Microbial Genetics-2023

Microbial Genetics
A.H.M. Nurun Nabi, PhD

Professor
Laboratory of Population Genetics
Department of Biochemistry and Molecular Biology
University of Dhaka
Dhaka-1000, Bangladesh.
E-mail: nabi@du.ac.bd
A. H. M. Nurun Nabi 02/04/2023
Microbial Genetics
Genetics is the study of heredity which is concerned with

how:
• information in nucleic acids is expressed

• nucleic acids are duplicated and transmitted to progeny
• these processes account for the characteristics of
progeny

Nucleic Acids
Made of monomer building blocks called

nucleotides.

What is DNA ???
DNA is a double stranded structure like a twisted

ladder. It is embedded in the nucleus of
eukaryotic cell but in prokaryotic it is lying in
cytoplasm because of the absence of nucleus.

• The basic structural units of DNA are nucleotides.
– Nucleotides form a gene

– Genes form DNA
– DNA forms chromosome
• Nucleotides are complex structures composed of three kinds of

molecules as
1. Nitrogenous Base
2. Deoxyribose sugar
3. Phosphate group
Deoxyribonucleic Acid
Is responsible for all cellular activity.
Directs the production of proteins.
Is double stranded and helical.
Is maintained by hydrogen bonds

(weak bonds)
Is very stable and can survive

• Temperatures as high as 70OC
• High salt concentrations
• Acid environments
Structure of

Chargaff's rules
One other key piece of information related to the structure of DNA came from
Austrian biochemist Erwin Chargaff. Chargaff analyzed the DNA of different
species, determining its composition of A, T, C, and G bases.
He made several key observations:

•A, T, C, and G were not found in equal quantities (as some models at the time
would have predicted)
•The amounts of the bases varied among species, but not between individuals of
the same species
•The amount of A always equalled the amount of T, and the amount of C always
equalled the amount of G (A = T and G = C)
These findings, called Chargaff's rules, turned out to be crucial to Watson and
Crick's model of the DNA double helix.
In the early 1950s, American biologist James Watson and British physicist Francis Crick
came up with their famous model of the DNA double helix. T
The structure of DNA, as represented in Watson and Crick's model, is a double-stranded,
antiparallel, right-handed helix. The sugar-phosphate backbones of the DNA strands make
up the outside of the helix, while the nitrogenous bases are found on the inside and form
hydrogen-bonded pairs that hold the DNA strands together.
In the model shown, the orange and

red atoms mark the phosphates of the
sugar-phosphate backbones, while the
blue atoms on the interior of the helix
belong to the nitrogenous bases.

Antiparallel orientation
Double-stranded DNA is an antiparallel molecule, meaning that it's composed of
two strands that run alongside each other but point in opposite directions. In a
double-stranded DNA molecule, the 5' end (phosphate-bearing end) of one
strand aligns with the 3' end (hydroxyl-bearing end) of its partner, and vice versa.

Right-handed helix
In Watson and Crick's model, the two strands of DNA twist around each other to form a right-
handed helix. All helices have a handedness, which is a property that describes how their grooves
are oriented in space.
The twisting of the DNA double helix and the

geometry of the bases creates a wider gap (called
the major groove) and a narrower gap (called
the minor groove) that run along the length of the
molecule, as shown in the figure above. These
grooves are important binding sites for proteins that
maintain DNA and regulate gene activity.

Base pairing
In Watson and Crick's model, the two strands of the DNA double helix are held together by
hydrogen bonds between nitrogenous bases on opposite strands. Each pair of bases lies flat,
forming a "rung" on the ladder of the DNA molecule.
Base pairs aren't made up of just any combination of bases. Instead, if there is an A found on one
strand, it must be paired with a T on the other (and vice versa). Similarly, an G found on one strand
must always have a C for a partner on the opposite strand. These A-T and G-C associations are
known as complementary base pairs.

