Microbial Molecular Genetics

BMS424
(TOPIC 1)

BY

Farizan binti Aris (Ph.D)

Faculty of Applied Sciences
Shah Alam Campus
Primary Source for figures and content:
iGenetics: A Molecular Approach, 2nd Edition, 2006, Peter J. Russell, Pearson, Benjamin Cummings.
Molecular Genetics of Bacteria, 2nd edition, 2013, Snyder, L. and Champness, W., ASM Press,.
DNA: The Genetic Material

• Search for genetic material---is it composed of nucleic acid or

protein/DNA
or RNA?

• Griffith’s Transformation Experiment

• Avery’s Transformation Experiment
• Hershey-Chase Bacteriophage Experiment
• Tobacco Mosaic Virus (TMV) Experiment
Frederick Griffith’s Transformation Experiment - 1928
“transforming principle” demonstrated with Streptococcus pneumoniae

Griffith hypothesized that the transforming agent was a “IIIS” protein.

But this was only a guess, and Griffith turned out to be wrong.
Oswald T. Avery’s Transformation Experiment - 1944
Determined that “IIIS” DNA was the genetic material responsible for Griffith’s results
(not RNA).

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Hershey-Chase Bacteriophage Experiment - 1953

Bacteriophage = Virus that attacks bacteria

and replicates by invading a living cell
and using the cell’s molecular
machinery.

Fig. Structure of T2 phage

Bacteriophages
are composed of
DNA & protein
Life cycle of virulent T2 phage:
 Hershey-Chase Bacteriophage Experiment - 1953

1. T2 bacteriophage is composed of DNA and

proteins:

2. Set-up two replicates:

• Label DNA with 32P

• Label Protein with 35S

3. Infected E. coli bacteria with two types of

labeled T2

4. 32P is discovered within the bacteria and

progeny phages, whereas 35S is not found
within the bacteria but released with
phage ghosts.
Tobacco Mosaic Virus (TMV) experiment
Demonstrated that RNA (not protein) is the genetic material of
TMV.
Conclusions about these early experiments:

1. Griffith 1928 & Avery 1944:

DNA (not RNA) is transforming agent.

2. Hershey-Chase 1953:

DNA (not protein) is the genetic material.

3. Tobacco Mosaic Virus (TMV) experiment

RNA (not protein) is genetic material of some viruses, but no known prokaryotes or
eukaryotes use RNA as their genetic material.

Alfred Hershey
Nobel Prize in Physiology or Medicine
1969
 1.1 Nucleic acid
Nucleotide = monomers that make up DNA and RNA

Three components

1. Pentose (5-carbon) sugar

DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)

2. Nitrogenous base

Purines (2 rings)
Adenine
Guanine

Pyrimidines (1 ring)
Cytosine
Thymine (DNA)
Uracil (RNA)

3. Phosphate group attached to 5’ carbon

5’ end

3’ end
Structure of DNA

James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on
two sources of information:

1. Base composition studies of Erwin Chargaff (Chargaff’s Rules)

• Discovered that there are always equal amounts of the bases Adenine and
Thymine, and equal amounts of Cytosine and Guanine.

• amount of A = amount of T and amount of G = amount of C

• %GC content varies from organism to organism

Examples: %A %T %G %C %GC

Homo sapiens 31.0 31.5 19.1 18.4 37.5

Zea mays 25.6 25.3 24.5 24.6 49.1
Drosophila 27.3 27.6 22.5 22.5 45.0
Aythya americana 25.8 25.8 24.2 24.2 48.4
The observation that [A] = [T] and [G] = [C]
became known as one of “Chargaff’s rules.”
Structure of DNA

James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on two
sources of information:

2. X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins

Conclusion-DNA is a helical structure with

distinctive regularities, 0.34 nm & 3.4 nm.
Double Helix Model of DNA: Six main features
1. Two polynucleotide chains wound in a right-handed (clockwise) double-helix.

