Microbial Genetics

Learning Objectives
• Define chromosome, plasmid, genotype, phenotype.
• Describe how DNA serves as genetic information.
• Describe mutation – types, mutagens.
• Describe Genetic variation in bacteria / gene transfer
in bacteria.
• Describe bacteriophages

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 Microbial Genetics
• Microbial genetics: the study of inheritance and the
variability of the characteristics of microorganisms
• Information is stored in chromosomes & plasmids.
• Phenotype: the observable structural traits produced by
the interaction of genes & environment
• Genotype: genetic “make-up” of the organism
Intergrons –
– Pieces of DNA that accumulate new genes
– They are specialized elements for the expression of
antibiotic resistance genes.

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 Microbial Genetics
Structure of Genetic Material (DNA)
• Double stranded (double helix)
• Chains of nucleotides
• 5’ to 3’ (strands are anti-parallel)
• DNA is composed of basic units - nucleotides
• Nucleotides are composed of
– Nitrogenous base: Purines & Pyrimidines (Adenine,
Thymine, Guanine, Cytosine)
– Phosphate – PO4
– Sugar - Deoxyribose
• Complimentary base pairing: A-T; G-C
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RNA: Types of RNA
1. Messenger RNA (mRNA): Contains 3 bases (codon)
2. Ribosomal RNA (rRNA): Comprises the 70S ribosome
3. Transfer RNA (tRNA): Transfers amino acids to
ribosomes for protein synthesis. Contains the anticodon
• Genetic information is transferred from genes to
the proteins they encode via a “messenger” RNA
intermediate
 Microbial Genetics
Protein synthesis
Central dogma of molecular genetics
DNA -------------  mRNA ------------  protein
transcription translation
Gene
• The unit of genetic information or hereditary material
contained in DNA molecule
• Most genes have their protein-coding information
interrupted by non-coding sequences called “introns”.
The coding sequences are then called “exons”.

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 exon 1 exon 2
intron
DNA GE NE

• x
Replication fork
 Microbial Genetics
1. Replication – new copy of DNA being made
2. Transcription – gene being copied from DNA sequence
into messenger RNA (mRNA)
a. This is the process of making a copy of gene (sequence
of DNA that codes for a protein or functional product)
b. The enzyme responsible for this process is RNA
polymerase
c. Copies the gene is a 5‘→ 3’ direction
d. Gene transcription begins at a site called the Promoter &
ends at another site called the Terminator.
3. Translation – mRNA read and protein produced
 Microbial Genetics

Central dogma of molecular biology

DNA replication
 Microbial Genetics
Gene expression
• The process by which a gene's information is converted
into the structures & functions of a cell by a process of
producing a biologically functional molecule of either
protein or RNA is made.
• The separation of eucaryotic & procaryotic genomes
is illustrated by comparing their mechanisms of gene
expression.
• Gene expression is controlled by repressor or
activator protein at various points in the sequence
leading to protein synthesis.

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 Chromosome
Bacterial Chromosome
• Usually circular, super coiled, double stranded DNA
molecule attached to cell membrane.
• Located/condensed into the nucleoid.
• No nuclear membrane.
• Replicate semi-conservatively – one DNA strand acts
as template for the synthesis of the other; bi-directional
• Replication starts from a specific site – Origin of
replication (Ori-C)
• The genome is haploid (only 1 chromosome per cell)
• Chromosome size differ between species
 Chromosome
• A chromosome is subdivided in to genes (segments).
• Genes are located along chromosomes in the nucleus.
• Genes control the properties of organisms.
• Chromosomes undergo replication prior to cell division.
• When the cell divides, each cell received an identical set
of chromosomes and genes.
 Plasmid
• Are extrachromosomal DNA elements capable of
autonomous replication.
• Smaller (<5% of the size) but similar to chromosomes,
Circular dsDNA – exept in B. burgdorferi (linear), inherited
by daughter cells
• More than 1 plasmid found in a bacterium
• Plasmids are most commonly found in gram-negative
bacteria
• Episome: a plasmid capable of integrating with the
bacterial chromosome.
• Plasmids can be removed from the host cell in the process
of curing.
 Plasmids
• Curing may occur spontaneously or may be induced by
treatments such as UV light, acridine dyes, ionizing
radiation, thymine starvation, growth above optimum
temperature.
Phenotypic properties of plasmids:
1.They are responsible for virulence (e.g. E. coli)
2.Antibiotic resistance
3.Production of antimicrobials (antibiotic, bacteriocins)
4.Metabolic pathway: Pseudomonas spp - catabolic
activity for salicylic acid
5.They are specific to 1 or a few particular bacteria
 Plasmid

