GENETICS OF BACTERIA

GENETICS OF BACTERIA
Bacteria fossil

Learning objectives
Describe the structure of a bacterial chromosome including the arrangement of DNA within bacterial cells.

Describe the process of binary fission, transformation, transduction and conjugation in bacteria and explain the role of F plasmids in bacterial conjugation. (Knowledge of Hfr is not required).

Distinguish between structural and regulatory genes. A structural gene is a region of DNA that codes for a protein or RNA molecule that forms part of a structure or has an enzymatic function (e.g. lacY, lacZ, lacA, but excludes lacI). A regulatory gene codes for a specific protein product that regulates the expression of the structural genes (e.g. lacI).

Distinguish between the concept of repressible and inducible systems of gene regulation using trp and lac operon as examples respectively (attenuation of trp operon is not required).

Describe the concept of a simple operon (using lac operon as an example).
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
GENERAL STRUCTURE OF PROKARYOTES
Learning objective 1
Describe the structure of a bacterial chromosome including the arrangement of DNA within bacterial cells.

THE BACTERIAL GENOME
The bacteria chromosome is part of the genome.

What is a genome?
the entire genetic information found in an organism. This includes both the genes and the non-coding sequences of the DNA

THE BACTERIAL GENOME
Bacterial chromosome
Bacterial chromosome
DNA binding proteins
Bacterial chromosome
THE BACTERIAL GENOME
Plasmids
Plasmids
Plasmids can also be transferred between bacterial cells during conjugation.
Plasmids
Every plasmid contains origin of replication which enables plasmid to be duplicated independently from the chromosomal DNA.
Roles of Plasmids
F plasmids (Learning objective 2) facilitate genetic recombination, which may be advantageous in a changing environment that no longer favours existing strains in a bacterial population.

Genetic recombination aids to increase genetic diversity

Compare to meiosis in eukaryotic cells  increase genetic diversity

Genes of Plasmids
Degrade complex macromolecules

Provide resistance to antibiotics (R plasmids)
Gene product coded by the gene in the R plasmid will protect the bacteria when it is exposed to antibiotics.

Produce toxins (bacteriocins – to inhibit growth of similar or closely-related bacteria)

Provide resistance to heavy metals

For genetic engineering,
Contain genetic markers, which confer well-defined phenotypes on the host cell. (see Appendix)
Plasmids can also serve function as vectors in genetic engineering (see Appendix).
Plasmids as vectors and markers
Plasmids as vectors and markers
Plasmids as vectors and markers
Plasmids as vectors and markers
CHECKPOINT pg 5
What is a genome?
the entire genetic information found in an organism. This includes both the genes and the non-coding sequences of the DNA

What are the different genetic elements that constitute the bacteria genome?
Bacterial chromosome, plasmid, transposon

List the main differences between these genetic elements.
only one bacterial chromosome vs many plasmids
chromosome contain many more genes than plasmids
plasmids can be transferred to other bacterial cells
transposons contain IS sequences for insertion into chromosome/plasmid
CHECKPOINT pg 5
What is a genome?
the entire genetic information found in an organism. This includes both the genes and the non-coding sequences of the DNA

What are the different genetic elements that constitute the bacteria genome?
Bacterial chromosome, plasmid, transposon

List the main differences between these genetic elements.
only one bacterial chromosome vs many plasmids
chromosome contain many more genes than plasmids
plasmids can be transferred to other bacterial cells
transposons contain IS sequences for insertion into chromosome/plasmid
CHECKPOINT
What is a genome?
the entire genetic information found in an organism. This includes both the genes and the non-coding sequences of the DNA

What are the different genetic elements that constitute the bacteria genome?
Bacterial chromosome, plasmid

List the main differences between these genetic elements.
only one bacterial chromosome vs many plasmids
chromosome contain many more genes than plasmids
plasmids can be transferred to other bacterial cells

Learning objective 1 – pg 5
Describe the structure of a bacterial chromosome including the arrangement of DNA within cells.