2. Nucleotide chains are anti-parallel: 5’  3’

3’  5’

3. Sugar-phosphate backbones are on the outside of the double helix, and the bases are
oriented towards the central axis.

4. Complementary base pairs from opposite strands are bound together by weak hydrogen
bonds.

A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).

5’-TATTCCGA-3’
3’-ATAAGGCT-5’

5. Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10
bases/turn).

6. Sugar-phosphate backbones are not equally-spaced, resulting in major and minor

grooves.
Location of DNA in cell

•GENOME
•CHROMOSOME
•GENE
1.2 Chromosome
 Chromosome
• A discrete cellular structure composed of a neatly packed DNA molecule
• Eukaryotic chromosomes
– DNA molecule tightly wound around histone proteins
– Located in the nucleus
– Vary in number from a few to hundreds
– Can occur in pairs (diploid) or singles (haploid)
– Appear linear
• Bacterial chromosomes
– Condensed and secured by means of histone-like proteins
– Single, circular chromosome

•The chromosome of E coli has a length of approximately 1.35 mm, several

hundred times longer than the bacterial cell, but the DNA is supercoiled and
tightly packaged in the bacterial nucleoid.
•The time required for replication of the entire chromosome is about 40
minutes
 E coli DNA

DNA length = 1000 X the cell length

The E. coli Chromosome
Compaction of DNA in a eukaryotic chromosome

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 Structure of DNA

• Structure of DNA consist of three forms

 Primary
 Secondary
 tertiary
-deoxyribose and phosphate
form linear strand, “backbone”
-nitrogenous bases hang off side
-two strands held together by H-bonding between bases,
forms a double helix, two strands wound around each other
-base pairing: A-T, G-C
-bases on one strand determine bases on the other: the strands are
complementary
-sequence contains genetic info
 Primary structure of DNA
The Primary structure consists of a linear sequence of nucleotides that are
linked together by phosphodiester bonds. It is this linear sequence of
nucleotides that make up the primary structure of DNA.

5’ end

5’

3’
Phosphodiester
linkage
3’ end 23
 Secondary structure of DNA

The Secondary structure is the set of interactions between

bases, i.e., which parts of strands are bound to each other.

Two anti-parallel polynucleotide

chains wound around the same axis.

Sugar-phosphate chains wrap

around the periphery.

Bases (A, T, C and G) occupy the core, forming

complementary A · T and
G · C Watson-Crick base pairs.

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 Tertiary structure of DNA
. • DNA double helical structure coils round histones.

• DNA bound to histones forms

nucleosomes (10nm fibres)

• Nucleosomes contain 146 nucleotides

• Nucleosomes- any of the repeating globular subunits of chromatin that
consist of a complex of DNA and histone

25
 Forms of DNA ON THE BASIS OF Double-helix
 Three major forms:
 B-DNA
 A-DNA
 Z-DNA
 B-DNA
is biologically THE MOST COMMON
 It is a -helix meaning that it has a Right handed, or
clockwise, spiral.
 Complementary base pairing
• A-T
• G-C
 Ideal B-DNA has 10 base pair per turn
 This structure exists when plenty
of water
 So complete rotation of molecule is 3.4 nm.

26
 A-DNA

 Right-handed helix
 Wider and flatter than B-DNA
 11 bp per turn
 Its bases are tilted away from
main axis of molecule
 Narrow Deep major Groove and
Broad, Shallow minor Groove.
 Observed when less water is
present. i.e.
Dehydrating condition.

27
  Z-DNA

 A left-handed helix
 Seen in Condition of High salt
concentration.
 In this form sugar-phosphate
backbones zigzag back and
forth, giving rise to the name
Z-DNA(for zigzag).
 12 base pairs per turn.
 A deep Minor Groove.
 No Discernible Major Groove.
 Part of some active genes form
Z-DNA, suggesting that Z-DNA
may play a role in regulating
gene transcription.

