long wavelength and thus low energy
- Forms pyrimidine dimers (Fig. 9.21)
Covalent cross-linking between adjacent pyrimidines

Front 14

• Ionizing radiation

Back 14

- Includes X-ray, gamma rays
- Characterized by very short wavelength and thus high energy
- Forms highly-reactive free radicals which break the DNA

Front 15

• Base Analogs =

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Structurally similar to nitrogenous bases and can be incorporated in the growing polynucleotide chains during replication
- Cause point mutations
- Example = 5-bromouracil

Front 16

• Base Modifiers =

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Change a base’s structure and thus alters its base-pairing properties
- Cause point mutations
- Example = Nitrous acid

Front 17

• Intercalating Agents =

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Planar molecules that insert themselves between the stacked bases
- Cause frame-shift mutations/ adding or removing of a base
- Example = ethidium bromide

Front 18

• Transposons =

Back 18

DNA elements that have the ability to move from one site of the genome to
another
- Are found in all 3 domains of life
- Cause HUGE frame-shift mutations
- Please refer to Figures 9.31 and 9.32

Front 19

DNA TRANSFER IN PROKARYOTES
1. Conjugation =

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Transfer of DNA from bacterium to another following cell-to-cell contact

Front 20

DNA TRANSFER IN PROKARYOTES
2. Transduction =

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Transfer of DNA from one bacterium to another via a bacteriophage

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DNA TRANSFER IN PROKARYOTES
3. Transformation =

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Transfer of free DNA from one bacterium to another via the “environment”

Front 22

• Competence =

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The particular physiological state in which cells can take up DNA and be genetically changed by it

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TRANSFORMATION

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• Transformation is the uptake of extracellular naked DNA by a cell
• It is mediated by chromosomal genes

Front 24

Natural Transformation
2) Haemophilus influenzae = A Gram--

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- The mechanism differs in several respects from that in Streptococcus
- To begin with, no competence factor is involved
- Changes in the cell envelope accompany the development of the competent state
- Numerous vesicles called transformasomes bud from the surface
- These contain proteins that react specifically with DNA from closely-related species
- The specificity is due to an 11-bp sequence (5′ AAGTGCGGTCA 3′) present at 4 Kb intervals in the Haemophilus genome
- DNA is taken into cells as intact duplex molecules
- However, only one strand participates in subsequent recombination

Front 25

Artificial competence can be induced by

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heatshock, electroporation
- Both perturb the membrane allowing the DNA to enter the cell

Front 26

TRANSDUCTION

Back 26

• Transduction is the unidirectional transfer of DNA from one bacterium to another via a bacteriophage
• It is mediated by phage genes

Front 27

PLASMIDS

Back 27

• Plasmids are typically double-stranded, circular, extra-chromosomal DNA molecules (Fig. 7.22)
- Linear plasmids have been found in certain bacteria
• Plasmids are characteristic of prokaryotes, although some eukaryotes possess them
• The plasmids are typically 1-5 %of the size of the host chromosome
• They are capable of autonomous replication
- However, they “borrow” the proteins involved from the host
- Only a small region surrounding the plasmid ori is required for replication

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Plasmids replicate in one of two basic ways (Fig. 7.23; p. 244)

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roling circle/ bi-directional

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1. Host range =

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The types of bacteria in which the plasmid can replicate

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2. Copy number =

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Average number of a particular plasmid per cell
- Please refer to Figure 1 (p. 245)

Front 31

1. Resistance plasmids

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- Confer resistance to antibiotics, and various other inhibitors of growth
- R100 plasmid of enteric bacteria
- Carries resistance-genes for a number of antibiotics and heavy metals

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2. Bacteriocinogenic plasmids

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- Carry genes that code for bacteriocins
- Proteins that are produced by bacteria and that adversely affect closely-related bacteria
- COL plasmids of E. coli encode colicins

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3. Metabolic plasmids

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- Code for proteins involved in the catabolism of unusual substances
-Pseudomonas has plasmids encoding genes for the degradation of octane, camphor and naphthalene

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4. Virulence plasmids

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- Code for proteins involved in pathogenesis

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4. Virulence plasmids
- Bacillus anthracis

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pX01 encodes the anthrax toxin
- pX02 encodes the capsule

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4. Virulence plasmids
- E. coli

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- ENT plasmids traveler’s diarhea

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5. Fertility plasmids

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- Found in Escherichia coli in 1-3 copies per cell
- Are about 95 Kb in size
- Contains genes responsible for DNA replication, DNA transfer, and episome function

Front 38

- The F plasmid of E. coli has two replication origins

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- orN is used to replicate and maintain the plasmid in non-conjugating cells
- orT is used to replicate the plasmid during conjugation