Arrangement of DNA within cells
Most of the genes found in chromosomal DNA are structural genes which codes for functional polypeptides.
Structural gene  functional polypeptide/protein  carry out a specific role
e.g. insulin, protein kinases, ATPase, collagen etc

Only a very small amount of non-coding DNA is present.
DNA is not transcribed into proteins


Contains a single origin of replication – which allows bacteria to replicate by binary fission (page 9; Learning objective 2).
Binary fission: asexual reproduction of bacteria cells to produce 2 genetically identical cells.



Arrangement of DNA within cells
Bacterial chromosome is commonly characterised with the presence of operons (pg30). These are regulatory regions with 2 or more structural genes and allow regulation of a group of genes that encode proteins with common functions.
Arrangement of DNA within cells
The typical E. coli cell has 4.6 x 106 bases.
This would make a strand of DNA >1000 µm long, but
E. coli is only 2-5 µm long.
How does it fit?

is achieved by
(1) formation of loop domains as well
(2)supercoiling of DNA.
Arrangement of DNA within cells
LOOP DOMAINS
Arrangement of DNA within cells
HU works with topoisomerase I to bind to DNA and introduce sharp bends in the chromosome to generate tension necessary for negative supercoiling.

The folded DNA is organised into a variety of conformations that are supercoiled and wound around tetramers of the HU protein.

The number of loops formed is dependent on the type of species and size of bacteria.




Arrangement of DNA within cells
SUPERCOILING
Arrangement of DNA within cells
SUPERCOILING

Negative supercoiling – DNA topoiosomerase II (DNA gyrase)
Type II topoisomerase (DNA gyrase) introduces negative supercoils by breaking TWO DNA strand and resealing the nick upon formation of a supercoil.
Topoisomerase II – introduce supercoils
Role of topoisomerases
As bacterial chromosome is made up of approximately 50 supercoiled DNA domains, topoisomerase prevents the entire bacterial chromosomes from becoming relaxed every time a nick is made.
I.e. a nick in the DNA in one of these domains does not relax the DNA in the others.
Role of topoisomerases

Action of topoisomerases allows DNA molecule alternate between supercoiled and relaxed state.
Supercoiling for packing the DNA into the confines of a cell
Relaxing for DNA to be replicated and transcribed.
Role of topoisomerases

Because topoisomerases play an important role in regulating the structure of DNA, it can be the target of antibiotics.

- quinolones (e.g.nalidixic acid)
- fluoroquinolones (e.g.ciprofloxacin)
- novobiocin

CHECKPOINT
What are the two methods used by the bacteria to compact its genome?
Loop Domain & Supercoiling

Name the protein (for each method) that plays a crucial role to ensure that bacterial DNA is highly compacted.
Loop Domain DNA binding proteins (HU and H-NS)
Supercoiling  DNA gyrase (Topoisomerase II)
CHECKPOINT
What are the two methods used by the bacteria to compact its genome?
Loop Domain & Supercoiling

Name the protein (for each method) that plays a crucial role to ensure that bacterial DNA is highly compacted.
Loop Domain DNA binding proteins (HU and H-NS)
Supercoiling  DNA gyrase (Topoisomerase II)
CHECKPOINT
What are the two methods used by the bacteria to compact its genome?
Loop Domain & Supercoiling

Name the protein (for each method) that plays a crucial role to ensure that bacterial DNA is highly compacted.
Loop Domain DNA binding proteins (HU and H-NS)
Supercoiling  DNA gyrase (Topoisomerase II)
QUICK CHECK: Comparing eukaryotic and prokaryotic cells…
Comparing eukaryotic and prokaryotic cells…
Linear double-stranded DNA molecules that wound around proteins called histones to form structures called nucleosomes.

As eukaryotic DNA is very long, supercoiling is important for DNA packaging. It reduces the space and allows for a lot more DNA to be packaged.

Solenoidal supercoiling is achieved with histones to form a 10nm fiber. This fiber is further coiled into a 30nm fiber, and further coiled upon itself numerous times more.



Learning objectives
Describe the structure of a bacterial chromosome including the arrangement of DNA within bacterial cells.

Describe the process of binary fission, transformation, transduction and conjugation in bacteria and explain the role of F plasmids in bacterial conjugation. (Knowledge of Hfr is not required).

Distinguish between structural and regulatory genes. A structural gene is a region of DNA that codes for a protein or RNA molecule that forms part of a structure or has an enzymatic function (e.g. lacY, lacZ, lacA, but excludes lacI). A regulatory gene codes for a specific protein product that regulates the expression of the structural genes (e.g. lacI).

