Bacterial Genetics G.Jamjoom 2005

Bacterial Genetics Lecture Outline : 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: Prokaryotic cells, Eukariotic cells, Archae

EUKARYOTES PROKARYOTES BACTERIA ARCHAEA

Prokaryotes Eubacter "True" bacteria Archaea 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 Lecture Outline: G.Jamjoom 2005 Lecture Outline: 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: - functions 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: 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 gal bio Site-specific recombination Phage coded enzyme Repression of the phage genome Repressor protein Specific Immunity to superinfection

Termination of Lysogeny Induction Adverse conditions Role of proteases recA protein Destruction of repressor gal bio gal bio gal bio Gene expression gal bio Excision Lytic growth

Specialized Transduction Lysogenic Phage Excision of the prophage gal bio Replication and release of phage Infection of the recipient Lysogenization of the recipient Homologous recombination also possible

Conjugation Donor 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 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 Pair formation Conjugation bridge F+ F- DNA transfer Origin of transfer Rolling circle replication

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

Mechanism of F’ x F- Crosses Pair formation Conjugation bridge F’ F- DNA transfer Origin of transfer Rolling circle replication

Conjugation Significance Gram - bacteria Gram + bacteria Antibiotic resistance Rapid spread Gram + bacteria Production of adhesive material by donor cells

Bacterial Genetics Lecture Outline: Transposons (jumping genes) : G.Jamjoom 2005 Lecture Outline: 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 ampicillin Tn5 kanamycin Tn6 Trimethoprim Tn9 Chloramphenicol Tn10 Tetracyclin Tn551 erythromycin

Structure of R Factors RTF R determinant 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 Regulatory gene Operator Operon 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 -Galactosidase Permease Transacetylase Protein

Lactose Operon Inducer -- lactose Negative control Absence Presence p z y a No lac mRNA Absence of lactose Active Inducer -- lactose Absence Active repressor No expression i p o z y a -Galactosidase Permease Transacetylase Presence of lactose Inactive Presence Inactivation of repressor Expression Negative control

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