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Variations in microbial population
Spontaneous and induced variation in microbial population
By Dr. Jagnoor Singh Introduction
The genome is a dynamic entity, subject to different types of heritable changes.
Mutation is a heritable genetic change in the genetic material of an organism that gives rise to alternate
forms of any gene.
The process by which mutation is produced is called mutagenesis .
An organism exhibiting a novel phenotype due to mutation is called a mutant .
A mutagen is an agent that leads to increase in frequency of mutations.
Mutation can include any change in chromosome number, chromosomal aberration, and changes in the
chemistry of genes. General characteristics and role of
# mutations
Ultimate source of all genetic
variation.
Provides raw material for evolution .
Results into the formation of alleles .
Without mutation, all genes would occur in
only one form.
Enables organisms to evolve and adapt to
environmental change.
Generally recessive , but dominant
mutations also occur.
Generally harmful .
Random , occur at any time and in
any cell of an organism.
Recurrent , the same mutation
may occur again and again. Molecular basis of
# gene mutation
Mutations can occur in two ways
1. Spontaneous mutations : that occur
without treatment of organism with any
exogenous mutagen. These can occur due to
replication errors , spontaneous
lesions.
2. Induced mutations : occur because a
mutagen has reacted with the parent DNA,
causing a structural change that affects the
base -pairing capability of the altered
nucleotide. Spontaneous
# mutations: replication
# errors
Each of the common bases in DNA can spontaneously
undergo a transient rearrangement of bonding.
A proton shift in nitrogenous base of DNA forms
one of its rare tautomeric forms (termed a
tautomeric shift).
Formation of the tautomer of any base alters its base
pairing properties.
The more stable keto forms of thymine and guanine
and amino forms of adenine and cytosine may
infrequently undergo tautomeric shifts to less stable
enol and imino forms, respectively. When the bases are present in their rare imino or enol states , they can
form A-C and G-T base pairs.
The net effect of such an event, is an AT to GC or a GC to AT base pair
substitution.
Error during replication may result in substitution mutation (also
called point mutation). Mistakes in DNA
# Replication
DNA polymerase enzymes are amazingly
particular with respect to their choice of
nucleotides during DNA synthesis, ensuring
that the bases added to a growing strand are
correctly paired with their complements on
the template strand.
Still, these enzymes do make mistakes
at a rate of about 1 per every 100,000
nucleotides.
The polymerase fixes most of these
mistakes but it only fixes about 99% of
these errors. Substitution mutation
A gene mutation that results from the
substitution of one base pair for another is
known as substitution mutation. Its of two
types
Transition: Transition, the most common
class, comprising the substitution of one
pyrimidine by the other, or of one purine by
the other. This replaces a G-C pair with an A-
T pair or vice versa.
Transversion : If purine is replaced by a
pyrimidine, or pyrimidine is replaced by a
purine then it is known as transversion.
Image:
http://www2.csudh.edu/nsturm/CHEMXL153/DNAMutationRe
p air.htm Most mispairing mutations are transitions. This is
likely to be because an AC or GT mispair does not
distort the DNA double helix as much as AG or CT
base pairs do. However , transversions also can
occur through mispairing. Deletions and
# Duplications
Large deletions (more than a few
base pairs) constitute a sizable fraction
of spontaneous mutations. The
majority, although not all, of the
deletions occur at repeated sequences.
Hot spots for deletions are in the
longest repeated sequences.
Duplications of segments of DNA
have been observed in many organisms.
Like deletions, they often occur at
sequence repeats.
Deletions may be generated as
replication errors. Alternatively,
deletions and duplications could be
generated by recombinational
mechanisms. Spontaneous mutations Naturally occurring damage to the DNA are called spontaneous
lesions. Two of the most frequent spontaneous lesions are
depurination/depyrimidination and oxidative
damage.
Depurination/ Depyrimidination: i.e the loss of a
purine or a pyrimidine. This results in the formation of an
apurinic/apyrimidinic site which does not base pair normally
and cause mutation.
