BIOL1020 Module 3 - Prokaryotic Genetics

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Dr Paul Ebert

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Prokaryotic Genetics Prokaryotes  Divided into bacteria and archaea  Able to adapt to any environment i.e. acidic, salty, cold, hot  Most are microscopic  Present in massive numbers Structural and functional adaptations  Most are unicellular but some form colonies  Most cells are 0.5-5 µm (20x smaller than eukaryotic cells)  Mostly spheres (cocci), rods (bacilli) or spirals  Cell wall maintains cell shape, provides protection and prevents cell from bursting in a hypotonic environment  Bacterial cell walls contain peptidoglycan, a network of sugar polymers cross-linked by polypeptides  Archaea lack peptidoglycan (unique to bacteria) Gram stain: classification into gram-positive and gram-negative based on cell wall  Gram-negative have less peptidoglycan, an outer membrane that can be toxic and are more likely to be antibiotic resistant Bacteria in the environment  Parasites - Plants - Animals  Mutualists (association between host & microbe) - 10x more bacteria than cells in human - In human guts to absorb nutrients - Nitrogen fixing rhizobia in legumes (uptake nitrogen from soil)  Saprophytes - Degrading organic matter - Recycling Antibiotics Antibiotics target peptidoglycan and damage bacterial cell walls  Do not target human cells due to lack of peptidoglycan Penicillin Discovered 1929 (Fleming)  Substance from contaminating fungus prevented staphylococcal growth Late 1930s-early 1940s (Florey)  Developed process for large scale production  Used to treat infectious disease i.e. WWII Antibiotic resistance Resistance to penicillin reported in some pathogens soon after its introduction  Mutation in bacteria which overcomes antibiotic – quickly multiplies  Chemical variants of original penicillin were produced  Resistance has developed against all other antibiotics  Approaching a situation where totally resistant organisms will emerge Mechanisms to achieve resistance: 1. Enzymes modify chemical structure of antibiotic 2. Antibiotic pumped out of cell 3. Modified target of antibiotic (in bacterium)  Similar to insects resistance to pesticides etc. Resistance to β-lactam antibiotics β-lactam exists in antibiotics i.e. penicillin  Inhibits enzyme for cell wall synthesis in bacteria β-lactamase cleaves the ring structure so no longer functional Genetic basis of antibiotic resistance Antibiotic resistance genes carried on plasmids  Double-stranded DNA circle(s) in most bacteria (approx 10 kbp)  Replicate independent of chromosome  Many contain genes for antibiotic resistance (R-plasmids)  Plasmids have no benefit to host Bacteria achieve resistance by activating antimicrobial resistance genes from eukaryotic cells Mechanisms of DNA transfer between bacteria Conjugation: transfer by direct cell to cell contact via conjugative plasmid Transformation: uptake and incorporation of naked DNA Transduction: transfer to chromosomal or plasmid DNA from cell to cell by a bacterial virus (bacteriophage) Conjugation 1. Pilus of donor cell attaches to recipient 2. Pilus retracts, pulling two cells together 3. Conjugation bridge formed between F+ bacterium to F-recipient 4. Plasmid makes a copy of itself 5. Copied DNA transferred to recipient Plasmids that promote conjugation are referred to as fertility factors (F-plasmids)  Broad host range  Contain multiple antibiotic resistance genes  Large (> 25 kbp) Transformation First reported by Griffith (1928) 1. Non-pathogenic cell takes up a piece of DNA carrying the allele for pathogenicity 2. Replaces own allele with foreign allele (exchange of homologous DNA segments) 3. Cell is now recombinant Harmless R strain pneumococcus became lethal S strain pneumococcus through horizontal gene transfer, transformation and genetic recombination Transduction 1. A phage infects a bacterial cell that carries A+ and B+ alleles on its chromosome (bacterium becomes donor cell) 2. Phage DNA is replicated and the cell makes copies of the proteins encoded by its genes 3. Certain phage proteins halt the synthesis of proteins encoded by the host cell’s DNA and it may be fragmented 4. As new phage particles assemble, a fragment of bacterial DNA carrying the A+ allele happens to be package in a phage capsid 5. The phage carrying the A+ allele from the donor cell infects a recipient cell with alleles A- and B-. Recombination between donor DNA and recipient DNA occurs at 2 places 6. Genotype of resulting recombinant cell (A+B-) differs from genotypes of both donor and recipient Transposon-mediated DNA transfer (within the cell) Transposon: length of DNA that can ‘jump’ from one site to another  Contain genes that promote integration and excision (cut through backbone)  Can integrate into and out of DNA  Gene for antibiotic resistance can be moved as a passenger as transposon is inserted at locations in the gene sequence When transposons are carried on a conjugative plasmid these resistance genes spread rapidly Control of Gene Expression Operon: a unit of linked genes that regulates other genes responsible for protein synthesis (Jacob & Monod 1961)  Consists of an operator (on-off switch), a promoter (adjacent to operator) and genes for proteins that work together  Corepressor and aporepressor combine to form repressor Lac operon: an inducible operon  Usually off but can be activated  Inducer molecule inactivates repressor and turns on transcription Trp operon: a repressible operon  Usually on but can be inhibited  Repressor molecule binds to operator and shuts off transcription Bacterial cell has somewhere between 2500 and 6000 genes – cannot all be expressed all the time  Respond to environmental change by regulating gene expression  RNA transcript covers all genes in the operon  Some regulatory proteins affect multiple genes Lac operon of e coli E. coli lives in the gut  Uses lactose as food  Requires enzymes to metabolise lactose Lactose in gut (environment) + lactose permease  lactose in E. Coli + β -galactosidase  glucose + galactose  Lactose catabolism induced  Increases rate of β-galactosidase synthesis Lactose absent 1. Regulatory protein (Lacl repressor) blocks lac transcription 2. Binds to upstream ‘operator’ sequence 3. Blocks RNA polymerase Lactose present 1. Lactose isomer (allolactose) binds to repressor protein 2. Bound repressor protein changes shape 3. Cannot bind to operator 4. RNA polymerase can bind and transcribe 5. LacZYA genes transcribed Catabolite repression: repression of synthesis of catabolic enzymes in order to use a preferred energy source first  Bacteria use glucose preferentially until it is exhausted  then move to lactose Positive control of lac operon For strong transcription of the lac operon: - Lactose must be present to prevent binding of repressor to the operator (negative control) - Level of cyclic AMP must be high enough so that catabolite activator protein (CAP) binds to the CAP binding site upstream of the promoter (positive control) - CAP stimulates binding of RNA polymerase to promotor  These conditions signal to cell that lactose is present and glucose is absent High glucose = low cAMP = CAP doesn’t bind Low glucose = high cAMP = CAP binds Meal consumed Lactose Glucose Lac operon None (starvation) Low Low CAP bound + operator bound = No transcription Milk High Low CAP bound + operator free = Strong transcription Powerade Low High CAP free + opera
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