Microbial Genetics & Mutation

Genetic
elements The bacterial genome Gene Flow Replication Transcription Translation Regulating of Gene Expression Genetic Variation Mutation Genetic Recombination Transposition
Circular
vs. linear Location of transcription and translation Single origin and termination of replication No introns/exons in prokayotic
Conjugative plasmid
Carries genes for sex pili and transfer of the plasmid

Encode enzymes for catabolism of unusual compounds
Dissimilation plasmids
R factors
Encode antibiotic resistance
Genetic variation: MUTATION
Is any heritable change in the genetic material Mutations may be neutral, beneficial, or harmful Mutant- organism or strain whose genome carries a mutation
Wild type- the usual (native) form of the organism

Occurrence of mutations:
Spontaneous - Occur in the absence of a mutagen Induced - Occur in the presence of a mutagen
Mutagen: Agent that causes or accelerates rate of mutations
Spontaneous
mutation rate = 1 in 109 replicated base pairs

1 in 106 replicated genes
Mutagens
increase to 105 or 103 per replicated gene
Mutagens
are chemical, physical or biological agents that increase the mutation rate i.e. induce mutations
Can
classify mutagens according to mode of action:

Incorporation of base analogues
Direct reaction with DNA

intercalation
Incorporated into polypeptide chain during replication like normal bases They have different base pairing properties and in subsequent replication events may form a stable mutation
eg. 5-bromouracil: incorporate into the growing DNA as if it were thymine
Mutagens acting directly on DNA
These mutagens change the structure of a base and alter the base pairing characteristics
e.g. methyl nitrosoguanidine (NTG), adds methyl groups to guanine, causing it to base pair with thymine
Intercalating Agents
These mutagens insert themselves between adjacent base pairs and push them apart During subsequent replication this abnormal structure leads to microinsertions/deletions and frameshifts e.g. acridine orange, ethidium bromide
Ionizing
radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone
Nucleotide excision repairs mutations
Non
ionizing radiation
UV radiation causes thymine dimers Light-repair separates thymine dimers
Biological
Segments
mutagen
of DNA that can move from one region of DNA to another insertion sequences for cutting and resealing DNA (transposase) transposons carry other genes
Contain
Complex
Base
substitution (point mutation) mutation

mutation mutation
Change in one base

Missense
Result in change in amino acid

Nonsense
Results in a nonsense codon

Frameshift
Insertion or deletion of one or more nucleotide
pairs
Mutants
in bacteria are mostly biochemical in nature, because we cant generally see the cell 3 major types:
Auxotroph
Chemoauxotroph
Antibiotic resistant
Other types are temperature sensitivity and viral resistance
An
auxotroph needs some nutrient that the wild type strain (prototroph) can make for itself. For example,
trp- auxotroph cant make its own tryptophan (an
amino acid). To grow trp- bacteria, you need to add tryptophan to the growth medium. Prototrophs are trp+; they dont need any tryptophan supplied since they make their own.
Chemoauxotrophs
are mutants that cant use some nutrient (usually a sugar) that prototrophs can use as food. For example:
lac- mutants cant grow on lactose (milk sugar),
but lac+ prototrophs can grow on lactose
confer
resistance antibiotics Example:

AmpR causes bacteria to be resistant to
ampicillin, a common antibiotic related to penicillin

Note: Auxotrophs
and chemoauxotrophs are usually recessive; drug resistance mutants are usually dominant
Positive
(direct) selection detects mutant cells because they grow or appear different (indirect) selection detects mutant cells because they do not grow
Negative
Recombination
Not
regularly occuring Cause mixing of genes\
The three bacterial sexual processes:

1. conjugation: direct transfer of DNA from one bacterial cell to
another. 2. transduction: use of a bacteriophage (bacterial virus) to transfer DNA between cells. 3. transformation: naked DNA is taken up from the environment by bacterial cells.
Conjugation is the closest analogue in bacteria to eukaryotic sex.

