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Chapter 08 Lecture Outline See separate PowerPoint slides for all figures and tables preinserted into PowerPoint without notes. Copyright © McGraw-Hill Education. Permission required for reproduction or display. 1 A Glimpse of History Barbara McClintock (1902–1992) • Observed that kernel colors in corn not inherited in a predictable manner • Concluded segments of DNA moved in and out of genes involved in color • Destroyed function of genes • Published results in 1950 • Met with skepticism • DNA believed stable • Idea established by 1970s • Awarded Nobel Prize in 1983 • Termed transposons • “Jumping genes” Antibiotic Resistance Staphylococcus aureus • Gram-positive coccus; commonly called Staph • Frequent cause of skin and wound infections • Since 1970s, treated with penicillin-like antibiotics • For example, methicillin • In 2004, over 60% of S. aureus strains from hospitalized patients were resistant to methicillin • ~2.3 million healthy people in U.S. harbor methicillinresistant S. aureus (MRSA) • Healthcare-associated MRSA (HA-MRSA) resistant to other antibiotics, including vancomycin • Vancomycin considered drug of last resort 8.1. Genetic Change in Bacteria Organisms adapt to changing environments • Natural selection favors those with greater fitness • Bacteria adjust to new circumstances • Regulation of gene expression (Chapter 7) • Genetic change (Chapter 8) • Bacteria excellent system for genetic studies • Rapid growth, large numbers • More known about E. coli genetics than any other • Change in organism’s DNA alters genotype • Sequence of nucleotides in DNA • Bacteria are haploid, so only one copy, no backup • May change observable characteristics, or phenotype • Also influenced by environmental conditions 8.1. Genetic Change in Bacteria Mutation and horizontal gene transfer 8.1. Genetic Change in Bacteria Mutations can change organism’s phenotype • Deletion of gene for tryptophan biosynthesis yields mutant that only grows if tryptophan supplied • Growth factor required; mutant termed auxotroph – Auxo = “increase”; troph = “nourishment” • Prototroph does not require growth factors – Proto = “first” • Geneticists compare mutants to wild type • Typical phenotype of strains isolated from nature • For example, wild-type E. coli strain is prototroph • Strains designated by three-letter abbreviations – for example, Trp– cannot make tryptophan – Streptomycin resistance designated StrR 8.2. Spontaneous Mutations Spontaneous mutations caused by normal processes Occur randomly at infrequent characteristic rates • Mutation rate: probability of mutation each cell division • Typically between 10–4 and 10–12 for a given gene • Mutations passed to progeny • Occasionally change back to original state: reversion • Large populations contain mutants (for example, cells in colony) • Environment selects cells that grow under its conditions • • • • For example, antibiotics select for resistant bacteria if present Environment does not cause mutations Single mutation rare; two even rarer Physicians may use two antibiotics to reduce resistance – Chance is product of mutation rate for each 8.2. Spontaneous Mutations Base substitution most common • Incorrect nucleotide incorporated during DNA synthesis • Point mutation is change of a single base pair 8.2. Spontaneous Mutations Base substitution leads to three possible outcomes • Silent mutation: wild-type amino acid • Missense mutation: different amino acid • Resulting protein may only partially function – Termed leaky • Nonsense mutation: Specifies stop codon – Yields shorter protein 8.2. Spontaneous Mutations Base substitutions • A mutation that inactivates gene is termed a null or knockout mutation • “Silent mutation” sometimes used to indicate mutation that does not alter protein function • Base substitutions more common in aerobic environments • Reactive oxygen species (ROS) produced from O2 • Can oxidize nucleobase guanine – DNA polymerase often mispairs with adenine 8.2. Spontaneous Mutations Deletion or addition of nucleotides • Impact depends on number of nucleotides • Three pairs changes one codon • One amino acid more or less • Impact depends on location within protein • One or two pairs yields frameshift mutation • Different set of codons translated • Often results in premature stop codon • Shortened, nonfunctional protein • Knockout mutation 8.2. Spontaneous Mutations Transposons (jumping genes) • Can move from one location to another • Process is transposition • Gene insertionally inactivated • Function destroyed • Most transposons have transcriptional terminators • Blocks expression of downstream genes in operon 8.2. Spontaneous Mutations Transposons (jumping genes) (continued…) • Classic studies carried out by Barbara McClintock • Observed color