Plasmid
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 Plasmid
Classification of Plasmids: Types of plasmids based on
I. Transfer properties (on their ability to transfer)
a. Conjugative plasmids
b. Non-conjugative plasmids
1. Conjugative plasmids (F factor):
– Self transmissible from one cell to another, have sex pili.
• These plasmids have 2 components
a.R Factors - associated with the transference of antibiotic
resistance
b.Resistance transfer factor (RTF) which initiates &
controls the conjugative transfer b/n cells.
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 Plasmids
• Have genes for construction of pili & can transfer copies of
plasmids to other during conjugation.
a. F or fertility factor: Promotes transfer of chromosome at
a higher frequency of recombination.
• F negative (F-): cells not containing the fertility factor
• F positive (F+)- F+ (free plasmid): male cells which effect
gene transfer
b. Hfr (high frequency of recombination): When F factor exists
in an integrated state with host chromosome
– The r determinant genes that confer resistance to specific
drugs, e.g – S. dysenteriae – to chloramphenicol,
sulfonamide, streptomycin
 Plasmid
2. Non conjugative
– Unable to initiate self transfer and don’t encode for a
sex pilus.
– Their transfer is mediated by co-resident conjugative
plasmids by the process of mobilization, E.g.
• From gonococcus to gonococcus.
• They are able to transfer the entire chromosome
of the cell. They arise from F+ cells.
 Plasmids
II. Types plasmid based on function
1. Fertility plasmid (F factor)
– Carry the fertility genes (tra-genes) for conjugation
– The transfer of genetic information is between 2 cells
2. Resistance plasmids (R plasmids)
– Contain genes that can build resistance to antibiotics
or poisons
– have genes that code for enzymes capable of
modifying antibiotics.
– Often resistance genes are within mobile genetic
materials known as transposones
 Plasmids
3. Bacteriocinogenic plasmids (Col plasmids)
– Contain genes that encode for the antibacterial
polypeptides called bacteriocines
– Contains genes for the synthesis of bacteriocins or
colicins (extracellular toxins or proteins) that inhibit
strains of same & different spp of bacteria
4. Virulence plasmids:
• Turn a bacterium into pathogen
5. Metabolic or Degradative plasmids
• Degrade or digest the dead organic matter from dead
animals or plants
 Transposons (Tn)
• Transposons are sequences (segments) of DNA that
can move around to different positions (b/n chromosome
& plasmids) within the genome of a single cell, a process
called transposition.
• These mobile segments of DNA are sometimes called
"jumping genes".
• Transposons are not self replicative, they depend on
chromosomal or plasmid DNA for replication.
• Transposable elements can cause genetic changes, and
have been involved in the evolution of both prokaryotic &
eukaryotic genomes
• In the process, they may cause mutations.
 Transposons
• The involvement of relatively short transposons (750-
2000 bp long), known as insertion elements (also called
insertion sequences, IS), produces the majority of
insertion mutations.
• Importance - Many antibiotic resistance genes are
located on transposons.
• Since transposons can jump from one DNA molecule to
another, these antibiotic resistance transposons are a
major factor in the development of plasmids which can
confer multiple drug resistance on a bacterium
harboring such a plasmid.
 Transposons
• Transposase: an enzyme that binds to the ends of
transposon & catalyses the movement of the transposon
to another part of the genome by a cut & paste
mechanism or a replicative transposition mechanism.
• There are 2 distinct types of transposons on the basis
of their mechanism for movement:
1.Class I transposons (retrotransposons):
– Encode reverse transcriptase for making DNA
copies of RNA transcripts;
– New DNA copies integrate at different sites.
– This type is found only in eukaryotes
 Transposons
2. Class II transposons (DNA transposons)
– Transposons consisting only DNA that moves directly
from place to place
• Encode proteins that move the DNA directly to a new
position or replicate the DNA to produce a new
element that integrates elsewhere.
• This type is found in both prokaryotes & eukaryotes.
Bacteria contain 2 types of transposons
1.Composite mobile genetic elements that are larger
than IS elements and contain 1 or more protein-coding
genes in addition to those required for transposition
2. Non-composite mobile genetic elements are those
which lack IS elements on its end, E.g is Tns
• Insertion sequence (IS element): a short DNA
sequence that acts as a simple transposable element.
Class I transposons (retrotransposons) that:
– First transcribe the DNA into RNA, then use RT to make
DNA copy of the RNA to insert in new location
 Transposons
• x