The ability to conjugate is conferred by the F plasmid.
A plasmid is a small circle of DNA that replicates independently of the
chromosome. Bacterial cells that contain an F plasmid are called F+. Bacteria that dont have an F plasmid are called F-.
F+ cells grow special tubes called sex pilli from their bodies. When an F+ cell bumps into an F- cell, the sex pilli hold them together, and a copy of the F plasmid is transferred from the F+ to the F-. Now both cells are F+.
Why arent all E. coli F+, if it spreads like that? Because the F plasmid can be spontaneously lost.
Conjugation
One bacterium passes some DNA (in a plasmid) to another bacterium
sometimes
the F plasmid can become incorporated into the bacterial chromosome, by a crossover between the F plasmid and the chromosome.
The resulting bacterial cell is called an Hfr, which stands for High frequency of recombination. bacteria conjugate just like F+ do, but they drag a copy of the entire chromosome into the F- cell.
Hfr
The process of making an Hfr from an F+ involves a crossover between the F plasmid and the chromosome. This process is reversible: an Hfr can revert to being F+ when the F plasmid DNA incorporated into the Hfr chromosome has a crossover and loops out of the chromosome forming an F plasmid once again. Sometimes the looping-out and crossing-over process doesnt happen at the proper place. When this happens, a piece of the bacterial chromosome can become incorporated into the F plasmid. This is called an F (F-prime) plasmid.
Exchange
genes between two DNA molecules

Crossing over
of
occurs when two chromosomes break and rejoin
Transduction
is the process of moving bacterial DNA from one cell to another using a bacteriophage. Bacteriophage or just phage are bacterial viruses.
Two forms of transduction:
1. generalized: any piece of the bacterial genome can be
transferred 2. specialized: only specific pieces of the chromosome can be transferred.
Definition
- Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery
Composition
Nucleic acid Genome size Modified bases Protein Protection Infection

Structure (T4)
Head/Capsid
Contractile Sheath Tail Fibers
Tail
Size Head or capsid Tail
Base Plate
Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA.
A phage containing bacterial DNA can infect a fresh host
After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the hosts chromosome). A double crossover event puts the exogenotes genes onto the chromosome, allowing them to be propagated.
Some
phages can transfer only particular genes to other bacteria.

lambda () has this property.
Phage
lambda has 2 distinct phases of its life cycle. The lytic phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage. it also has lysogenic phase
Then
Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover
Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the prophage. After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs. When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell.
Phage protein coat Bacterial chromosome
Recombinant
A phage infects the donor bacterial cell. Phage DNA and proteins are made, and the bacterial chromosome is broken down into pieces.
Bacterial DNA Phage DNA Donor bacterial DNA Recipient bacterial DNA
Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.
Recombinant cell
A phage carrying bacterial DNA infects a new host cell, the recipient cell. Recombinant can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.

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Genetic

Carries genes for sex pili and transfer of the plasmid

Encode antibiotic resistance

Genetic variation: MUTATION

Wild type- the usual (native) form of the organism

Mutagen: Agent that causes or accelerates rate of mutations

mutation rate = 1 in 109 replicated base pairs

increase to 105 or 103 per replicated gene

classify mutagens according to mode of action:

Direct reaction with DNA

Mutagens acting directly on DNA

UV radiation causes thymine dimers Light-repair separates thymine dimers

substitution (point mutation) mutation

Change in one base

Result in change in amino acid

Results in a nonsense codon

Insertion or deletion of one or more nucleotide

Other types are temperature sensitivity and viral resistance

but lac+ prototrophs can grow on lactose

resistance antibiotics Example:

ampicillin, a common antibiotic related to penicillin

regularly occuring Cause mixing of genes\

The three bacterial sexual processes:

Conjugation is the closest analogue in bacteria to eukaryotic sex.

genes between two DNA molecules

occurs when two chromosomes break and rejoin

Two forms of transduction:

1. generalized: any piece of the bacterial genome can be

transferred 2. specialized: only specific pieces of the chromosome can be transferred.

Nucleic acid Genome size Modified bases Protein Protection Infection

Contractile Sheath Tail Fibers

Size Head or capsid Tail

phages can transfer only particular genes to other bacteria.

Phage protein coat Bacterial chromosome

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