variation in corn kernels resulting from transposons moving into and out of genes controlling pigment synthesis 8.3. Induced Mutations Induced mutations result from outside influence • Agent that induces change is mutagen • Geneticists may use mutagens to increase mutation rate • Two general types: chemical, radiation 8.3. Induced Mutations Chemical mutagens may cause base substitutions or frameshift mutations Some chemicals modify nucleobases • Change base-pairing properties • Increase chance of incorrect nucleotide incorporation • Nitrous acid (HNO2) converts cytosine to uracil – Base-pairs with adenine instead of guanine 8.3. Induced Mutations Some chemicals modify nucleobases (cont…) • Alkylating agents add alkyl groups onto nucleobases • Nitrosoguanidine adds methyl group to guanine – Base-pairs with thymine 8.3. Induced Mutations Base analogs resemble nucleobases • Have different hydrogen-bonding properties • Can be mistakenly incorporated by DNA polymerase • 5-bromouracil resembles thymine, often base-pairs with guanine 2-amino purine resembles adenine, often pairs with cytosine 8.3. Induced Mutations Intercalating agents cause frameshift mutations • Flat molecules that intercalate (insert) between adjacent base pairs in DNA strand • Pushes nucleotides apart, produces space • Causes errors during replication • If in template strand, a base pair added to synthesized strand • If in strand being synthesized, a base pair deleted • Often results in premature stop codon • Ethidium bromide is common intercalating agent • Likely carcinogen • Chloroquine, used to treat malaria, is another example 8.3. Induced Mutations Transposition • Transposons can be used to generate mutations • Transposon inserts into cell’s genome • Generally inactivates gene into which it inserts 8.3. Induced Mutations Radiation: two types • Ultraviolet irradiation forms thymine dimers • Covalent bonds between adjacent thymines – Cannot fit into double helix; distorts molecule – Replication and transcription stall at distortion – Cell will die if damage not repaired – Mutations result from cell’s SOS repair mechanism • X rays cause single- and double-strand breaks in DNA – Double-strand breaks often produce lethal deletions • X rays can alter nucleobases 8.4. Repair of Damaged DNA Enormous amount of spontaneous and mutageninduced damage to DNA • If not repaired, can lead to cell death; cancer in animals • For example, in humans, two genes associated with breast cancer code for DNA repair enzymes; mutations in either result in 80% probability of breast cancer • Mutations are rare; alterations in DNA generally repaired before being passed to progeny • Several different DNA repair mechanisms 8.4. Repair of Damaged DNA 8.4. Repair of Errors in Nucleotide Incorporation During replication, DNA polymerase sometimes incorporates wrong nucleotide • Mispairing slightly distorts DNA helix • Recognized by enzymes • Mutation prevented by repairing before DNA replication • Two mechanisms: proofreading, mismatch repair • Proofreading by DNA polymerase • • • • Verifies accuracy Can back up, excise nucleotide Incorporate correct nucleotide Very efficient but not flawless 8.4. Repair of Errors in Nucleotide Incorporation • Mismatch Repair • Fixes errors missed by DNA polymerase • Enzyme cuts sugarphosphate backbone • Another enzyme degrades short region of DNA strand • Methylation of DNA indicates template strand – Methylation takes time, so newly synthesized strand is unmethylated • DNA polymerase, DNA ligase make repairs 8.4. Repair of Modified Nucleobases in DNA Modified nucleobases lead to base substitutions • Glycosylase removes oxidized nucleobase • Another enzyme cuts DNA at this site • DNA polymerase removes short section; synthesizes replacement • DNA ligase seals gap 8.4. Repair of Thymine Dimers Several methods to repair damage from UV light • Photoreactivation: light repair • Enzyme uses energy from light • Breaks covalent bonds of thymine dimer • Only found in bacteria • Excision repair: dark repair • Enzyme removes damage • DNA polymerase, DNA ligase repair Repair of Thymine Dimers Several methods to repair damage from UV light (continued…) • SOS repair: last-ditch repair mechanism • • • • Induced following extensive DNA damage Photoreactivation, excision repair unable to correct DNA and RNA polymerases stall at unrepaired sites Several dozen genes in SOS system activated – Includes a DNA polymerase that synthesizes even in extensively damaged regions – Has no proofreading ability, so errors made – Result is SOS mutagenesis 8.5. Mutant Selection Mutants rare, difficult to isolate • Two main approaches • Direct selection: cells inoculated onto medium that supports growth of mutant but not parent • for example, antibiotic-resistant mutants exposed to antibiotic • Indirect selection: