Bacterial Genetics G.Jamjoom 2005. Bacterial Genetics Lecture Outline : 1.The study of bacterial genetics helped illustrate: - the nature of genetic material.

Bacterial GeneticsG.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 4,000 bp ~10 genes

- Bacteriophage T2 Virus 21,000 bp ~ 200 genes

- Escherichia coli Bacterium 4,000,000 bp ~4288 genes

- Saccharomyces Yeast 14,000,000 bp

- C. elegans Nematode 100,000,000 bp

- A. thaliana plant 100,000,000 bp - Drosophila Insect 165,000,000 bp - Mouse Mammal 3,000,000,000 bp - Human Mammal 3,500,000,000 bp ~ 40,000genes

Bacterial Genetics

Lecture Outline:

2. Prokaryotic cells, Eukariotic cells, Archae

PROKARYOTESPROKARYOTES

BACTERIABACTERIA ARCHAEAARCHAEA

EUKARYOTESEUKARYOTES

ProkaryotesProkaryotes

• EubacterEubacter "True" bacteria "True" bacteria – human pathogenshuman pathogens

– clinical or environmentalclinical or environmental

– one kingdomone kingdom

• ArchaeaArchaea – Environmental organisms Environmental organisms

– second kingdom 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

Lecture Outline:

6. Bacteriophages (bacterial viruses):

- virulent

- temparate:

lysogey

G.Jamjoom 2005

Phage Composition and Structure

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath

Genomic DNA

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 FunctionsPlasmid- 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

– Recombination

• Steps– Uptake of DNA

• Gram +• Gram -

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

• Release of phage

• Phage replication and degradation of host DNA

• Assembly of phages particles

• Infection of recipient• Homologous recombination

• Infection of Donor

Potentially any donor gene can be transferred

Events Leading to Lysogeny

• Site-specific recombination

–Phage coded enzyme

• Repression of the phage genome

– Repressor protein– Specific– Immunity to superinfection

gal bio

gal bio

gal

bio

Termination of Lysogeny• Induction

– Adverse conditions

• Role of proteases– recA protein– Destruction of

repressor

• Excision• Lytic growth

gal

bio

gal bio

gal bio

gal bio

• Gene expression

Specialized TransductionLysogenic Phage

• Excision of the prophage

gal

bio

gal bio

gal bio

gal

bio

bio

gal

• Replication and release of phage

• Infection of the recipient

• Lysogenization of the recipient– Homologous

recombination also possible

Conjugation

• Definition: Gene transfer from a donor to a recipient by direct physical contact between cells

• Mating types in bacteria– Donor

• F factor (Fertility factor)– F (sex) pilus

Donor

Recipient

– Recipient• Lacks an F factor

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

• DNA transfer– Origin of

transfer– Rolling circle

replication

• Pair formation

– Conjugation bridge

F+ F- F+ F-

F+ F+F+ F+

Mechanism of Hfr x F- Crosses

• DNA transfer– Origin of transfer– Rolling circle

replication

• Homologous recombination

• Pair formation

– Conjugation bridge

Hfr F- Hfr F-

Hfr F-Hfr F-

Mechanism of F’ x F- Crosses

• DNA transfer– Origin of transfer– Rolling circle

replication

• Pair formation

– Conjugation bridge

F’ F’F’ F’

F’ F- F’ F-

Conjugation

• Significance– Gram - bacteria

• Antibiotic resistance• Rapid spread

– Gram + bacteria• Production of adhesive material by donor cells

Bacterial Genetics

Lecture Outline:

5. Transposons (jumping genes) :

- role in antibiotic resistance

G.Jamjoom 2005

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 ampicillin• Tn5 kanamycin• Tn6 Trimethoprim• Tn9 Chloramphenicol• Tn10 Tetracyclin• Tn551 erythromycin

Structure of R Factors

• RTF– Conjugative

plasmid– Transfer genes

Tn 9

Tn

21

Tn 10

Tn 8

RTF

R determinant

• R determinant– Resistance genes– Transposons

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

i

Operon

RegulatoryGene

p o z y a DNA

m-RNA

-GalactosidasePermease

Transacetylase

Protein

Lactose Operon

• Inducer -- lactose– Absence

• Active repressor• No expression

i p o z y a

No lac mRNA

Absence of lactose

Active

i p o z y a

-Galactosidase Permease Transacetylase

Presence of lactose

Inactive

– Presence• Inactivation of

repressor• Expression

• Negative control

Bacterial Genetics

Lecture Outline:

8. Genetic Engineering

- Synthesis of human proteins in

bacteria, e.g. insulin, interferon

- DNA vaccines