Microbial Genetics

By: Shimelis Teshome (BSc MLS)

 Transfer of DNA between Bacterial Cells
• Genetic exchange in bacteria is unidirectional

• The transfer of genetic information from one cell to another can occur
by three methods

1. Conjugation,
2. Transduction,

3. Transformation
• The most important consequence of DNA transfer is that antibiotic
resistance genes are spread from one bacterium to another by these
processes.
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 1. Conjugation
• Is the mating of two bacterial cells during
which DNA is transferred from the donor to
the recipient cell through tube F-Pilli (sex
pilus)
• Described by Lederberg and Tatum in 1946.
• Equivalent to sexual polarity in bacteria.
• Studied in E. coli K12 strain.

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 2. Transduction

• Bacteriophage mediated transfer of genetic

material from one bacteria to another.
• Penicillinase production in Staphylococci.
• Generalised and restricted transduction.
• Lambda phage in E.coli transfers a particular
gene present between gal and bio gene.

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 3. Transformation

 is taking up fragments of naked DNA released from dead cells in

the vicinity.
 Transformation will only happen at a specific stage in the bacterial
life cycle, when cells are in a physiological state known as
competence.
 commonly during late log phase

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 Mutation
• In the living cell, DNA undergoes frequent
chemical change, especially when it is being
replicated.
• Most of these changes are quickly repaired
those that are not result in a mutation.
• Mutation is a failure of DNA repair.

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 Mutation

• Mutant: an organism, population, gene, chromosome etc which differs

from the corresponding WILD TYPE by one or more MUTATIONS.
• Mutation result from three types of molecular changes:

• Base Substitution
• Frameshift Mutation
• Transposons or Insertion Sequences (IS)

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 Mutations
1. Base Substitution
 Occurs when one base is inserted in place of another.
 Takes place at the time of DNA replication

 Called point mutations

 Two reasons, either because :

 the DNA polymerase makes an error

 a mutagen alters the hydrogen bonding of the base being used as a
template in such a manner that the wrong base is inserted

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 Mutations
1. Base Substitution
A. Missense mutation: When the base substitution results in a
codon that simply causes a different amino acid to be inserted
 may be without discernible phenotypic effect.

Ex. sickle-cell disease:-GAG (glutamic acid) to GTG (valine)

B. Nonsense mutation: when the base substitution generates

a termination codon that stops protein synthesis prematurely
 almost always destroy protein function

Ex. (TAA, TAG, or TGA)

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 Mutations
2. Frameshift Mutation
 when one or more base pairs are added or deleted
 shifts the reading frame on the ribosome
 incorporate the wrong amino acids "downstream" from the mutation
 produce an inactive protein.

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 Mutations
3.Transposons or Insertion Sequences (IS)
 Transposable Element (TE; ‘Jumping gene’): A discrete DNA

segment, which can effectively translocate to another (target) site in the

same replicon, to a target site in another replicon in the same cell

 Encodes not only those functions necessary for transposition but also

functions that are unrelated to transposition – e.g., resistance to

antibiotics

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 Mutations

Insertion Sequence (IS Element): A transposable element which

contains no genetic information other than that necessary for its
transposition.

Transposons or Insertion Sequences (IS)

 are integrated into the DNA
 can cause profound changes in the genes into which they insert and in
adjacent genes.

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 Causes of Mutation

a. Chemicals
b. Radiation
c. Viruses

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 Causes of Mutation

a. Chemicals
 Nitrous acid (HNO2) and alkylating agents:

 alter the existing base so that it forms a hydrogen bond preferentially

with the wrong base

e.g., adenine would no longer pair with thymine but with cytosine

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 Causes of Mutation

a. Chemicals
Benzpyrene: is found in tobacco smoke, bind to the existing
DNA bases and cause frameshift mutations.
 intercalate between the adjacent bases, thereby distorting and
offsetting the DNA sequence.
 carcinogenic

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 Causes of Mutation
.
b. Radiation
 X-rays: have high energy and can damage DNA in three
ways:
a. by breaking the covalent bonds that hold the ribose
phosphate chain together
b. by producing free radicals that can attack the bases

c. by altering the electrons in the bases and thus changing their

hydrogen bonding.
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 Causes of Mutation
.
b. Radiation, cont’d
 Ultraviolet radiation
 has lower energy than X-rays
 causes the cross-linking of the adjacent pyrimidine bases
to form dimers
 e.g., cross-linking of adjacent thymines to form a thymine
dimer, results in inability of the DNA to replicate properly
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 Causes of Mutation
C. Virus
 Bacterial virus Mu (mutator bacteriophage)
 Cause a high frequency of mutations when their DNA is inserted
into the bacterial chromosome.
 Since the viral DNA can insert into many different sites, mutations
in various genes can occur
 These mutations are either frameshift mutations or deletions.