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 Mutation
Genetic variation
Genetic variation in bacteria
– Bacterial variation may be Phenotypic or genotypic
Genetic variation is produced in 2 ways:
– Through Mutation: heritable changes in DNA sequence
– Through Gene transfer: acquiring genes from another
member of a spp
Phenotypic variations:
– Are non-hereditary & reversible which occur under the
influence of environment; e.g. Loss of flagella by
treatment with phenol; variation in culture (rough &
smooth); due to enzyme action, etc.
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 Genetic variation
• Mutation: Defined as a rare random, discontinuous,
heritable variation in the coded message of DNA molecule.
• It is a change in the genetic material/DNA (i.e. nucleotide
base sequence of a genome.
• Flow of information within a cell involves transcription of
DNA to mRNA & the translation of mRNA to protein.
• Mutation is brought about by chemical alteration in DNA
• Rare event: Mutation occurs at 1 out of 106-1012 bacterial
population
• Mutants are variants in which 1 or more bases in their
DNA are changed.
Mutation

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Effects of mutations
• This change is lethal, heritable & irreversible, but
sometimes repair mechanism may occur depending on
the extent of the original mutation.
• Rarely leads to a protein that improves ability of
organism to survive
• Mutations affect morphology, nutritional
requirements, susceptibility to antibiotics &
bacteriophage.
• Mutation can be: Harmful, Lethal, Helpful, Silent
• Almost always deleterious
• Significance of mutations
1. Most are neutral
– Eye color
– Birth marks
2. Some are harmful
– Sickle cell anemia
– Down syndrome: Chrom 21 doesn’t separate
3. Some are beneficial
– Sickle cell anemia to malaria
– Immunity to HIV
 Mutation
Mutation can be
1. Spontaneous - Due to shift of electrons in Pu/Py
(natural; occur in the absence of a mutagen)
2. Induced – Physical & chemical agents induce mutation

Causes of mutation
• Mutagens: Physical & chemical agents that change
DNA
Mutagens can be:
1. Physical – Radiation (UV, X-ray, ionizing)
2. Chemical – 5-bromouracil, nitrous acid
• Overview of some of the major chemical & physical
mutagens & their modes of action
 Mutation

Types of mutations
I. Gene mutations: the allele of a gene changes
• Point mutations: Base substitution - Can be
– Silent Mutations
– Non-sense Mutations
– Missense Mutations
• Substitution, Insertion, Deletion
II. Chromosomal mutations
• Segment of chromosome or whole chromosome or
entire sets of chromosomes change