Proof that DNA is a genetic material
Frederick Griffith: Bacterial transformation
In 1928, British bacteriologist Frederick Griffith conducted a series of

experiments using Streptococcus pneumoniae bacteria and mice. Griffith
wasn't trying to identify the genetic material, but rather, trying to develop a
vaccine against pneumonia. In his experiments, Griffith used two related
strains of bacteria, known as R and S.
•R strain. When grown in a petri dish, the •S strain. S bacteria formed colonies that were
R bacteria formed colonies, or clumps of rounded and smooth (hence the abbreviation
related bacteria, that had well-defined "S"). The smooth appearance was due to a
edges and a rough appearance (hence polysaccharide, or sugar-based, coat produced
the abbreviation "R"). The R bacteria by the bacteria. This coat protected the S
were nonvirulent, meaning that they did bacteria from the mouse immune system,
not cause sickness when injected into a making them virulent (capable of causing
mouse. disease). Mice injected with live S bacteria
A. H. M. Nurun Nabi developed pneumonia and died. 02/04/2023
As part of his experiments, Griffith tried injecting mice with heat-killed S
bacteria (that is, S bacteria that had been heated to high temperatures,
causing the cells to die). Unsurprisingly, the heat-killed S bacteria did not
cause disease in mice.
The experiments took an unexpected turn, however, when harmless R

bacteria were combined with harmless heat-killed S bacteria and injected
into a mouse. Not only did the mouse develop pnenumonia and die, but
when Griffith took a blood sample from the dead mouse, he found that it
contained living S bacteria!

Experiment by
Frederick Griffith

Avery, McCarty, and MacLeod: Identifying the transforming principle
In 1944, three Canadian and American researchers, Oswald Avery, Maclyn McCarty, and Colin
MacLeod, set out to identify Griffith's "transforming principle."
To do so, they began with large cultures of heat-killed S cells and, through a long series of
biochemical steps (determined by careful experimentation), progressively purified the
transforming principle by washing away, separating out, or enzymatically destroying the other
cellular components. By this method, they were able to obtain small amounts of highly purified
transforming principle, which they could then analyze through other tests to determine its identity.

Several lines of evidence suggested to Avery and his colleagues that the
transforming principle might be DNA
•The purified substance gave a negative result in chemical tests known to

detect proteins, but a strongly positive result in a chemical test known to
detect DNA.
•The elemental composition of the purified transforming principle closely

resembled DNA in its ratio of nitrogen and phosphorous.
•Protein- and RNA-degrading enzymes had little effect on the transforming

principle, but enzymes able to degrade DNA eliminated the transforming
activity.

In the mid-twentieth century, scientists were still unsure as to
whether DNA or protein was the genetic material of the cell
•It was known that some viruses consisted solely of DNA and
a protein coat and could transfer their genetic material into
hosts

In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to

prove that DNA was the genetic material.
• Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order
to radioactively label a specific viral component
• Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is

present in proteins but not DNA)
• Viruses grown in radioactive phosphorus (32P) had

radiolabeled DNA (phosphorus is present in DNA but not proteins)

The viruses were then allowed to infect a bacterium (E. coli) and then
the virus and bacteria were separated via centrifugation
•The larger bacteria formed a solid pellet while the smaller viruses
remained in the supernatant
The bacterial pellet was found to be radioactive when infected by

the 32P–viruses (DNA) but not the 35S–viruses (protein)
•This demonstrated that DNA, not protein, was the genetic material
because DNA was transferred to the bacteria

Hershey and Chase experiment

Central Dogma Theory
The central dogma theory of
molecular biology is represented
by a simple pathway:
DNA—>RNA-->protein, which
demonstrates the flow of genetic
information in a living cell.
The major processes involved in

this pathway are replication,
transcription, and translation.

In DNA replication, the DNA polymerase

enzyme replicates all the DNA in the
nuclear genome in a semi-
conservative manner,
meaning that the double stranded DNA
is separated into two and a template
is made by DNA polymerase.
This allows genomic material to be

duplicated so it can be evenly
partitioned between two somatic
cells (daughter cells) upon division.

The process in which DNA is copied into RNA by

RNA Polymerase is called transcription.
Three forms of RNA are produced here: messenger

RNA (mRNA), ribosomal RNA (rRNA), and transfer
RNA (tRNA).

Central Dogma Theory Summary
1. DNA guides the synthesis of mRNA which

in turn directs the order in which amino
acids are assembled into proteins.
2. DNA directs its own replication by giving

rise to two
complete, identical DNA molecules.
This replication is necessary because each cell

must inherit a complete set of all genes in
order to carry out the cell’s life processes.

DNA Replication in Bacteria
Bacteria contain 1 chromosome
Many contain plasmids
When bacterial chromosomes replicate both strands are

duplicated. Each strand functions as a template.
During replication, enzymes known as polymerases

transport nucleotides from the cytoplasm that are
complimentary to the template and fit them into place,
resulting in two strands, one parental and one new one

DNA Replication in Bacteria
During replication, enzymes known as

polymerases transport nucleotides from
the cytoplasm that
are complimentary to the template and fit
them into place, resulting in
two strands, one parental and one new
one.
The replication is said to be semi-

conservative because the parental strand
is conserved (remains the same ).

