Genetics of Prokaryotes

Dr. Valerie B. O‘Leary

Lecture No 1004
 Prokaryotes are unicellular organisms
that lack a membrane-bound nucleus, mitochondria
or any other membrane-bound organelle.

Chromosome Plasmid

Nucleoid

Prokaryotes are divided into two distinct groups:

1) the bacteria and 2) the archaea,
which scientists believe have unique evolutionary lineages.
Characteristics of bacteria:
Size: Typically a few m in length.
Shape: Spheres – Rods- Spirals.

Details:
-They were amongst the first life forms on earth.
-They live in most habitats eg.soil, water, acidic hot springs,
radioactive waste and deep portions of the earths crust.
-They live in symbiotic and parasitic relationships with plants and animals.
-Virtually all animal life depends on bacteria for their survival
because they possess the genes and enzymes necessary to
synthesize vitamin B12 and provide it through the food chain.

Facts: There are 40 million bacteria in a gram of soil.

There is 1 million bacteria in 1 milliliter of water.
There are 5x 1030 bacteria on earth.

Bacteria as a biomass exceeds all plants and animals.

Molecular characteristics of bacteria

• Bacteria have a primitive chromosome

called a nucleoid – composed of
1) DNA (double stranded, circular, supercoiled)
2) Proteins (histone like).
• Bacterial genes do not have introns.
• RNA transcription and protein translation occur in the same compartment
(ie. there is no nucleus in bacteria only a nuclear region).
• There is no cytoskeleton in bacteria.
• Bacteria have ribosomes, each consisting of a small (30S) and a large
(50S) subunit.
• Division is binary and non-sexual (process known as binary fission).
 The bacterial colony growth curve.
The bacterial growth curve represents the number of live cells
in a bacterial population over a period of time.

There are four distinct phases of the growth curve: lag, log, stationary and death.
Lag phase - bacteria adapt to growth conditions. They are not dividing.
Log phase (also called logarithmic phase or exponential phase)
-bacterial cell number doubling.
Stationary phase - growth reaches a plateau as the number of dying cells
equals the number of dividing cells.
Death phase - an exponential decrease in the number of living cell.
Characteristics of Archaea:
Size: Typically a few m in length.
Shape: Spheres, Rods, Spirals, Flat square.

Details:
-They have a unique biochemistry eg. Ether lipid cell membrane.
-They use more sources for energy compared to eukaryotes eg. Ammonia,
metal ions, hydrogen gas.
-First observed in extreme environments eg. Hot springs, salt lakes etc
-Better detection methods has shown that Archaea live in most habitats.
-They are important for human microbiota in the gut, mouth and skin.

Facts:
1977 – Archaea classified as a separate group
from bacteria.

No evidence to date that they cause disease.

Molecular characteristics of archaea

• Archaea have chromatin composed of

1) DNA (double stranded, circular, supercoiled)
2) Histones (similar to eukaryotic histones)
• Introns are present in the genes encoding tRNA and rRNA.
• RNA transcription and protein translation are similar to eukaryotic
processes.
• 15% of proteins are unique to archaea.
• There is an actin cytoskeleton in archaea.
• Archaea have ribosomes, each consisting of
a small (30S) and a large (50S) subunit.
• Archaea reproduce by binary division,
fragmentation or budding.
 Genetics of prokaryotes

In order to understand this topic the following must be known:

1)Plasmid
2)Replication
3)Transfer of genes:
4)Transformation
5)Transduction
6)Conjugation
7)Operon
Transformation Transduction Conjugation
 1) Plasmid
2) Replication
Plasmid 3)
4)
Transfer of genes:
Transformation
5) Transduction
6) Conjugation
• A circular double stranded DNA molecule. 7) Operon

• 1000 base pairs (bps) to >1,000,000 bps.

• Plasmids are found mainly in bacteria but can be also present in archaea.
• A bacteria may contain one to several hundred plasmids. The number of
copies of a plasmid is called the Plasmid Copy Number.
• Plasmids often carry genes that benefit the survival of the organism.
• Plasmids encode genes for resistance to antibiotics or heavy metals.
• Plasmids encode genes for metabolic enzymes involved in nitrogen
fixation.
Note: Plasmids typically provide a selective advantage under a given
environmental state.
 A plasmid can replicate (ie. make an exact copy of itself). 1) Plasmid
2) Replication
It can do this independently. 3) Transfer of genes:
4) Transformation
But they need the following to replicate: 5) Transduction
6) Conjugation
7) Operon
1)A stretch of DNA from which replication is initiated/started
– An origin of replication (Ori).

Note: A few types of plasmids can also insert into the host chromosome, and these
integrative plasmids are sometimes referred to as episomes in prokaryotes.
 DNA replication of chromosome/plasmid
(circular DNA molecules in prokaryotes)

Beginning:
 DNA replication of chromosome/plasmid
(circular DNA molecules in prokaryotes)

Process:

https://youtu.be/jmWuju1S9_E
1) Plasmid
2) Replication
3) Transfer of genes:
4) Transformation
5) Transduction
6) Conjugation
7) Operon
Prokaryotes transmit DNA both vertically and horizontally
1) Plasmid
2) Replication
3) Transfer of genes:
4) Transformation
5) Transduction
6) Conjugation
7) Operon
Bacterial Transformation
The first evidence that bacteria could undergo transformation:
• Some bacteria take up foreign genetic material from the environment.
Griffith‘s experiment (1928): Infection of mice by virulent and non-virulent
strains of S. pneumoniae - Identified a ´Transforming Principle´.
Avery, McLeod, McCarthy (1944): Griffith experiment repeated but with
isolated DNA → identified the ´Transforming Principle´ to be DNA.