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 Mutation
I. Point mutation (substitution):
• The most common type of mutation.
• A single base at 1 point in the DNA sequence is inserted,
deleted, substituted by another base.
• Involve changing a single nucleotide base or a few base
pairs in a single gene
• Such mutations cannot be detected without sequencing the
gene (or its mRNA).
Two kinds of base pair substitution –
a. Transition – Purine /pyrimidine orientation preserved,
E.g. GC  change to AT
b. Transversion – Pu/Py orientation altered, e.g GC  CG
 – Base Pair Insertions and deletions
• Triplet Repeats
• Frame Shift mutations

Triplet Repeats
• Change of one base to 1 of the other 3 bases as a result
of error during replication - results in change in the triplet
of the code.
• Occurs when DNA is not copied correctly and a segment
is repeated. E.g. Huntington Disease - CAG Repeat
 Mutation
• Point Mutations / Base Pair Substitutions can be:
• Silent mutation - causes no change in the activity
of the protein.
• Missense mutation – new protein (amino acid
substitutions). A nucleotide substitution that changes
a codon so that it codes for a different amino acid in
the protein.
– E.g. CG changed by TA – Ser for Gly (1 Amino
acid substituted for another)
• Nonsense Mutation - The same as a missense
mutation except the resulting codon codes for a
STOP signal (TAA, TAG, or TGA).
 Mutation
• Therefore, translation of the mRNA transcribed
from this mutant gene will stop. The result is a
premature termination of translation.
• The protein is shorter than usual &doesn’t contain all
the amino acids that it should = protein is most likely
nonfunctional.
Frame shift mutation – shifts the translational reading
frame - a type of point mutation caused by:
1.Insertion – An additional base alters the reading frame
2.Deletion of a base pair - Loss of a base,

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• An inserted or deleted nucleotide alters the triplet
grouping of nucleotides into codons & shifts the reading
frame so that all nucleotides downstream from the
mutation will be improperly grouped. The result is a
protein with extensive missense ending sooner or later in
nonsense.
• Frame shift insertions and deletions can lead to
missense, same sense & non-sense mutations
• The result is a protein with extensive missense ending
sooner or later in nonsense.
• Frame shift insertions and deletions can lead to
missense, same sense & non-sense mutations.
Mutation

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 Chromosomal mutations
• Changes in number & structure of entire chromosomes
• Sometimes, chromosomes fail to separate during 1 st or 2nd
stages of meiosis
Mutations involving changes in chromosome structure
occur in 4 common types:
a. Deletions: Eg. AC - DEF
b. Duplications: Occurs during crossing over and one
chromosome ends up with more genes than it received
c. Inversions (changing orientation of a DNA segment): A
reversal in the order of a segment of a chromosome
d. Translocations: When one piece of a chromosome breaks
off & attaches to another chromosome
 Mutation

Identification of mutants
Ames test
• The test employs a sensitive bacterial assay system for
detecting chemical mutagens in the environment
• It is used to test whether a particular substance can
induce mutation or not
• Special strains of salmonella that have lost their ability to
synthesize amino acid histidine are used in this test
• If a substance is a mutagen, it will increase the rate at
which these organisms revert to histidine synthesizers.

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• Significance of mutations
 Mutation

Ames Test for Chemical Carcinogens

 Mutation
Mutant Isolation
• How can you tell if there are any mutant colonies in a
culture?
• By either positive (direct) or negative (indirect) selection
• Penicillin selection – penicillin kills growing but not
mutant (non growing) cells in minimal medium
• Nutritionally defective mutants can also be detected by
the technique of replica plating
Positive Selection:
• Positive selection entails growing the culture on media
that will allow for the growth of only the mutant colonies.
 Mutation
– If, for example, we want to find mutants that resistant to
penicillin, we grow the culture on a medium that contains
penicillin. Only those colonies that are resistant to
penicillin will grow and we ca identify them directly.
Negative Selection:
• Negative selection is used to identify mutants that have
lost the ability to perform a certain function that their
parents had.
– Auxotrophic mutants, for example, are bacteria that
have lost the ability to synthesize an essential nutrient
• The replica-plating technique is used to identify
mutants by negative selection.
Kinds of mutants
 Gene Transfer in Bacteria
Genetic recombination
• A process by which genetic information is re-assorted
(exchanged) between chromosomes.
• Recombination involves the cutting & covalent joining of
DNA sequences (homologous, non-homologous).
Homologous Recombination
• In bacteria, bacterial recombination takes place by
• Transformation
• Transduction
• Conjugation.
• Genetic transfer-results in genetic variation
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 Gene Transfer in Bacteria