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Anti-Microbial Agents

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Introduction
• Groups of antimicrobial agents
Antibiotics- are natural substances produced
by certain groups of microorganisms as a
means of inhibiting other organisms
Semi-synthetic antibiotics
synthetic antibiotics
chemotherapeutic agents- synthetic origin
e.g. sulfonilamides, isoniazid, ethambutol,
AZT, chloramphenicol
Microbial Sources of Antibiotics
 Fleming and Penicillin

1928: Fleming discovered penicillin, produced by Penicillium notatum

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 MECHANISMS OF ACTION OF
ANTIBACTERIAL DRUGS
• Mechanism of action include:
– Inhibition of cell wall synthesis
– Inhibition of protein synthesis
– Inhibition of nucleic acid synthesis
– Inhibition of metabolic pathways
– Interference with cell membrane integrity

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Anti-Microbial resistance

Human Behavior Change

• Reasons for antibiotics resistance
 Antibiotic resistance continues to expand for a
multitude of reasons, including
Over-prescription of antibiotics by physicians
 Non-completion of prescribed antibiotic
treatments by patients
 Use of antibiotics in animals as growth
enhancers (primarily by the food industry)
 Increased international travel
 Poor hospital hygiene
 Mechanism of Resistance to Antimicrobial agents
con’ted
• Main mechanisms by which microorganisms
exhibit resistance to antimicrobials are:
1.Drug inactivation or modification: e.g. enzymatic
deactivation of Penicillin G in some penicillin-
resistant bacteria through the production of β-
lactamases.
2. Altered target sites of antibiotic : e.g. alteration
of PBP—the binding target site of penicillins—in
penicillin-resistant bacteria.
3.Alteration of metabolic pathway: e.g. some
sulfonamide- resistant bacteria do not require para-
aminobenzoic acid (PABA), an important precursor
for the synthesis of folic acid and nucleic acids in
bacteria inhibited by sulfonamides.

4. Reduced drug accumulation: by decreasing drug

permeability and/or increasing active efflux
(pumping out) of the drugs across the cell surface

= Decreased cell wall permeability to antibiotics

5. by new mutation or by gene transfer (e.g.
acquisition of a plasmid)
6. The organism may lack the structure an
antibiotic inhibits
 Mechanism of Drug Resistance
Biology of antibiotic resistance

Biochemical Genetic aspect

aspect
Antibiotic Mutation(Spontaneous
inactivation mutation ,Hyper
mutation, Adaptive
Target modification mutation)

Efflux pump Horizontal gene

transfer(Transformation
Membrane , Transduction,
permeability change Cogugation)

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 Principles of disinfection and sterilization

Decontamination:- killing, elimination, inhibition or

reduction in number of the microorganisms on
vehicles or reservoirs involved in transmission by
applying physical conditions or using chemical
agents
Disinfection:- Disinfection is the elimination or
inhibition of pathogenic microorganisms in or on an
object so that they no longer pose a threat.
• A disinfectant is a chemical agent used to disinfect
inanimate objects such as work surfaces and floors
The effect produced can be described as
• cidal, if the organisms are killed
• static if microbial growth is only inhibited
Sterilization
Kills all forms of microbial life
 Desirable but not always feasible
 Is one of the tasks that must be completed to ensure customers and
patients have
clean, sterile and complete instrument sets
 There are many types and ways to achieve sterilization for an object
Chemical sterilization :- Chemical Agents that
Damage the Cell Membrane
– Phenolic compounds
– Alcohols
Physical agents
– Heat
– Freezing and thawing
– Radiation
– Filtration
Moist heat
1. Steam sterilization
Quite efficient to eliminate
Resistant bacterial endospores
viruses
 The autoclave
=Is the most widely used method for
sterilizing heat stable material.
=High pressure is used to increase the
temperature to which the water can be
heated
2. Tyndallization
 A fractional sterilization method
 Heat to 80°-100°C for 30 minutes on 3
consecutive days
 Spores are activated during each heating cycle,
and then killed during the next cycle
3. Pasteurization (63 oC/30’ or 72 oC/15’or 141
oC/3’)

 Reduces the number of microorganisms and

kills most pathogens
 Used for milk (developed by Pasteur for wine)
4. Boiling
Not efficient to remove resistant structures and
thermophiles
Requires extremely long exposure
Dry heat
Oxidizes essential cell components
Slower and requires temperatures higher than those
used in moist heat sterilization
 Flaming
 Hot air (160 oC/2h or 170 oC for at least an hour)
 Incineration
B. Ultraviolet irradiation: mechanism
• Physical process
• Energy absorbed UV
by DNA
– pyrimidine dimers,
strand breaks, C
A
T
other damages C A A T G G
DNA
G T T A C
– inhibits replication
 Physical agents con’ted
D. Membrane Filtration
 Gases and liquids can be sterilized
Whether the effect is sterilization or merely
disinfection will depend on the pore size of the filter
 A filter pore size of 0.2 um will trap most
bacterial cells but will not trap the smaller viruses
 Removal of viruses requires ultrafiltration, which
is feasible only for highly specialized materials
 Requires high pressure because of the small pore
sizes of the membranes