Polysaccharides Lipids RNA Proteins DNA

Destroyed Destroyed Destroyed Destroyed Destroyed
1) Plasmid
2) Replication
3) Transfer of genes:
4) Transformation
5) Transduction
6) Conjugation
7) Operon
 Transduction:

A process whereby foreign DNA is introduced into another cell by a virus.

A virus that infects bacteria is called a bacteriophage.

This leads to genetic recombination in bacteria.

The bacteriophage can transfer DNA from one bacterium to another bacterium.
General, non-specific – a transfer of any part of host DNA (only bacterial genes)
4. General transduction
• Bacterium is infected by phage DNA.
• Phage replicates and bacterial DNA is broken down into pieces.
• Sometimes a fragment of bacterial DNA is packaged into phage capsid
instead of phage DNA.
• The donor cell lyses and phage carrying bacterial DNA is released to
infect a new host cell.
• DNA in the new host cell become recombined after infection.

Nonspecifically transducing particles comprise only bacterial DNA. -

any genes of bacteria could be transferred.
Specialized, specific – the transfer of a particular part of host DNA along with viral genes.
 Special transduction

E.g.. gene gal of E. coli

(galactose utilization)
gene bio (biotin synthesis)

The recipient cell receives

a new metabolic ability
(from the transferred bacterial genes).
1) Plasmid
2) Replication
3) Transfer of genes:
4) Transformation
5) Transduction
6) Conjugation
7) Operon
 Conjugation
This is the transfer of genetic material between bacterial cells via:
1)direct cell to cell contact
or
2) a bridge-like connection between two cells.

It is a method of horizontal gene transfer.

It is not sexual reproduction (ie. there is no transfer of gametes).

Transferred DNA is known as a transposon/conjugative plasmid.

The transferred DNA is often beneficial to the recipient.

Benefits may include antibiotic resistance, xenobiotic (chemical) tolerance
or the ability to use new metabolites.
Conjugation from F+ into F- bacterium F+ bacterium

F- bacterium
Mechanism of rolling circle replication (unidirectional replication)

is observed during conjugation

3´ OH a) An endonuclease cut one DNA strand.

5´ P

New strand b) A new DNA strand is formed in direction

3´ OH
5´→3 and transmitted to the recipient cell.
5´

c) A new strand is formed made up of

Okazaki fragments
Okazaki fragments.
5´
Formation of High Frequency of Recombination (Hfr) strain
Formation of F- from F+ bacteria
1) Plasmid
2) Replication
3) Transfer of genes:
4) Transformation
5) Transduction
6) Conjugation
7) Operon
An operon is a functional unit of transcription and genetic regulation.

It enables organisms (eg. prokaryotes) to regulate gene expression depending on

the environmental conditions.

An operon is made up of 3 basic DNA components:

1) Promoter – a nucleotide sequence that enables a gene to be transcribed.

The promoter is recognized by RNA polymerase, which then initiates transcription.

2) Operator – a segment of DNA to which a repressor binds.

The repressor protein obstructs the RNA polymerase from transcribing the genes.

3) Structural genes – the genes that are regulated within the operon.

First identified in prokaryotes by Jacob and Monod.

They won the Nobel Prize in Physiology 1965 for this discovery.
Operons in prokaryotes:

1)The Lac (Lactose) operon

2)The Trp (Tryptophan) operon

The Lac operon (lactose operon) is required for the transport and metabolism
of lactose in Escherichia coli (and other bacteria).

Although glucose is the preferred carbon source, the Lac operon enables
bacteria to digest lactose when glucose is not available.
 The Lac Operon

RNA polymerase Inactive

Repressor

Structural genes

Promoter Operator

Active

Structural genes

LacZ gene LacY gene LacA gene

= Lactose
 cAMP
CAP

-galactosidase -gal transacetylase

(-gal)
-gal permease
The structural genes are orientated in the same direction immediately adjacent
to each other on the chromosome and are co-transcribed into a single mRNA molecule.
Transcription starts with the binding of the enzyme RNA polymerase (RNAP)
which binds to the promoter, immediately upstream of the genes.
Binding of RNAP to the promoter is aided by the cAMP-bound catabolite activator protein
(CAP, also known as the cAMP receptor protein)

* when glucose is present – cAMP concentration is low; when glucose absent – cAMP levels high
 The Trp Operon

The Trp operon (tryptophan operon) is required for the production of tryptophan
in Escherichia coli (and other bacteria).

When tryptophan is present in the environment, the genes for tryptophan synthesis
are not expressed.
 Repressive negative regulation

This gene allows for graded regulation

Structural genes (required for tryptophan synthesis) are not transcribed as

tryptophan (Trp) is already present in the environment.
* Trp sometimes referred to as a ‘co-repressor´ in this situation
 Operator (O) is accessible in the absence of tryptophan,
thereby enabling transcription of structural genes.

The structural genes are orientated in the same direction immediately adjacent
to each other on the chromosome and are co-transcribed into a single mRNA molecule.
Antranilate synthase
Phosphoribosylantranilate isomerase
Indole-3-glycerol-phosphate synthase
Tryptophan synthase subunits (x 2)
+ Serine

= Tryptophan
 Regulation of gene expression

• Genes constitutively expressed = constitutive (housekeeping) genes

• Genes expressed only in certain environmental condition (presence of

effector molecules)

- inducible genes - codes for catabolic enzymes, produced only in

presence of substrate = inductive regulation

- repressible genes = genes for anabolic enzymes - negative feedback

by product of anabolic pathway = repressive regulation

 Dr. Valerie B. O‘Leary
Assistant Professor

Email: valerie.oleary@lf3.cuni.cz

Consulting hours: Tuesday 10 – 11

Tuesday 12 – 13

Please send an email to make an appointment.

Thank you.