Recombination can occur between

• 2 different DNA molecules (intermolecular
recombination) or
• 2 regions of a single DNA molecule (intramolecular
recombination).
• Intermolecular recombination can occur with DNA
templates: the DNA templates may be linear DNA
acquired via transduction, transformation, conjugation or
linear chromosomes / plasmids in bacteria
• The recombination may occur between linear & circular
dsDNA or between 2 circular dsDNA templates
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 Gene Transfer in Bacteria
How do bacteria become resistant?
• Bacteria can gain resistance over time through:
• Acquired resistance
• Vertical gene transfer
• Horizontal gene transfer
Acquired resistance
• Either modification of existing genetic material or
acquisition of new genetic material from another source.
• Development of resistance through spontaneous
mutation is called primary resistance.
– Errors in DNA synthesis during replication
– Occasional failures in the DNA repair systems
 Gene Transfer in Bacteria

Vertical Gene Transfer

• Genes directly transferred from parent to offspring
(progeny) during DNA replication
• Resistant genes are developed
• This is directly related to Darwin’s theory of evolution.
Horizontal gene transfer (HGT)- “Lateral gene transfer”
• Lateral transfer to same generation - process of
swapping genetic material between neighboring bacteria
• Genetic material is contained in small packets of DNA.
• Packets can be transferred between individual bacteria
of the same or different spp
 Gene Transfer in Bacteria

Gene transfer in bacteria: 3 Mechanisms of HGT

1. Transformation
• Uptake of “naked” DNA from the environment into its
chromosome.
• 1%, random, occur naturally in certain spp, e.g. Bacillus,
Neisseria, Haemophilus, Pseudomonas, Streptococcus
(pneumococcus), Staphylococcus
• When fragments of exogenous bacterial DNA are taken
up and absorbed into recipient cells from the external
environment due to the death of other bacteria
 Transformation

Transformation

Donor cell

Cell lysis
and free DNA

Recipient cell
Transformation
Transformation
 Gene Transfer in Bacteria

2. Transduction
• Bacteriophage-mediated transfer of genetic material
results from the infection of a cell with a virus that
contain DNA from bacteria.
• DNA or plasmid DNA is transferred from one bacterium
to another by a virus or viral vector.
• Transduction happens through either the lytic cycle or
the lysogenic cycle.
• Phages are virulent or temperate
e.g. Staphylococci, Shigella, Salmonella, E. coli

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• Transduction
 Gene Transfer in Bacteria

bacterial Transduction
cell

lytic cycle
phage
transduced
cell

transducing
phage

• Transduction
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BACTERIOPHAGES (phages)
• Bacteriophages: groups of viruses that infect bacteria
composed of either RNA or DNA & are highly host-
specific.
• They are obligate intracellular parasites that multiply
inside bacteria by making use of some or all of the host
biosynthetic machinery.
• A typical phage particle consists of a head & tunnel tail.
• The head represents a tightly packed core of nucleic
acid (DNA or RNA) surrounded by capsid (protein coat)
• The viral DNA is injected through the tail into host cell
• Every known type of bacterium may serve as host to one
or more phages.
 Bacteriophage
Viruses that Infect Bacteria
• Bacteriophages (Robot virus) are viruses that infect
bacteria
• Bacteriophage are excellent model for other bacteria.
• Bacteriophage contain dsDNA, ssDNA, RNA; most are
dsDNA viruses.
• At present, over 5000 bacteriophages studied by
electron microscopy divided into 13 virus families.
• Temperate phages - special DNA phages that undergo
adsorption & penetration but are not replicated or
released immediately.
• Instead the viral DNA enters an inactive prophage stage