The process of bacterial chromosome/DNA replication initiation and
replisome assembly. The scheme presents replication initiation and
replisome assembly at chromosomal E. coli origin, oriC.
The DNA replication initiation starts with binding a replication initiator DnaA
to the DnaA boxes (Stage I).
Origin Recognition Complex (ORC) formation induces local destabilization

and pre-Replication Complex (pre-RC) formation and melting of the DNA
Unwinding Element (DUE) region (Stage II).
Then, assisted by replication initiators and the DnaC helicase loader, the
DnaB helicase is recruited and loaded onto the single-stranded DUE
(Stage III).
Association of DnaG primase triggers the release of helicase loader,

helicase activation and primers synthesis (Stage IV).
Next, the holoenzyme of DNA Polymerase III, which comprises clamp

loader, DNA Polymerase III core (Pol III core), and β-clamp is assembled
and conducts DNA synthesis (Stage V).

The experiment done by
Meselson and Stahl

Meselson & Stahl first grew bacteria for several generations in a medium containing
only 15N ("heavy" nitrogen).
When examined in an analytical centrifuge, DNA isolated from these bacteria produced a
single "heavy" band. Meselson & Stahl then transferred a portion of the culture to a new
medium that contained only 14N ("light" nitrogen).
When DNA was isolated from these bacteria after one generation, they observed a single
band that was "lighter" than the one obtained before; the "heavy" band was not observed
in these bacteria.
When DNA was isolated from the same culture after two generations, they observed two
distinct bands of equal intensity, one with the same weight as seen in the previous
experiment, and a new one still "lighter." When DNA was isolated from the same culture
after three generations, this lightest band became the predominant one, and the middle
band faded.

Meselson & Stahl reasoned that these experiments showed that DNA replication was semi-conservative:
The DNA strands separate and each makes a copy of itself, so that each daughter molecule comprises one
"old" and one "new" strand. Bacteria grown in "heavy" Nitrogen have been labeled on both strands entirely
with "heavy" Nitrogen.
After one generation in "light" Nitrogen, all of the DNA molecules comprise one "old heavy" and one "new
light" strand, and have the same "heavy / light" molecular weight, which is less than that of "heavy / heavy"
molecules.
After two generations in "light" medium, the "heavy" and "light" strands separate, and both replicate with
"light" nitrogen. Half therefore become "light / light", and half become "heavy / light" as in the previous
experiment. In each successive generation, the proportion of “heavy” strands is reduced by half, and the
“heavy / light” band gradually fades.

Steps of DNA Replication
• DNA unwound with enzyme (replication fork)
• Complementary bases added to template (parent strand) using

enzyme
• Replication fork moves down strand
• Newly replicated DNA rewinds
• Process called Semiconservative Replication

Steps of DNA Replication
Copied in 5’ to 3’ direction Polymerase can only add nucleotides to 3’ end
In Prokaryotes, replication begins at specific site in chromosome called the origin of

replication
Replication of DNA begins a specific site on the DNA template termed the origin and
proceeds in both directions from the
origin until nuclear division and cytokinesis take place.
Replication speed = 1000 nucleotides/sec

RNA Synthesis in Bacteria
Transcription is the synthesis of RNA and involves the
assembly of nucleotides by an enzyme, RNA
polymerase.
1. RNA polymerase binds to DNA at a promoter site

near the gene to be transcribed.
2. RNA polymerase travels the length of the DNA

using it as a template to duplicate.
3. The RNA polymerase continues until it reaches a

termination site at which time the transcription is
complete.

Transcription uses one of the two
exposed DNA strands as a template;
this strand is called the template
strand. The RNA product is
complementary to the template
strand and is almost identical to the
other DNA strand, called
the nontemplate (or coding) strand.
However, there is one important
difference: in the newly made RNA,
all of the T nucleotides are replaced
with U nucleotides.
The site on the DNA from which the first RNA nucleotide is transcribed is called
the +1+1plus, 1 site, or the initiation site. Nucleotides that come before the
initiation site are given negative numbers and said to be upstream. Nucleotides
that come after the initiation site are marked with positive numbers and said to
be downstream.
Micrograph of gene transcription of ribosomal RNA
illustrating the growing primary transcripts. "Begin"
indicates the 5' end of the coding strand of DNA, where
new RNA synthesis begins; "end" indicates the 3' end,
where the primary transcripts are almost complete.

