Welcome to Bacterial Genetics (BIO 4443/6443)

Professor: Dr. Justin Thornton Text: Molecular Genetics of Bacteria, 3rd Edition. (ASM Press) Snyder and Champness

Meets: Mon, Wed, Fri (8:00 - 8:50 a.m.)

Prerequisites: General Microbiology (BIO3304) and recommended General Biochemistry (BCH 4603) Will utilize: MyCourses and Turning Technologies clicker system

Why study bacterial genetics??

Bacteria are relatively simple organisms Many types are easy to manipulate in the lab Central cellular functions have remained largely unchanged throughout evolution Studies of DNA damage and mutagenesis in bacteria have allowed for understanding of such processes in higher organisms DNA repair responses originally identified in bacteria are highly similar in all organisms cancer research Essential for ecology of the Earth: N2-fixation, degradation of recalcitrant natural polymers, detoxification of poisonous compounds, production of greenhouse gases

Why study bacterial genetics??

Some bacteria are capable of surviving in extreme environments Symbiotic bacteria- perform functions which allow other organisms to survive Many bacteria are pathogens of humans, plants, and animals Antibiotics, medicines, foods, chemicals, molecular biology tools Of the 1014 cells in a human, only 10% are human!

The Biological Universe

All organisms on Earth belong to one of three divisions:
Eubacteria
Familiar- Escherichia coli, Staphylococcus aureus Most are single celled and rod shaped or spherical Some are multicellular with complicated developmental cycles Distinguished by biochemical criteria: ribosomal RNA sequence

Archae
Single celled organisms- differ biochemically from Eubacteria Majority are extremeophiles- high temperature, pressure, osmolarity May be more closely related to eukaryotes than eubacteria

Eukaryotes
Diverse organisms- plants, animals, and fungi Can be single or multicellular Very similar at the biochemical level (macromolecular synthesis)

Evolutionary Divergence

Classical vs Reverse Genetics

Genetics- the study/manipulation of DNA to characterize cellular and organismal functions
Classical genetics: mutants that are altered in the function being studied are isolated. The changes in the DNA (mutations) are localized in the chromosome by genetic crosses. Functions of the genes affected can sometimes be deduced by the phenotype displayed. MUTANTS- Individuals that differ from the normal, or wild type, members of the species by an observable attribute, or phenotype

Classical vs Reverse Genetics

Reverse genetics: a gene is first cloned from an organism and altered in a test tube before reintroducing it back into cells to determine the effect of the alteration.
Made possible by the development of modern molecular genetic techniques
Bacteria expressing firefly luciferase

Both classical and reverse genetics have strengths and weaknesses and the two approaches often complement each other

Advantages to studying genetics in bacteria

Bacteria are haploid one copy or allele of each gene
Most higher organisms are diploid (two copies of each gene). With haploid organisms the effects of the mutation are immediate.

Short generation times E. coli can replicate every 20 mins. Asexual reproduction all progeny are genetically identical to parents (clones) Colony formation - ability to isolate clones Can be serially diluted- large numbers of bacteria can be diluted to manageable numbers by series of dilutions

Advantages to studying genetics in bacteria

Selection- (a major advantage)- allows us to identify rare individual mutants among billions of normal bacteria
Selective conditions: nutrients/nutrient limitation, temperature, antibiotics Extremely powerful: 1 in a billion

Storing stocks of bacteria- bacteria can be stored in a dormant state

prevents the need to continuously propagate them Methods: dormant spores, frozen in glycerol, or dried down Useful for accumulating a variety of different clones for genetic experiments

Genetic exchange- transformation, conjugation, transduction

HISTORY Rosalind Franklin and Maurice Wilkins (early 1950s)- X-ray diffraction studies identified that DNA was a double helical structure Based on these and other results in 1953 Francis Crick and James Watson proposed the now famous model for the structure of DNA

Fig 1.1

Composition and Structure of DNA: 1) Right-handed double helix 2) Sugar-phosphate backbone 3) Helixes are connected by hydrogen bonding between nucleobases (purines, pyrimidines) 4) Spacing between helices results in major and minor grooves 5) 1 helical turn = 10 base pairs (3.4 nm length)

Fig 1.2

Bases

Purines two ring structures (A, G) Pyrimidines one ring structures (T, G, and U) Carbons and nitrogens making up rings are numbered sequentially (Know the numbering!)

