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MUTATIONS
•Stable, heritable, alterations in the gene sequence which
usually produce phenotypic changes (ie mutant)
•Morphological mutations-change microorganisms
appearance, colony or cellular morphology
•Lethal mutations-result in death of microorganism
•Conditional mutations-expressed under certain
environmental conditions eg. not expressed at a low
temperature (permissive temp), but expressed at high
temperature (non-permissive temp)
•Biochemical mutations-alter a biosynthetic pathway
and the organisms ability to grow on minimal media
*AUXOTROPH- requires nutrient supplement
cannot grow on minimal media
*PROTOTROPH- wild-type, not a mutant thus
can grow on minimal media
•Resistant mutations- a mutant that is now resistant to
an antibiotic or virus
How Do Mutations Occur?
Spontaneous mutation-occurs randomly, may result from
errors during DNA replication
Transition-changing a purine for the other purine or a
Pyrimidine for the other pyrimidine
Transversion-purine is substituted for a pyrimidine
Frameshift- deletion or addition of a base causes an altered
Codon reading frame and the synthesis of a non functional
protein
Induced Mutations
Occurs after exposure to a physical or chemical agent called
a mutagen
Modes of action
1. Incorporation of base analogs-structurally similar to the
normal bases that get incorporated into the growing
chain during replication. Typically they base pair with
different bases and thus replace the normal base pair.
eg. 5-Bromouracil
2.
Specific mispairing-mutagen changes a base structure
eg. Methylnitrosoguanidine puts a CH3 group on
guanine causing it to mispair with thymine
3.
Intercalating agents-distort DNA to induce single
nucleotide pair insertions and deletions
4.
UV radiation-generates thymine dimers between
adjacent pyrimidines. Usually this triggers repair
mechanisms, but repair may not always occur.
Types of Mutations
Wild-type = No mutation
Forward mutation = a mutation that alters phenotype
Reverse mutation = a second mutation which may make the
mutant appear wt (in same gene)
Suppressor mutation = a mutation in a different gene which
overcomes the effect of the 1st mutation resulting
in a wt phenotype
Point mutation = affects only 1 bp at a single lociation
Silent mutation = a point mutation that has no visible effect
because of code degeneracy
Missense mutation = a single base substitution in the DNA
that changes a codon from one amino acid to another
Nonsense mutation = converts a sense codon to a nonsense or
stop codon, results in shortened polypeptide
Frameshift mutation = arise from insertion or deletion of one
or two bp within coding region of gene, results in the
synthesis of nonfunctional protein
Mutant Detection
In order to study microbial mutants, one must be able to
detect them and isolate them from the wt organisms
Detection Systems in bacteria are straightforward because
there is only 1 copy of the gene therefore any new allele
should be seen immediately
Screening vs Selection Systems
Screening for auxotrophic mutants
A lysine auxotroph will only grow on media that is
supplemented with lysine because it cannot synthesize
the amino acid
Use of replica plating to find mutant
Selection for antibiotic resistant mutants
Technique uses conditions under which only the mutant
will grow
Bacterial Viruses
Just like we have viruses that infect us causing the common
cold to meningitis, bacteria also have viruses that infect
them called: Bacteriophages
Bacteriophage cannot reproduce and survive on it’s own,
it must take over it’s host cell
Virulent bacteriophages = lytic life cycle (eg T4)
1. Adsorption of phage to host
2. Penetration of virus genetic material
3. Replication, synthesis of virus DNA and proteins
4. Cell lysis and release of phage particles
Temperate bacteriophages = lysogenic life cycle (eg l)
1. Adsorption of phage to host
2. Penetration of virus genetic material
3. Integration of virus into host chromosome = prophage
4. Replication along with host chromosome, maintained
as a prophage
Temperate Bacteriophages
Instead of destroying host to produce virus progeny, the
viral genome remains within the host cell and replicates
with the bacterial chromosome.
This relationship between phage and host is called
lysogeny
Lambda (l), family Siphoviridae, is the best understood
Temperate phage
DNA is double stranded with cohesive ends (cos sites)
which are ss stretches of DNA that are complementary
to each other so it circularizes immediately after
injection into the host
Once a closed circle is formed transcription by host RNA
polymerase is initiated
The BIG Decision: Lytic or Lysogenic life cycle?
Battle between two repressors, cI or cro which compete for
The same binding sites (operators)
•if cI binds represses synthesis of all genes = Lysogeny
•If cro binds represses synthesis of cI = Lytic
Temperate phage l
If cI repressor wins the circular DNA is inserted into the
chromosome via a process called integration
At this stage it is called a Prophage
The l prophage can decide to leave the host genome
and begin the the production of new phage
This is triggered by a drop in cI levels
Environmental factors such as UV light or chemical
Mutagens that damage host DNA causes a host protein,
recA, to act as a protease and cleave the cI repressor
Now the balance will be shifted to cro and the lytic cycle
l is smarter than we think!
Recombination
Process in which nucleic acid molecules are rearranged
or combined to produce new sequences
This occurs in a process called crossing-over
In bacteria General Recombination involves exchange
between a pair of homologous DNA sequences
Since bacteria don’t have sex (reproduce via meiosis)
Recombination takes places in several ways following
Horizontal Gene Transfer
1.
Direct transfer between 2 bacteria temporarily in
physical contact [conjugation]
2.
Transfer of naked DNA fragments [transformation]
3.
