Transcript bacterial genetics

Bacterial Genetics
G.Jamjoom 2005
Bacterial Genetics
Lecture Outline :
1.
The study of bacterial genetics helped illustrate:
- the nature of genetic material as DNA
- the genetic code
- the nature of mutations (changes in nucleotide
sequences)
- regulation of gene function (repressors,
activators)
DNA Forms the Genetic Information:
- The Griffith experiment (1928,1944):
DNA fragments from capsulated
pneumococcus can give noncapsulated
strain the ability to make capsule “ transformation”
DNA Forms the Genetic Information:
- The Hershey and Chase experiment
Bacteriophage DNA alone enters the
bacterial cell and makes new progeny
phages. Phage protein coat remains
outside and is not involved in this process.
Length of DNA In Different Organisms
- Bacteriophage MS2 Virus
- Bacteriophage T2
Virus
- Escherichia coli
- Saccharomyces
- C. elegans
- A. thaliana
- Drosophila
- Mouse
- Human
Bacterium
Yeast
4,000 bp
21,000 bp
~10 genes
~ 200 genes
4,000,000 bp
14,000,000 bp
~4288 genes
Nematode
100,000,000 bp
plant
100,000,000 bp
Insect
165,000,000 bp
Mammal
3,000,000,000 bp
Mammal
3,500,000,000 bp ~ 40,000genes
Bacterial Genetics
Lecture Outline:
2. Prokaryotic cells, Eukariotic cells,
Archae
EUKARYOTES
PROKARYOTES
BACTERIA ARCHAEA
Prokaryotes
• Eubacter "True" bacteria
– human pathogens
– clinical or environmental
– one kingdom
• Archaea
– Environmental organisms
– second kingdom
Bacterial Genetics
Lecture Outline:
3. The bacterial chromosome :
- structure, genes, operons
- mapping
- complete sequences of selected bacteria
- replication, transcription, translation
The Bacterial Chromosome
• Most bacterial chromosomes are circular
• Many have been fully sequenced
• Many genes have been identified and
mapped using gene transfer techniques
such as conjugation, transduction, and
transformation
The Complete
Sequence of
Escherichia
coli
Chromosome
Echerichia coli chromosome
• Size 4,600,000 base pairs (4.6 megabases)
• Contains 4288 genes ( 62% identified)
• Many genes code for the following :
- Cell structure
- Energy metabolism
- Proteins form DNA replication
- Proteins for transcription, translation, RNA
synthesis
- Synthesis of amino acids , nucleotides, etc.
- Synthesis of enzymes
• Contains transposons and plasmid and phage sequences
Bacterial Genetics
G.Jamjoom 2005
Lecture Outline:
6. Bacteriophages (bacterial viruses):
- virulent
- temparate:
lysogey
Phage Composition and Structure
Head/Capsid
Genomic
DNA
Contractile
Sheath
Tail
Tail Fibers
Base Plate
Types of Bacteriophage
Lytic or virulent :
Phage that multiply within the host cell,
lyse the cell and release progeny phage (e.g. T4)
Lysogenic or temperate phage:
Phage that can either multiply via the lytic cycle or enter
a quiescent state in the bacterial cell. (e.g., )
– Expression of most phage genes repressed
– Prophage – Phage DNA in the quiescent state
– Lysogen – Bacteria harboring a prophage
Bacterial Genetics
Lecture Outline:
4. Plasmids (extrachromosomal elements):
- functions
- role in antibiotic resistance (R plasmids)
Plasmids
• Definition: Extrachromosomal genetic
elements that are capable of autonomous
replication (replicon)
• Episome - a plasmid that can integrate into
the chromosome
Plasmid- Coded Functions
•
Fertility
• Resistance to:
- antibiotics
- irradiation
- phages
• Production of :
- exotoxins
- enterotoxins
- bacteriocins
- Proteases
(cheese)
•
Metabolism of :
- various sugars
- hydrocarbons
• Tumergenesis in
plants
Bacterial Genetics
Lecture Outline:
7. Mechanism of gene transfer in bacteria:
- Transformation
- Transduction
- Conjugation
Transformation
• Steps
– Uptake of DNA
• Gram +
• Gram -
– Recombination
Transduction
• Types of transduction
– Generalized Transduction : in which
potentially any dornor bacterial gene can
be transferred.