Distinguish between the concept of repressible and inducible systems of gene regulation using trp and lac operon as examples respectively (attenuation of trp operon is not required).

Describe the concept of a simple operon (using lac operon as an example).
BINARY FISSION
Bacteria cells divide by binary fission.

Asexual reproduction (offspring arise from one parent)
Leads to genetically identical cells.

Bacterial chromosome must be replicated before actual division of the cell into daughter cell.
BINARY FISSION
Replication and partitioning (separation) of the chromosome occur as a concerted process.

Compare to eukaryotic cell division:
Replication of chromosomes during S phase THEN
Mitosis takes place to separate the chromosome.

BINARY FISSION

As DNA is replicated, cell elongates as it grows larger in size.

Plasma membrane invaginates when cell has grown to an appropriate size. Cell wall is then deposited on the membrane.
Mechanism - BINARY FISSION
Mechanism -BINARY FISSION
Mechanism - BINARY FISSION


Binary fission leads to formation of genetically identical bacteria.

Is it advantageous for bacteria to stay genetically the same always?

If the environment changes, what will happen to the population of bacteria which is genetically identical?

Q: How to generate genetic diversity in prokaryotes?

MUTATION
GENETIC RECOMBINATION
Mutation  Genetic Diversity
radiation, UV rays and various chemicals damages DNA  increase the likelihood of mutation.
This will result in some of the offspring possessing a slightly different genetic makeup.
Because E.coli reproduces via binary bission very quickly, occasional mutations may lead to significant impact on genetic diversity.
Genetic Recombination
Genetic recombination can also generate diversity within bacterial populations.
Bacteria can exchange DNA between different cells (with different genetic composition).
Conjugation -requiring cell-to-cell contact
Transduction - by means of viruses
Transformation- bacteria can also pick up material from the environment
Let’s compare to eukaryotes!
What gives rise to genetic diversity in eukaryotes?
Any genetic recombination events?
Mutation
Meiosis  prophase I (crossing over at chiasma)

Recap:
Binary fission will lead to genetically identical cells.
One cell giving rise to another cell; Bacteria chromosome replicate; Plasmids replicate;
Asexual Reproduction

What is the evidence that to prove that recombination is possible in bacteria?
Detection of recombination
E.coli requires both tryptophan and arginine (amino acids) to survive.
It needs to synthesise these two enzymes, and hence requires the genes to code for the amino acid.
Detection of recombination
Detection of recombination

Incubate the
mutant strain arg+trp- and the
mutant strain arg-trp+ together.

Results in a recombinant strain
arg+trp+
Detection of recombination
Bacterial colonies

When bacteria from the two strains were incubated together, cells emerged that could grow on minimum medium (containing only glucose and salts), indicating that they made both tryptophan and arginine.

These cells that could synthesise both amino acids must have acquired one or more genes from the other strain, by genetic recombination
Learning Objective 2
Describe the process of binary fission, transformation, transduction and conjugation in bacteria and explain the role of F plasmids in bacterial conjugation.
Transformation
Transformation
Transformation is the alteration of a bacterial cell’s genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment. There is no requirement for cell to cell contact.

This occurs naturally for some bacteria only e.g. Haemophilus, Streptococcus (not for E.coli) as a mean of gene transfer.
Transformation
In biotechnology, artificial transformation can be applied to introduce foreign genes into E.coli genome – genes coding for valuable proteins, such as human insulin or growth hormone.
Evidence for transformation
Griffith’s experiment – proves that naked DNA can indeed be taken up by other cells.

Streptococcus pneumoniae has 2 forms
R strain – is benign; does not kill host
S strain – virulent; kills host
Evidence for transformation
Evidence for transformation

Does R strain kill the mouse?
Does not kill mouse
Does S strain kill the mouse?
Not treated with heat – kill mouse
Heat treated S strain – does not kill mouse

Does R strain kill the mouse?
Does not kill mouse
Does S strain kill the mouse?
Not treated with heat – kill mouse
Heat treated S strain – does not kill mouse
Evidence for transformation
Evidence for transformation
Evidence for transformation
Evidence for transformation

How does Strain R (Streptococcus pneumoniae) pick take up DNA when it was released from heat-killed S-strain?