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 Bacteriophage
• Types of Bacteriophage: 2 possible outcomes
following viral infection of a bacterial cell
1. Lytic Infection (virulent phage): viruses multiply inside
the cells they invade. Phage that can only multiply within
bacteria & kill the cell by lysis (e.g. T4).
2. Lysogenic Infection (temperate phage): integrate viral
DNA into host cell chromosome; the virus DNA
replicates as the bacterial chromosome replicates.
– They have a protein tail piece with tail fibers that attach
to the bacterium.

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 Bacteriophage
Replication of lytic phage
 Adsorption → Penetration → Replication → Maturation
→ Release → Reinfection
• Phage nucleic acid enters the bacterium & capsid
remains outside
• Nucleic acid replicated along with phage proteins

• Many virions are formed.

• Phages exit by bursting the cell

• Phages that go through this life cycle are called virulent.

• Virulent: has the ability to overcome host defenses and

cause disease
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 Bacteriophage
• At present, over 5000 bacteriophages have been studied
by EM and can be divided into 13 virus families.
• Some:
 T4 (lytic phage)
 Lambda (temperate phage)
• T4 has a DNA core within a protein coat, and tail with tail
fibers to attach to bacteria
• Composition & structure of bacteriophage
 Head – capsid & DNA
 Tail – with fibers to attach to bacteria

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 Bacteriophage

Structure of bacteriophage
Bacteriophage  65
 Bacteriophages
• Phages are used in the lab in studying host–parasite
relationships & virus multiplication.

Phage multiplication cycle (Life cycle)

• There are at least 3 phases in the life cycle of a
bacteriophage:
– Attachment
– Replication
– Release
 Bacteriophages

Structure of T4 bacteriophage
 Bacteriophages
– The phage nucleic acid is called vegetative phage.
A. Lytic or virulent infection
– The injected vegetative phage material may be
reproduced forming many replicas  mature  Lysis of
host cells  free phages liberated.
B. Lysogenic or Temperate Phage: Prophage
– Temperate bacteriophage:  one whose genetic
material (prophage) becomes an intimate part of the
bacterial genome
– The affected bacterial cell is known as a
lysogenic bacterium.
 Bacteriophages
– Temperate phages fail to lyse the cells they infect (lytic
cycle are not completed)
– They are reduced to prophage as alternative to
producing a lytic infection.
– The bacterium is now lysogenic.
 Its progency may later lyse & liberate infective phage

Loss of prophage
– Occasionally a lysogenic bacterium may lose its
prophage, remaining viable as an uninfected cell
 Bacteriophages
Methods of Study
• Culture
– Plaque assay: Phage particles when added into
dividing bacteria in nutrient agar plate will produce clear
zone of lysis (plaque) which results from the lysis of
bacteria
– Lytic phage are enumerated by a plaque assay.
 Bacteriophages

Bacteriophage life cycle

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 Gene Transfer in Bacteria

3. Conjugation
– Plasmids that enable bacterial conjugation encode a
pilus that is expressed from the donor cell and binds to
the recipient cell, mediating DNA transfer [between
bacterial cells by direct cell-to-cell contact or by a
tubular bridge-like connection between two cells].
– This is the major way of gene transfer in nature.
– Donor cell (male)  recipient (fem) bacteria conjugative
plasmids (sex, or F – factor)

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x Sex pilus
 Gene Transfer in Bacteria

• Conjugation
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Hfr conjugation (High frequency recombination)
• Hfr bacteria conjugate like F+ do, but they drag a copy of
the entire chromosome into the F- cell.
 Gene Transfer in Bacteria

Conjugation
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 Gene Transfer in Bacteria

– It is the main mode of horizontal gene transfer that

spreads antibiotic resistance among bacteria
– E.g. In E. coli.

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 Gene Transfer in Bacteria

Bacterial acquisition of resistance genes

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