Microbe diversity is enormous' Wildtype and mutant bacteria are haploid

1
Microbe diversity is enormous. Wildtype and
mutant bacteria are haploid (only one set of
genes) so any changes quickly change the
organisms. Natural selection - bacterial
populations are forever changing, special bugs
forever being devised. When nucleotides of an
organisms genome is changed the organism is
said to have mutated or represent a
mutant. Mutants can occur be derived by gene
transfer as well as by mistakes (can be induced)
in DNA replication. Properties of cell is
determined by overall DNA composition - genotype
- in addition to regulation of gene expression -
phentoype.
2
Fig. 08.01

• Base substitution (point mutation)
• leading to mutation
• missense mutation(substitution of amino acid)
• nonsense mutation (stop codon)
• silent mutations - no alteration in phenotype
• observed.

3
Fig. 08.02
Removal or addition of nucleotides leads to
frameshift mutation. Frameshift affects all
amino acids incorporated beyond original site at
which deletion or addition occurred.
4
Fig. 08.04
Base analogs - increased probability that analog
(once incorporated into DNA) will pair with an
inappropriate base. AZT - does not have an -OH
on the third carbon of deoxyribose - elongation
does not proceed.
5
Fig. 08.05
Thymine-dimers - Covalent bonds occur between
adjacent thymine molecules on the same strand of
DNA when exposed to UV light.
6
Table 08.01
7
Fig. 08.06

• Wrong bases can be inserted (either
• spontaneously of following inducing
• with mutagen - causes
• distortion in DNA helix and if allowed
• to remain leads to mutation
• DNA polymerase is a multi-functional
• enzyme and has proofreading
• capabilities i.e. it can correct errors
• that it makes.
• Second mechanism - Mismatch repair
• involves enzyme (endonuclease) that
• cuts out errors in base incorporation

8
Fig. 08.07

• Repair of thymine dimers
• 1. Enzyme that is active in presence of light
• source - photoreactivation.
• 2. Excision repair (no light) - 3 enzymes 1 to
• excise damage, DNA polymerase
• and DNA ligase to ligate brokensingle strand ends

9

• 3. SOS repair - extensive damage, many thymine
  dimers
• Consists of 20 genes
• One is an important DNA polymerase that bypasses
  damage. but can make mistakes,
• adding mutations unlike normal error-free
  (mostly) DNA-replicating machinery.
• But at least cell has a chance to survive, unless
  of course mutation incorporated
• by SOS-repair is Lethal.

10
Table 08.02
11
Fig. 08.08
Direct selection of mutants - the mutant is
already there in a population of 109. Antibiotic
resistant bacteria (to some antibiotics are easy
to isolate, clinically resistant bacteria that
is).
12
Fig. 08.09
Indirect selection - for auxotrophic mutants -
these methods have been used extensively to
determine the metabolic pathways of the cell.
This figure is only the first part of a
journey. Replica plating - amazing technique.
13
Fig. 08.12

• Ames test to screen for mutagens.
• 1. Reversion of mutant gene in a biosynthetic
• pathway can be easily measured.
• 2. Frequency of reversions is increased by
• mutagens.
• 3. Most carcinogens are mutagens.
• 4. Gives you an idea of how mutagenic
• chemicals are.
• 5. Animals cost more, but REALLLY.
• 6. May need to transform some suspected
  substances
• with liver extracts, since modifications of
• chemicals within the body, turns them into
• mutagens.
• -Thus far no compound with negative Ames
• test has been shown to be mutagen in animal.

14
Fig. 08.13
Gene transfer in bacteria Transformation - naked
DNA taken up by cells. Transduction - bacterial
DNA transferred by bacterial virus
(bacteriophage). Conjugation - DNA transferred
from one bacteria to another when cells are in
contact with one another. Proof of.
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Transformation - requires homologous
recombination Competent cells have the ability
to take up and integrate DNA into their
chromosomes or maintain the DNA in plasmid
form.
16
Fig. 08.15
Competence - natural and artificially induced
17
Streptococcus pneumoniae - transforming principle
- led to discovery of DNA as stuff of genes
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Fig. 08.16a
Transduction
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• Plasmids
• 1. covalently closed circular ds DNA
• 2. most prokaryotes contain one or more
• 3. traits (genes) coded on plasmids are useful
  but not always
• indispensable.
• 4. plasmids vary is size - some only have
  information necessary
• to replicate.
• 5. number of copies per cell varies low-copy or
  high-copy
• 6. Plasmids can be transferred via conjugation,
  transduction and
• transformation

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Fig. 08.18
Conjugation -transfers lots of different kinds
of plasmids. F- fertility plasmid - codes for
sex pilus and information required for its own
transfer. 1. Contact between donor and
recipient 2. Mobilization or activation of DNA
transfer, enzyme (endonuclease) cleaves one
strand at specific nucleotide sequence -origin
of transfer. 3. Plasmid transfer - single strand
is transferred. 4. Complementary strand is
synthesized in recipient.
21
Fig. 08.22
22
Table 08.04
23
Fig. 08.24
Transposable elements
24
Fig. 08.23
25
Table 08.03