– Specialized Transduction : in which only
certain donor genes can be transferred
Generalized Transduction
• Infection of Donor
• Phage replication and degradation of host DNA
•
•
•
•
Assembly of phages particles
Release of phage
Infection of recipient
Homologous recombination
Potentially any donor gene can be transferred
Events Leading to Lysogeny
• Site-specific
recombination
Phage coded –
enzyme
gal
• Repression of the
phage genome
– Repressor protein
– Specific
– Immunity to
superinfection
bio
gal
bio
gal
bio
Termination of Lysogeny
• Induction
– Adverse
conditions
• Role of proteases
bio
gal
– recA protein
– Destruction of
repressor
• Gene expression
• Excision
• Lytic growth
bio
gal
gal
bio
gal
bio
Specialized Transduction
Lysogenic Phage
• Excision of the
prophage
• Replication and
release of
phage
• Infection of the
recipient
• Lysogenization
of the recipient
– Homologous
recombination
also possible
bio
gal
gal
gal
bio
gal
bio
bio
bio
Conjugation
• Definition: Gene transfer from
a donor to a recipient by
direct physical contact
between cells
• Mating types in bacteria
Donor
– Donor
• F factor (Fertility factor)
– F (sex) pilus
– Recipient
• Lacks an F factor
Recipient
Physiological States of F Factor
• Autonomous (F+)
Characteristics of F+ x F- –
crosses
• F- becomes F+ while F+ remains F+
• Low transfer of donor
chromosomal genes
F+
Physiological States of F Factor
• Integrated (Hfr)
– Characteristics of
Hfr x F- crosses
• F- rarely becomes
Hfr while Hfr
remains Hfr
• High transfer of
certain donor
chromosomal
genes
F+
Hfr
Physiological States of F Factor
• Autonomous with
donor genes (F’)
– Characteristics of
F’ x F- crosses
• F- becomes F’
while F’ remains
F’
• High transfer of
donor genes on
F’ and low
transfer of other
donor
chromosomal
genes
Hfr
F’
Mechanism of F+ x F- Crosses
• Pair formation
– Conjugation
bridge
• DNA transfer
– Origin of
transfer
– Rolling
circle
replication
F+
F-
F+
F-
F+
F+
F+
F+
Mechanism of Hfr x F- Crosses
• Pair formation
– Conjugation
bridge
• DNA transfer
Hfr
F-
Hfr
F-
– Origin of transfer
– Rolling circle
replication
• Homologous
recombination
Hfr
F-
Hfr
F-
Mechanism of F’ x F- Crosses
• Pair formation
– Conjugation
bridge
• DNA transfer
F’
F-
F’
F-
F’
F’
F’
F’
– Origin of transfer
– Rolling circle
replication
Conjugation
• Significance
– Gram - bacteria
• Antibiotic resistance
• Rapid spread
– Gram + bacteria
• Production of adhesive material by donor cells
Bacterial Genetics
G.Jamjoom 2005
Lecture Outline:
5. Transposons (jumping genes) :
- role in antibiotic resistance
Transposons
(Transposable Genetic Elements)
• Definition: Segments of DNA that are able
to move from one location to another
• Properties
–
–
–
–
Inverted terminal repeat sequences (loop formation)
“Random” movement from one DNA site to another
Not capable of self replication (not a replicon)
Transposition mediated by site-specific recombination
• Transposase
– Transposition may be accompanied by duplication
Examples of Antibiotic Resistance
Transposons
•
•
•
•
•
•
Tn 1
Tn5
Tn6
Tn9
Tn10
Tn551
ampicillin
kanamycin
Trimethoprim
Chloramphenicol
Tetracyclin
erythromycin
Structure of R Factors
• RTF
RTF
– Conjugative
plasmid
– Transfer genes
• R determinant
– Resistance
genes
– Transposons
R determinant
Mechanism of Plasmid-Mediated Resistance
• Production of enzymes for :
- Hydrolysis of β-lactam ring
- phosphorylation
- adenylation
- acetylation
- methylation
- modification of permeability
- other
Control of Gene Expression
• Transcriptional control
• Clustering of genes with related function
• Coordinate control of genes with related
function
• Polycistronic mRNA
Inducible Genes - Operon Model
• Definition: Genes whose expression is
turned on by the presence of some
substance
– Lactose induces expression of the lac genes
– An antibiotic induces the expression of a
resistance gene
Lactose Operon
• Structural genes
– lac z, lac y, & lac a
– Promoter
– Polycistronic
mRNA
• Regulatory gene
– Repressor
• Operator
• Operon
• Inducer - lactose
Regulatory
Gene
i
Operon
p
o
z
y
a
DNA
m-RNA
Protein
-Galactosidase
Transacetylase
Permease
Lactose Operon
• Inducer -lactose
Absence of lactose
i
p
y
a
No lac mRNA
• Active repressor
• No expression
• Inactivation of
repressor
• Expression
z
Active
– Absence
– Presence
o
Presence of lactose
i
p
o
z
y
a
Inactive
• Negative control
-GalactosidasePermease Transacetylase
Bacterial Genetics
Lecture Outline:
8. Genetic Engineering
- Synthesis of human proteins in
bacteria, e.g. insulin, interferon
- DNA vaccines