Termination in bacteria
There are two major termination strategies found in bacteria:
Rho-dependent and Rho-independent

In Rho-dependent termination, the RNA contains a binding site for
a protein called Rho factor. Rho factor binds to this sequence and
starts "climbing" up the transcript towards RNA polymerase.
When it catches up with the polymerase at the transcription bubble, Rho

pulls the RNA transcript and the template DNA strand apart, releasing the
RNA molecule and ending transcription. Another sequence found later in
the DNA, called the transcription stop point, causes RNA polymerase to
pause and thus helps Rho catch up.

Rho-independent termination depends on specific sequences in the DNA template
strand. As the RNA polymerase approaches the end of the gene being transcribed, it hits
a region rich in C and G nucleotides. The RNA transcribed from this region folds back on
itself, and the complementary C and G nucleotides bind together. The result is a stable
hairpin that causes the polymerase to stall.

In a terminator, the hairpin is followed by a stretch of U
nucleotides in the RNA, which match up with A nucleotides in the
template DNA. The complementary U-A region of the RNA
transcript forms only a weak interaction with the template DNA.
This, coupled with the stalled polymerase, produces enough
instability for the enzyme to fall off and liberate the new RNA
transcript.

Protein Synthesis in Bacteria
Protein synthesis is carried out in the cytoplasm.
It begins with DNA duplication by mRNA

(Transcription)
mRNA then migrates to the ribosome where tRNA

transfers information from mRNA to rRNA
(Translation).

Protein synthesis is continuous and takes place

in three stages:
1. Initiation
2. Elongation
3. Termination

Initiation
The beginning of protein synthesis starts

methionine which is the start codon.
Start codon is known as formylmethoinine (f-

met).
It is coded as AUG.

Initiation
The initiation of protein synthesis begins with the formation of an initiation complex.
In E. coli, this complex involves the small 30S ribosome, the mRNA template, three
initiation factors that help the ribosome assemble correctly, guanosine triphosphate
(GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-
methionine (fMet-tRNAfMet).
The initiator tRNA interacts with the start codon AUG of the mRNA and carries a
formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at
the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E.
coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-
Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts
through complementary base pairing with the rRNA molecules that compose the
ribosome.
This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA
template. At this point, the 50S ribosomal subunit then binds to the initiation complex,
forming an intact ribosome.
Elongation
In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms
three functionally important ribosomal sites: The A (aminoacyl) site binds incoming
charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino
acids that have formed peptide bonds with the growing polypeptide chain but have not
yet dissociated from their corresponding tRNA.
The E (exit) site releases dissociated tRNAs so that they can be recharged with free
amino acids. There is one notable exception to this assembly line of tRNAs: During
initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the
P site directly without first entering the A site, providing a free A site ready to accept the
tRNA corresponding to the first codon after the AUG.

Elongation
 Elongation proceeds with single-codon movements of the ribosome each called a translocation
event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P
site, and then finally to the E site for removal.
 Ribosomal movements, or steps, are induced by conformational changes that advance the
ribosome by three bases in the 3′ direction. Peptide bonds form between the amino group of the
amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the
P-site tRNA.
 The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based
ribozyme that is integrated into the 50S ribosomal subunit.
 The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the
ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the
amino acid, and is expelled.
 Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A
site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific
elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add
each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination
 The termination of translation occurs when a nonsense codon (UAA,

UAG, or UGA) is encountered for which there is no complementary tRNA.
 On aligning with the A site, these nonsense codons are recognized by release
factors in prokaryotes and eukaryotes that result in the P-site amino acid
detaching from its tRNA, releasing the newly made polypeptide.
 The small and large ribosomal subunits dissociate from the mRNA and from
each other; they are recruited almost immediately into another translation
initiation complex.

Translation in Bacteria
Property Bacteria
70S
Ribosomes •30S (small subunit) with 16S rRNA subunit
•50S (large subunit) with 5S and 23S rRNA subunits
Amino acid carried by
fMet
initiator tRNA
Shine-Dalgarno sequence
Present
in mRNA
Simultaneous transcription
Yes
and translation

BACTERIAL CONJUGATION, BACTERIAL TRANSDUCTION, BACTERIAL
TRANSFORMATION
• In transformation, a bacterium takes up a piece of DNA floating in its

environment.
• In transduction, DNA is accidentally moved from one bacterium to

another by a virus.
• In conjugation, DNA is transferred between bacteria through a tube

between cells.