Transport of bacterial DNA by a bacteriophage
[transduction]
Plasmids
Plasmids are small ds DNA molecules, usually circular that
can exisit independently of the host chromosome. They
have their own replication origin so can replicate
automonously (Replicon) and have relatively few genes (<30)
that are not essential to the host.
Conjugative plasmids have genes for pili and can transfer
copies of themselves to other bacteria during conjugation
Fertility factor or F factor - These plasmids can also
intergrate into the host chromosome or be maintained
as an episome (independent of chromosome)
R factor - Also conjugative plasmids which have genes that
code for antibiotic resistence for the bacteria harboring them.
These do not integrate into the host chromosome.
Col Plasmids - harbor Bacteriocins which are proteins that
destroy other bacteria (eg cloacins kill Enterobacter species)
Virulent plasmids - have genes which make bacteria more
pathogenic because the bacteria is better able to resist host
defenses or produce toxins/invasins
Transposable Elements
Transposons - DNA segments that carry genes that allow
them to move about the chromosome (transposition)
•Behave somewhat like a lysogenic phage
•Unlike plasmids or phages, they are unable to reproduce
or exist apart from the host chromosome
Insertion sequences - IS elements- short sequence of DNA
Containing only genes required for transposition
•Flanked by inverted repeats (IR) - identical or similar
sequences 15-25 bp in reversed orientation
•Transposase - enzyme that recognizes the IR and promotes
transposition
Composite transposon (Tn)- contains other genes in addition
to transposase like antibiotic resistance genes or toxins
Importance
•Can insert within a gene to cause a mutation or stimulate
DNA rearrangement leading to deletions of genetic material
•Can have stop codons or termination sequences to block
translation or transcription
•Can have promoters which activate genes near pt of insertion
•Can be on plasmids to aid in insertion of F plasmids into
host chromosome
•Some bear transfer genes (Tn916) and can move between
bacteria through conjugation (conjugative transpososn)
Conjugation
Conjugation - transfer of genetic material by direct cell
contact.
First evidence by Lederberg and Tatum 1946 - mixed two
auxotrophic strains for several hours in rich medium than
plated on minimal medium. Only recombinants which are now
Prototrophic will be able to grow
Second evidence- U tube experiment proved direct cell contact
was required
During this process there is a definite donor strain , F+ (F factor)
and a recipient strain, F- (does not have the F factor)
The F factor codes for pilus formation which joins the donor
and recipient and for genes which direct the replication and
transfer of a copy of the F factor to the recipient
The F factor can remain as an episome or it can integrate into
the bacterial chromosome via IS sequences. This type of donor
is called and Hfr strain (High frequency recombination)
F’- When the F factor in an Hfr strain leaves the chromosome,
sometimes is makes an error in excision and picks up some
bacterial genes
Transformation
Transformation- uptake by a cell or a naked DNA molecule
or fragment from medium and incorporation of this DNA into
the recipient chromosome. This process is random and any
portion of the genome may be transferred.
For this process to occur the bacterial recipient must be able
to take up the DNA, called competency
Competent bacteria must be in a certain stage of growth
(usually exponential) and secrete a small protein (competency
factor) that stimulates production of new protein required
for DNA uptake
Gene transfer by this process occurs in soils and marine
environments so it is an important route of genetic exchange
in nature
Artificial transformation- carried out in laboratory to transfer
plasmid DNA, a common method for introducing recombinant
DNA into bacterial cells. eg CaCl2 or electroporation
Transduction
Transduction is the transfer of bacterial genes by phage.
Bacterial genes are incorporated into a phage capsid due to
errors made during the virus life cycle. The virus containing
these genes then injects them into another bacteria
*most common mechanism for gene exchange and
recombination in bacteria.
Generalized Transduction- occurs during the lytic cycle of
virulent or temperate phages. When the viral chromosomes
are being packaged into phage heads, random fragments of
partially degraded bacterial chromosome may be packaged by
mistake.
Transducing particle- the phage which injects bacterial DNA
into a new recipient. Simply a carrier of genetic information
Specialized Transduction- occurs when an error is made
during the lysogenic life cycle. When a prophage in induced
to leave the chromosome inaccurate excision results in
specific bacterial genes packaged into the phage head and
subsequently transferred
Antibiotic Resistance
Resistance to antibiotics can be acquired as genes are
transferred between bacteria (eg R plasmids or transposons)
Many infections are treated with antibiotics and the increasing
number of drug resistant pathogens is a serious public
problem
Antimicrobial agents kill or stop growth of various pathogens
1. Inhibit cell wall synthesis
2. Affect protein or nucleic acid synthesis
3. Disrupt membrane structure and function
4. Block metabolic pathways
Mechanisms by which bacteria resist antibiotic treatment
1. Inactivation of antibiotic through chemical modification
2. Change in target of antibiotic to inhibit action
3. Change in permeability by reducing uptake
How do we break the cycle of antibiotic resistance?
Recombinant DNA technology
Genetic Engineering - the deliberate modification of an
organisms genome. The methods used to accomplish this
are know as recombinant DNA technology
Recombinant DNA - DNA with a new sequence formed by
joining fragments from different sources
Restriction enzymes - bacterial enzymes (endonucleases) that
recognize and cleave specific sequences (4-8 bp long).
Normally bacteria use them to destroy foreign DNA like
incoming phage DNA
Type II Restriction enzymes cleave DNA at specific recognition
site which is a palendrome (read same forward and backward)
and cleavage leaves single stranded or “sticky” ends.
Methodology used to construct a genomic library (bank of all
genes in a given organism) or express foreign genes in
bacterial cells