Front 39

CONJUGATION

Back 39

• Conjugation is the unidirectional transfer of DNA from one bacterium to another following cell-to-cell contact
• Conjugation is mediated by plasmid genes

Front 40

• F+ cell

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- A cell that has an unintegrated F-plasmid
- Also termed male and donor

Front 41

1. Resistance plasmids

Back 41

- Confer resistance to antibiotics, and various other inhibitors of growth
- R100 plasmid of enteric bacteria
- Carries resistance-genes for a number of antibiotics and heavy metals

Front 42

2. Bacteriocinogenic plasmids

Back 42

- Carry genes that code for bacteriocins
- Proteins that are produced by bacteria and that adversely affect closely-related bacteria
- COL plasmids of E. coli encode colicins

Front 43

3. Metabolic plasmids

Back 43

- Code for proteins involved in the catabolism of unusual substances
-Pseudomonas has plasmids encoding genes for the degradation of octane, camphor and naphthalene

Front 44

4. Virulence plasmids

Back 44

- Code for proteins involved in pathogenesis

Front 45

4. Virulence plasmids
- Bacillus anthracis

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pX01 encodes the anthrax toxin
- pX02 encodes the capsule

Front 46

4. Virulence plasmids
- E. coli

Back 46

- ENT plasmids traveler’s diarhea

Front 47

5. Fertility plasmids

Back 47

- Found in Escherichia coli in 1-3 copies per cell
- Are about 95 Kb in size
- Contains genes responsible for DNA replication, DNA transfer, and episome function

Front 48

- The F plasmid of E. coli has two replication origins

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- orN is used to replicate and maintain the plasmid in non-conjugating cells
- orT is used to replicate the plasmid during conjugation

Front 49

CONJUGATION

Back 49

• Conjugation is the unidirectional transfer of DNA from one bacterium to another following cell-to-cell contact
• Conjugation is mediated by plasmid genes

Front 50

• F+ cell

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- A cell that has an unintegrated F-plasmid
- Also termed male and donor

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• F– cell

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- A cell that does not contain an F-plasmid
- Also termed female and recipient

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• F+ X F–

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- The tip of the F pilus attaches to a receptor on the F– cell
- The pilus contracts to bring cells closer
- The two cell envelopes fuse and generate a conjugation bridge
- This triggers the synthesis of an F plasmid helicase/endonuclease that nicks the
phosphodiester backbone at OriT
- DNA POL III is recruited to the nicked site, where replication begins
- It occurs unidirectionally by rolling circle replication
- The 5′ end of the nicked F plasmid strand moves through a pore in the conjugation bridge
- In the donor cell, DNA synthesis restores the double-stranded plasmid
- In the recipient cell, DNA synthesis converts the transferred single-stranded into double-stranded DNA
- The F factor then circularizes to reform the plasmid
=> F– cell becomes F +
- Finally, the conjugation complex spontaneously comes apart, and the membranes seal

Front 53

• Hfr (High Frequency of Recombination) cell

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- A bacterium that has a chromosome-integrated F plasmid
- Hfr cells can transfer chromosomal genes at a much higher frequency than F+ cells do
- Nick in the Hfr chromosome at OriT
=> The F plasmid is now divided into two portions separated by the entire bacterial chromosome
- First, a part of F is transferred, then chromosomal genes in order
- Conjugation does not last long enough for the second part of the F to be transferred
=> F– cell stays F-
- In the donor cell, DNA synthesis restores the double-stranded Hfr chromosome
- In the recipient cell, DNA synthesis converts the transferred single-stranded into double stranded DNA
- Recombination occurs between the endogenote and exogenote

Front 54

• F′ cell

Back 54

- Once an F factor has been integrated into a host chromosome, it can also be excised
- Usually the F factor is excised completely and restored to its original form
- In rare cases, an aberrant excision takes place
- The result is a plasmid which carries adjacent bacterial chromosomal DNA
- This plasmid is called the F′ plasmid
- Please refer to Fig. 9.5

Front 55

• F′ X F–

Back 55

- The process is very similar to that of F+ X F–
- One difference is that it leads to the formation of a stable merozygote
- A cell that contains two copies of some genes
One set on the chromosome and the other on the F′ factor

Front 56

• Enterococcus faecalis

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Sex pili are not involved
- Recipient cells excrete pheromones
- These are small peptides which enter the donor cell through special oligopeptide permeases
- Once inside, the pheromone stimulates transcription of the plasmid transfer genes
- The proteins produced include an AS- Aggregation substances
- AS of the Donor Cell combines with Binding sub. S of the Recipient Cell
- This results in the formation of donor-recipient mating aggregates
- Plasmids are transferred by a mechanism that is not very well understood
- However, plasmid transfer results in repression of pheromone specific for that plasmid