Oxidative damage: ROS such as free radicals or
peroxides produced during anaerobic oxidation cause
these lesions. Example: Guanine can be converted to 8-
oxo -7,8 -dihydrodeoxyguanine, which pairs with adenine
instead of cytosine, during replication. Induced mutations These are caused by mutagens.
Mutagens have been divided according to their mode
of action.
There are 3 common types of chemical mutagens
i.e.
1. Base analogues,
2. DNA -modifying agents and
3. intercalating agents.
Biological mutagens
Then there are also physical mutagens such as
1. UV light
2. X-rays. Induced mutations
types of chemical mutagens -
Base Analogues :
These are chemicals that resemble normal DNA bases and
can be mistakenly incorporated into the DNA during
replication.
Example: 5 -bromouracil, which can pair with both adenine
and guanine, causing mutations like base -pair substitutions.
DNA -Modifying Agents :
These chemicals alter the structure of DNA bases, causing
them to mispair during replication.
Example: Nitrous acid, which deaminates adenine and
cytosine, leading to incorrect base pairing.
Intercalating Agents :
These chemicals insert themselves between DNA base
pairs, distorting the DNA structure.
Example: Ethidium bromide, which can cause insertions or
deletions, leading to frameshift mutations. Base analogues
Base analogues are structurally similar to
normal nitrogenous bases and can be
incorporated into the growing polynucleotide
chain during replication.
Once in place, these compounds typically exhibit
base -pairing properties different from the bases
they replace and can eventually cause a
stable mutation.
A widely used base analogue is 5-bromouracil ,
analog of thymine. It undergoes a tautomeric
shift much more frequently than a normal base.
The enol tautomer forms hydrogen bonds like
cytosine pairing with guanine rather than
adenine.
> Image: https://www.sciencedirect.com/topics/biochemistry -gene
> tics -and -molecular -biology/5 -bromouracil
# DNA modifying
# agents
These are mutagens that change a
base's structure and therefore
alter its base -pairing specificity.
Some of these mutagens
preferentially react with certain
bases and produce a particular kind
of DNA damage.
For example, methyl -
nitrosoguanidine adds methyl groups
to guanine, causing it to mispair with
thymine. A subsequent round of
replication can then result in a GC -AT
transition .
Hydroxylamine is another example
of a DNA -modifying agent. It attaches
a hydroxyl group to cytosine causing
it to base pair like thymine. Intercalating agents
Intercalating agents distort DNA to
induce single nucleotide pair insertions
and deletions.
These mutagens are planar and insert
themselves between the stacked bases
of the helix.
The result is a mutation, possibly by
the formation of a loop in DNA Other mutagens
Deaminating Agents: These agents
remove amino groups on nucleotide bases.
They produce an adenine species that
pairs with cytosine and a cytosine
species (Uracil) that pairs to adenine.
Alkylating agents: Agents like ethyl
methanesulfonate and dimethyl
nitrosoguanidine alter the nucleotide base
by adding alkyl groups . The nature and
position of the alkylation can vary but usually
leads to point mutations through base
mispairing. Biological
# mutagens
Biological agents of mutation are
sources of DNA from elements like
transposons and viruses.
Transposons are sequences of DNA that
can relocate and replicate
autonomously.
Insertion of a transposon into a DNA
sequence can disrupt gene
functionality.
There are three types of transposons :
Replicative transposons keep the
original locus and translocate a copy.
Conservative transposons occur
when the original transposon
translocates . Retrotransposons
transpose via RNA intermediates Physical Mutagens
Many mutagens, and carcinogens damage bases so
severely that hydrogen bonding between base pairs is
impaired or prevented and the damaged DNA can not
act as a template for replication.
Radiations are of 2 types: Ionising and non -
ionising radiations .
For instance , ultraviolet (UV) radiation (non -ionising ),
often generates thymine dimers between adjacent
thymines . Image: https://i.pinimg.com/originals/6c/86/f6/6c86f654e229ee12f42cf4 b4000e809a.png Ionising radiations such as X-rays and Gamma -rays induce mutations by the
following ways
They cause single stranded or double stranded breaks in DNA backbone through the
formation of hydroxyl radicals on radiation exposure.