Mechanism of transformation
Learning objective 2: Describe the process of binary fission, transformation.
Mechanism of transformation
Mechanism of transformation
Mechanism of transformation
Mechanism of transformation
Mechanism of transformation
Crossing over in Prophase I
Transformation
http://del.icio.us/tpjcbiology

http://www.sinauer.com/cooper/4e/animations0402.html

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_1.html
Natural transformation
For bacteria that carry out natural competence, they possess cell-surface proteins (known as competence-specific proteins) that first recognise and transport DNA from closely related species into the cell, which can then integrate the foreign DNA into the genome.
Natural transformation
Natural transformation
1-3
DNA binding proteins on surface binds to fragment of DNA

Autolysins degrade cell wall.

DNA penetrates through cell wall.
Natural transformation
4
Nuclease cut bound DNA into fragments as DNA penetrates through the cell wall.

Natural transformation
5
Other nucleases destroy one strand of DNA and allow one strand to enter the bacterium.

Natural transformation
5
Other nucleases destroy one strand of DNA and allow one strand to enter the bacterium.

Natural transformation
6
Single-stranded DNA is bound to a competence-specific protein, and reaches the bacteria chromosome where Rec A takes over.

Natural transformation
7
Rec A protein promotes genetic exchange btw fragment of donor’s DNA and bacteria chromosome, allowing donor DNA to be integrated into it.
Natural transformation
7
Rec A protein promotes genetic exchange btw fragment of donor’s DNA and bacteria chromosome, allowing donor DNA to be integrated into it.
Natural transformation
8.
Uncombined DNA will be degraded.
Natural transformation
Natural transformation
Natural transformation
Competent cells
Natural competence-
A small percentage of bacteria are naturally capable of taking up DNA, either in laboratory conditions, or in their natural environments. Such species carry sets of genes specifying machinery for bringing DNA across the cell's membrane or membranes  natural transformation

The mechanism discussed on page 18 is natural transformation.
Competent cells
Artificial competence –
induced by laboratory procedures in which cells are passively made permeable to DNA, using conditions that do not normally occur in nature, e.g. treating the cells with CaCl2 followed by heat, or via electroporation.
The cell membrane is made temporarily permeable to DNA  articificial transformation.
Transformation - animation
CHECKPOINT
What is transformation?
Uptake of naked DNA from the cell’s environment.

What is the aim of transformation?
Uptake of foreign DNA into the cell will promote genetic recombination between the foreign DNA and host cell’s DNA  leads to genetic diversity

Using the information from page 17 (figure 14) and 18 (description), identify the step that is critical in order to fulfill the aim of transformation.
Step 7: integration of foreign DNA into host cell’s DNA – genetic exchange with the help of Rec A
CHECKPOINT
What is transformation?
Uptake of naked DNA from the cell’s environment.

What is the aim of transformation?
Uptake of foreign DNA into the cell will promote genetic recombination between the foreign DNA and host cell’s DNA  leads to genetic diversity

Using the information from page 17 (figure 14) and 18 (description), identify the step that is critical in order to fulfill the aim of transformation.
Step 7: integration of foreign DNA into host cell’s DNA – genetic exchange with the help of Rec A
CHECKPOINT
What is transformation?
Uptake of naked DNA from the cell’s environment.

What is the aim of transformation?
Uptake of foreign DNA into the cell will promote genetic recombination between the foreign DNA and host cell’s DNA  leads to genetic diversity

Using the information from page 17 (figure 14) and 18 (description), identify the step that is critical in order to fulfill the aim of transformation.
Step 7: integration of foreign DNA into host cell’s DNA – genetic exchange with the help of Rec A
CHECKPOINT
What is transformation?
Uptake of naked DNA from the cell’s environment.

What is the aim of transformation?
Uptake of foreign DNA into the cell will promote genetic recombination between the foreign DNA and host cell’s DNA  leads to genetic diversity

Using the information from page 17 (figure 14) and 18 (description), identify the step that is critical in order to fulfill the aim of transformation.
Step 7: integration of foreign DNA into host cell’s DNA – genetic exchange with the help of Rec A