DNA Mutation

Changes in the DNA molecules can Cause Mutations
One base pair is exchanged for another in the DNA molecule
One or more base pairs are inserted in the DNA molecule.
One or more base pairs are deleted in the DNA molecule
There is a rearrangement of sections in the DNA molecule.
There is an exchange of DNA region with another DNA molecule (Recombination).
Some mutations harmful, some beneficial, some neutral

Recombinant DNA Technology

Techniques used in recombinant DNA Technology

Vectors used in recombinant DNA Technology

Plasmid Vectors

Lambda Phage Vector

Cosmid Vectors

Expression Vectors

Yeast Artificial Chromosomes (YACS)

Bacterial Artificial Chromosomes (BACS)

Advantages of BAC Technology
BAC has been a promising tool that should greatly facilitate our ability to manipulate
gene expression in oncolytic virus construction. It showed many advantages compared
with traditional cloning vectors.
•BACs can accept transgenes up to 300 kb in length, which is sufficient to allow the
genomes of oncolytic viruses to be maintained. What's more, the large size of BAC helps
to minimize site of integration effects.
•Endogenous gene expression more accurately than other cloning systems.
•BAC maintains stability and high fidelity of insert propagation over multiple generations.
•BAC clones permit an assessment to be made not only of the genomic variation that
exists within a virus isolate, but also how these differences impact on viral biology.
•BAC clone helps ameliorate the effects of in vitro passage of virus isolates.
•BAC can be transfected and expressed in numerous mammalian cell lines even if
transfection efficiency and copy numbers are low.
Gel Electrophoresis

Cloning libraries

Restriction Enzyme Mapping

Nucleic acid hybridization

DNA microarray

Applications of recombinant DNA Technology

Microbial Genetics-2023

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Microbial Genetics

A.H.M. Nurun Nabi, PhD

Genetics is the study of heredity which is concerned with

• information in nucleic acids is expressed

A. H. M. Nurun Nabi 02/04/2023

Made of monomer building blocks called

A. H. M. Nurun Nabi 02/04/2023

DNA is a double stranded structure like a twisted

A. H. M. Nurun Nabi 02/04/2023

– Nucleotides form a gene

• Nucleotides are complex structures composed of three kinds of

Directs the production of proteins.

Is double stranded and helical.

Is maintained by hydrogen bonds

Is very stable and can survive

A. H. M. Nurun Nabi 02/04/2023

He made several key observations:

In the model shown, the orange and

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

The twisting of the DNA double helix and the

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

In 1928, British bacteriologist Frederick Griffith conducted a series of

The experiments took an unexpected turn, however, when harmless R

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

•The purified substance gave a negative result in chemical tests known to

•The elemental composition of the purified transforming principle closely

•Protein- and RNA-degrading enzymes had little effect on the transforming

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to

• Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is

• Viruses grown in radioactive phosphorus (32P) had

A. H. M. Nurun Nabi 02/04/2023

The bacterial pellet was found to be radioactive when infected by

A. H. M. Nurun Nabi 02/04/2023

A. H. M. Nurun Nabi 02/04/2023

The major processes involved in

A. H. M. Nurun Nabi 02/04/2023

In DNA replication, the DNA polymerase

This allows genomic material to be

A. H. M. Nurun Nabi 02/04/2023

The process in which DNA is copied into RNA by

Three forms of RNA are produced here: messenger

A. H. M. Nurun Nabi 02/04/2023

1. DNA guides the synthesis of mRNA which

2. DNA directs its own replication by giving

This replication is necessary because each cell

A. H. M. Nurun Nabi 02/04/2023

Bacteria contain 1 chromosome

Many contain plasmids

When bacterial chromosomes replicate both strands are

During replication, enzymes known as polymerases

A. H. M. Nurun Nabi 02/04/2023

During replication, enzymes known as

The replication is said to be semi-

A. H. M. Nurun Nabi 02/04/2023

Origin Recognition Complex (ORC) formation induces local destabilization

Association of DnaG primase triggers the release of helicase loader,

Next, the holoenzyme of DNA Polymerase III, which comprises clamp

A. H. M. Nurun Nabi 02/04/2023