The radiations can also modify bases. E.g. Deamination of cytosine to uracil. Types of Mutations
Forward Mutations : A mutation that changes the phenotype from wild type to a mutant
phenotype .
Reverse Mutations : A mutation that causes a change of phenotype from mutant to a
wild type .
Suppressor mutations : a suppressor is a second mutation that restored a function lost by
the first mutation . They may be within the same gene (intragenic suppressor mutation) or in a
different gene (extragenic suppressor mutation) Mutations in protein
# coding genes
Point mutations in protein -coding genes
can affect protein structure in a variety
of ways.
Point mutations are named according to if
and how they change the encoded
protein.
The most common types of point
mutations are
1. silent mutations,
2. missense mutations,
3. nonsense mutations and
4. frameshift mutations
5. Conditional mutations Silent mutations
Silent mutations change the nucleotide sequence of a
codon but do not change the amino acid encoded by
that codon.
When more than one codons code for the same amino
acid, a single base substitution may result in the
formation of a new codon for the same amino acid. For
example, if the codon CGU were changed to CGC, it
would still code for arginine, even though a mutation had
occurred.
When there is no change in the protein , there is no
change in the phenotype of the organism. Missense Mutation
These involve a single base substitution that changes a
codon for one amino acid into a codon for another.
For e.g , the codon GAG, which specifies glutamic acid, could
be changed to GUG, which codes for valine.
The effects of missense mutations vary. They alter the primary
structure of a protein , which may cause complete loss of
activity to no change at all.
The effect on protein function depends on the type and location
of the amino acid substitution .
Replacement of a nonpolar AA in the protein's interior with a
polar AA can drastically alter the protein's 3-D structure and
function.
Missense mutations play a very important role in
providing new variability to drive evolution because
they often are not lethal and therefore remain in the gene
pool. Nonsense mutation
Nonsense mutations convert a sense codon to a nonsense codon
(stopcodon ).
This causes the early termination of translation and therefore results in a
shortened polypeptide.
Depending on the location of the mutation, the phenotype may be more or less
severely affected.
Most proteins retain some function if they are shortened by only one or two amino
acids.
Complete loss of normal function usually results if the mutation occurs closer to the
beginning or middle of the gene. Frameshift mutation Frameshift mutations arise from the insertion or deletion of
base pairs within the coding region of the gene.
Since the code consists of precise sequence of triplet codons, the
addition/deletion of fewer than three base pairs causes the
reading frame to be shifted for all subsequent codons
downstream.
Frameshift mutations usually are very deleterious and yield
mutant phenotypes resulting from the synthesis of
nonfunctional proteins.
In addition , frameshift mutations often produce a stop codon
so that the peptide product is shorter as well as different in
sequence. Conditional mutations Conditional mutations are those that are expressed
only under certain environmental
conditions .
For e.g , a conditional lethal mutation might not be expressed
when a bacterium is cultured at a low temperature but would
be expressed high temperature. Thus, the mutant would grow
normally at cooler temperatures but would die at high
temperatures.
Other common mutations inactivate a biosynthetic pathway,
frequently eliminating the capacity of the mutant to make an
essential molecule such as an amino acid or nucleotide.
A strain bearing such a mutation has a conditional phenotype:
it is unable to grow on medium lacking that molecule but
grows when the molecule is provided. Such mutants are called
auxotrophs , and they are said to be auxotrophic for the
molecule they cannot synthesize. The wild -type strain from which the mutant arose is called a prototroph .
Another interesting mutant is the resistance mutant .
These mutants have acquired resistance to some pathogen e.g. ,
bacteriophage) , chemical (e.g. , antibiotic), or physical agent.
Auxotrophic and resistance mutants are quite important in microbial
genetics due to the ease of their detection and their relative abundance. Detection and isolation of mutants Why isolate mutants? Mutations are of practical importance to microbial
geneticists .
