Bacterial Genetics G Jamjoom 2005 Bacterial Genetics Lecture

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 MS 2 Virus - Bacteriophage T 2 Virus - Escherichia coli - Saccharomyces - C. elegans - A. thaliana - Drosophila - Mouse - Human Bacterium Yeast 4, 000 bp 21, 000 bp 4, 000 bp 14, 000 bp ~10 genes ~ 200 genes ~4288 genes Nematode 100, 000 bp plant 100, 000 bp Insect 165, 000 bp Mammal 3, 000, 000 bp Mammal 3, 500, 000 bp ~ 40, 000 genes

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 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. T 4) 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

Termination of Lysogeny • Induction – Adverse conditions • Role of proteases bio gal – rec. A protein – Destruction of repressor • Gene expression • Excision • Lytic growth 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 bio l ga gal 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+

Mechanism of Hfr x F- Crosses • Pair formation – Conjugation bridge • DNA transfer Hfr F- – Origin of transfer – Rolling circle replication • Homologous recombination Hfr F-

Mechanism of F’ x F- Crosses • Pair formation – Conjugation bridge • DNA transfer 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 Tn 5 Tn 6 Tn 9 Tn 10 Tn 551 ampicillin kanamycin Trimethoprim Chloramphenicol Tetracyclin erythromycin

Structure of R Factors • RTF Tn 21 T n 9 – Resistance genes – Transposons 0 8 • R determinant 1 n T R determinant Tn – Conjugative plasmid – Transfer genes RTF

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 m. RNA

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 m. RNA • 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 • Negative control y a No lac m. RNA • Active repressor • No expression • Inactivation of repressor • Expression z Active – Absence – Presence of lactose i p o z y a Inactive -Galactosidase. Permease Transacetylase

Bacterial Genetics Lecture Outline: 8. Genetic Engineering - Synthesis of human proteins in bacteria, e. g. insulin, interferon - DNA vaccines