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Z-DNA B-DNA A-DNA
1.3 Extrachromosomal genetic elements
 transposon ( transposable element or ‘jumping genes’)
 plasmid

30
 Plasmid
Definition: Extrachromosomal genetic elements that are capable of
autonomous replication (replicon)

Episome - a plasmid that can integrate into the chromosome

They are usually much smaller than the bacterial chromosome,

varying from less than 5 to more than several hundred kbp.

 Most plasmids are supercoiled, circular, double-stranded DNA

molecules, but linear plasmids have also been demonstrated in
Borrelia and Streptomyces.
 Classification of plasmid

 Transfer properties

 Conjugative (This plasmids code for functions that promote

transfer of the plasmid from the donor bacterium to other recipient

bacteria)

 - Nonconjugative (do not)

 Phenotypic effects

 Fertility

 Bacteriocinogenic plasmid

 Resistance plasmid (R factors)

Phenotypic effects
The average number of a given plasmid per bacterial chromosome
is called its copy number.

Large plasmids (40 kilobase pairs) are often conjugative, have

small copy numbers (1 to several per chromosome).

Plasmids smaller than 7.5 kilobase pairs usually are

nonconjugative, have high copy numbers (typically 10-20 per
chromosome), rely on their bacterial host to provide some
functions required for replication, and are distributed randomly
between daughter cells at division.

Some plasmids are cryptic and have no recognizable effects on the

bacterial cells that harbor them.
 Transposable Genetic Elements

• Definition: Segments of DNA that are able to move from one location
to another
• Properties
 “Random” movement
 Not capable of self replication
 Transposition mediated by site-specific recombination
-Transposase
 Transposition may be accompanied by duplication
 Types of Transposable Genetic Elements
 Insertion sequences (IS)
 Definition: Elements that carry no other genes except those
involved in transposition
 Nomenclature - IS1
 Structure
 Importance
 Mutation
Plasmid insertion
Phase variation
 The known insertion sequences vary in length from approximately 780 –
1500 nucleotide pairs, have short (15-25 base pair) inverted repeats at
their ends, and are not closely related to each other.

ABCDEFG Transposase GFEDCBA

 Phase Variation in Salmonella H
Antigens

H1 gene IS H2 gene

H1 H2
flagella flagella
 Types of Transposable Genetic Elements

• Transposons (Tn)
– Definition: Elements that carry other genes except those involved
in transposition
– Nomenclature - Tn10
– Transposons can move from one site in a DNA molecule to other
target sites in the same or a different DNA molecule.
– Structure

IS Resistance Gene(s) IS

IS Resistance Gene(s) IS

Transposons are not self-replicating genetic elements, however, and they must
integrate into other replicons to be maintained stably in bacterial genomes
Most transposons in bacteria can be separated into 4 major classes.
1 - insertion sequences and related composite transposons.
2- transposons consists of the highly homologous TnA family (ampicillin
resistance transposon Tn3 and Tn1000 (the gamma-delta transposon)
found in the F plasmid.
3- third class of transposons consists of bacteriophage and related
temperate phages.
4- transposons, discovered in Gram-positive bacteria and represented
by Tn917, consists of conjugative transposons (Gram-positive bacteria
the host strain carrying the transposon can act as a conjugal donor).
Tn917 encodes tetracycline resistance
 The importance of transposon
 they cause mutations,
 mediate genomic rearrangements,
 function as portable regions of genetic homology, and acquire new genes,
 contribute to their dissemination within bacterial populations,
 insertion of a transposon often interrupts the linear sequence of a gene and
inactivates it,
 transposons have a major role in causing deletions, duplications, and
inversions of DNA segments as well as fusions between replicons.
In medically important bacteria, genes that determine production of
adherence antigens, toxins, or other virulence factors, or specify resistance to
one or more antibiotics, are often located in complex transposons.
Well-known examples of complex transposons are Tn5 and Tn10, which
determine resistance to kanamycin and tetracycline, respectively.
Transposon

Complex transposons - in length from

about 2,000 to > 40,000 bp.
Contain insertion sequences at each end,
usually as inverted repeats.
 The entire complex element can
transpose as a unit.