Fig 1.2

All 4 bases are attached to the 5-carbon sugar deoxyribose Deoxyribose - identical to ribose (found in RNA) except lacks oxygen at 2nd carbon Carbons of sugar are also numbered and designated as prime () to distinguish from carbons of the base

Sugars

Fig 1.2

Deoxynucleotides (nucleotides) = base + sugar + one or more phosphates Deoxynucleosides = base + sugar but no phosphate - No phosphate- deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine - One, two, or three phosphates- deoxynucleoside monophosphates, diphosphates, triphosphates - Individual deoxynucleoside monophosphates are called deoxyadenosine monophosphate, deoxyguanosine monophosphate, etc. (dAMP, dGMP, etc.) - Deoxynucleoside diphosphates and triphosphates (dADP, dGDP, dATP, dGTP, etc.) - Collectively the four deoxynucleoside triphophates are dNTPs

Fig 1.3

Deoxynucleotides are linked together by phosphodiester bonds. The phosphate attached to the 5 carbon of the deoxyribose sugar of one nucleotide is attached to the third (3) carbon of the sugar of the next nucleotide.

Fig 1.3

Antiparallel construction

Free phosphate (5 phosphate end)

Free hydroxyl group (3 hydroxyl end)

Free hydroxyl group (3 hydroxyl end)

Free phosphate (5 phosphate end)

Fig 1.4

Bases pair via hydrogen bonds

Hydrogen bonds (2)

Complimentary base pair Chargaffs Rules: No matter source of DNA, Concentration of C = G Concentration of A = T Complimentary base pair

Hydrogen bonds (3)

Fig 1.6

Mechanism of DNA Replication

Very similar for all organisms on Earth Process involves polymerizing deoxynucleotides using sequence of opposite strand as a template Deoxynucleotides are precursors of DNA synthesis and must be synthesized by a series of enzymatic steps prior to being incorporated into DNA Reductase removes oxygen Kinase adds phosphates Phosphatase removes phosphates Synthetase links together to molecules

Deoxyribonucleotide Precursor Synthesis

Fig 1.5

1. Ribonucleotide reductase reduces ribose sugar to deoxyribose by changing the OH at the 2 position to hydrogen 2. A kinase adds a phosphate to the deoxynucleotide diphosphate to make the dNTP precursor

For dTTP additional steps are required and dUTP serves as a precursor:
3. Phosphatase removes 2 phosphates from dUTP to make dUMP 4. dUMP is converted to dTMP by thymidylate synthetase using tetrahydrofolate to donate a methyl group 5. Kinases then add 2 phosphates to dTMP to make dTTP Replaces donated methyl groups

Fig 1.6

DNA polymerization
Requires enzymes known as DNA polymerases join deoxynucleotides together to make long chains DNA polymerase attaches the 1st phosphate ( phosphate) of one dNTP to the 3 carbon of the sugar of the dNTP previously added to the growing chain Releases the last 2 phosphates ( and phosphates) to produce the energy for the reaction

Replication Requirements
Template strand- T in template strand = A in new strand, etc. DNA polymerase- two types
DNA polymerase III DNA polymerase I

Nucleases- degrade DNA strands by breaking phosphodiester bonds

Endonucleases- initiate breaks in the middle of DNA strand Exonucleases- can remove nucleotides only from the ends of DNA
3 exonucleases- degrade DNA only from the 3 end in the 3-to-5 direction 5 exonucleases- degrade DNA only from the 5 end in the 5-to-3 direction

Ligases- form phosphodiester bonds between the ends of two chains of DNA Primases - make RNA primers to initiate synthesis of new strands of DNA; DNA polymerases cannot initiate the synthesis of new strands of DNA, they can only attach dNTPs to a free 3 OH group

RNA Primers

Replication Requirements
Accessory proteins- travel with DNA polymerase as part of the DNA replication complex. (with the polymerase, they make up the DNA polymerase III holoenzyme)
One protein forms a clamp ( clamp) to keep the DNA polymerase from falling off the DNA Other proteins form a clamp loader to allow the polymerase to release when required (example, during lagging strand synthesis) Additional proteins traveling with the DNA polymerase are exonucleases and serve editing functions to correct any mistakes made by the polymerase

Fig 1.8

Meselson-Stahl Experiment
Density determines position in gradient

E. coli grown in media containing heavy isotope 15N

DNA extracted and density analyzed by density gradient centrifugation

E. coli are transferred to media containing lighter isotope 14N

After 1 generation time, DNA is extracted an analyzed intermediate weight

E. coli divide again in media containing 14N

After 1 generation time, DNA is extracted an analyzed intermediate and light weight DNA is present

SEMICONSERVATIVE REPLICATION

NEXT LECTURE: DNA Replication

READ AHEAD:
Mechanism of DNA replication Replication Errors Chromosomal Replication, Cell Division, Segregation Molecular Biology Applications