Mutant strains have been used to reveal mechanisms of
complex processes such as DNA replication, endospore
formation, and regulation of transcription.
They are also useful as selective markers in recombinant
DNA procedures.
To study microbial mutants, they must be readily detected
and then efficiently isolated from wild -type organisms and
other mutants that are not of interest.
The likelihood of obtaining mutants can be increased by
using mutagens to increase the rate of mutation .Mutant detection:
# screening of
# mutants with an
# observable
# phenotype
To collect mutants of a particular
organism,
1. the wild -type characteristics must be
known so that an altered phenotype can
be recognized.
2. A suitable detection system for the
mutant phenotype also is needed. The
use of detection systems is called
screening .
Some screening procedures require
only examination of colony
morphology.
Other screening methods are more
complex. E.g. Replica plating
technique for screening of auxotrophs Replica plating technique
It distinguishes between mutants and the
wild -type strain based on their ability to
grow in the absence of a particular
biosynthetic end product.
E.g. A lysine auxotroph, grows on
lysine -supplemented media but when the
colonies that grow on this medium are
transferred to a medium lacking lysine,
they will not grow, because they cannot
synthesize this amino acid. Image:
https://www.researchgate.net/profile/Usha_Mina/publication/310995415/figure/fig10/AS:43
3360712540173@1480332653816/Replica -plating -For -the -detection -of -muta nts -cells -are -
transferred -on -to -successive.png Mutant selection:
# using conditions to
# inhibit growth
Mutant selection techniques use
incubation conditions under which
the mutant grows due to mutation, but
the wild type doesnt.
Selection methods often involve
reversion or suppressor mutations
that restore the wild -type
phenotype.
For e.g., if we want to isolate a
prototroph from a lysine auxotroph
(Lys), a large population of lysine
auxotrophs is plated on minimal
medium lacking lysine, incubated, and
examined for colony formation.
Only cells that are prototrophs will grow
on minimal medium. Thus, many cells
can be tested for mutations. Other selection methods are used to identify mutants resistant to a
particular environmental stress such as virus attack, antibiotic treatment,
or specific temperatures.
So , it is possible to grow the microbe in the presence of the stress and
look for surviving organisms.
For e.g. Consider an antibiotic -sensitive wild -type bacterium. When it is
cultured in medium lacking the antibiotic and then plated on selective
medium containing the antibiotic, any colonies that form are resistant to the
antibiotic carry a mutated gene that confers antibiotic resistance. Isolation of rare mutants
Isolating rare mutants is achieved by using enrichment , a method that
favors the growth of desired mutants relative to non -mutant bacteria.
Penicillin enrichment is a classical example of this approach.
Penicillin disrupts the growth of bacterial cell wall, so it only kills growing
bacteria.
Eg. penicillin enrichment can be used to isolate a rare auxotrophic mutant
from a population of bacteria.
If a mix of wild -type and auxotrophic bacteria is suspended in a
nonpermissive medium containing penicillin, the auxotrophic mutants will
not grow and thus will survive, while 99% of wild type bacteria will grow
and be killed by penicillin. References
Prescotts Microbiology (10th edition) Willey/ Sherwood/ Woolverton
Life Sciences Fundamentals and Practices II (6th edition) - Pranav Kumar, Usha Mina
https://www.researchgate.net/publication/275965123_Bacterial_Mutation_Types_Mech
anisms_ and_Mutant_Detection_Methods_a_Review
https://www.ncbi.nlm.nih.gov/books/NBK459274/
https://www.ncbi.nlm.nih.gov/books/NBK21897/#:~:text=Spontaneous%20mutations%
20can% 20be%20generated,the%20absence%20of%20mutagenic%20treatment .
https://courses.lumenlearning.com/microbiology/chapter/mutations/
https://www.sciencedirect.com/science/article/pii/B9780123847195004317
https://www.nature.com/scitable/topicpage/dna -replication -and -causes -of -mutation -
409/#:~:text
=When%20Replication%20Errors%20Become%20Mutations,longer%20recognizes%20t
hem%2 0as%20errors .Thank You