isolates auxotroph from prototrophic parent strain • More difficult since parents will grow on any media on which auxotroph can grow • Replica plating 8.5. Mutant Selection 8.5. Mutant Selection Penicillin enrichment of mutants sometimes used • Increases proportion of auxotrophs in broth culture • Penicillin kills growing cells • Prototrophs • Auxotrophs survive • Penicillinase then added • Cells plated on rich medium 8.5. Mutant Selection Screening for Possible Carcinogens • Carcinogens cause many cancers; most are mutagens • Animal tests expensive, time-consuming • Mutagens increase low frequency of spontaneous reversions • Ames test measures effect of chemical on reversion rate of histidine-requiring Salmonella auxotroph • Uses direct selection • If mutagenic, reversion rate increases relative to control • Rat liver extract added since non-carcinogenic chemicals often converted to carcinogens by animal enzymes • Additional tests on mutagenic chemicals to determine if carcinogenic 8.5. Mutant Selection Horizontal Gene Transfer as a Mechanism of Genetic Change Microorganisms commonly acquire genes from other cells: horizontal gene transfer • Can demonstrate recombinants with auxotrophs • Combine two strains – for example, His–, Trp– with Leu–, Thr– • Spontaneous mutants unlikely • Colonies that can grow on glucose-salts medium most likely acquired genes from other strain Horizontal Gene Transfer as a Mechanism of Genetic Change Genes naturally transferred by three mechanisms • Transformation: naked DNA uptake by bacteria • Transduction: bacterial DNA transfer by viruses • Conjugation: DNA transfer during cell-to-cell contact Horizontal Gene Transfer as a Mechanism of Genetic Change DNA replicated only if replicon • Has origin of replication • Plasmids, chromosomes • DNA fragments added to chromosome via homologous recombination • Only if sequence similar to region of recipient’s genome 8.6. DNA-Mediated Transformation • Naked DNA is not within cell or virus • Cells release when lysed • Addition of DNase prevents transformation • Demonstration of transformation in pneumococci • Only encapsulated cells pathogenic 8.6. DNA-Mediated Transformation Transformation • Recipient cell must be competent • Most take up regardless of origin • Some accept only from closely related bacteria (DNA sequence) • Process tightly regulated • Bacillus subtilis has twocomponent regulatory system • Recognizes low nitrogen or carbon • High concentration of bacteria (quorum sensing) • Only a fraction of population becomes competent 8.7. Transduction Transduction: transfer of genes by bacteriophages • Specialized transduction: specific genes (Chapter 13) • Generalized transduction: any genes of donor cell • Rare error during phage assembly • Transfer of DNA to new bacterial host 8.8. Conjugation Conjugation: DNA transfer between bacterial cells • Requires contact between donor, recipient cells • Conjugative plasmids direct their own transfer • Replicons • F plasmid (fertility) of E. coli most studied • Other plasmids encode resistance to some antibiotics • Spread resistance easily 8.8. Conjugation Conjugation (continued…) • F plasmid of E. coli • F+ cells have, F– do not • Encodes proteins including F pilus • • • • • Sex pilus Brings cells into contact Enzyme cuts plasmid Single strand transferred Complementary strands synthesized • Both cells are now F+ 8.8. Conjugation Chromosomal DNA transfer less common • Involves Hfr cells (high frequency of recombination) • F plasmid is integrated into chromosome via homologous recombination • Process is reversible • F′ plasmid results when small piece of chromosome is removed with F plasmid DNA • F′ is replicon 8.8. Conjugation Chromosomal DNA transfer less common (continued…) • Hfr cell produces F pilus • Transfer begins with genes on one side of origin of transfer of plasmid (in chromosome) • Part of chromosome transferred to recipient cell • Chromosome usually breaks before complete transfer (full transfer would take ~100 minutes) • Recipient cell remains F– since incomplete F plasmid transferred 8.9. The Mobile Gene Pool Genomics reveals surprising variation in gene pool of even a single species • Perhaps 75% of E. coli genes found in all strains • Termed core genome of species • Remaining make up mobile gene pool • Plasmids, transposons, genomic islands, phage DNA 8.9. The Mobile Gene Pool Plasmids found in many bacteria and archaea • • • • • Some eukarya Usually dsDNA with origin of replication Generally nonessential; cells survive loss Few to many genes Low-copy-number • One or a few per cell • High-copy-number • Many up to 500 • Most have narrow host range • Single species • Some broad host range • Includes Gram– and Gram+ 8.9. The Mobile Gene Pool Resistance plasmids (R plasmids) • Resistance to antimicrobial medications, heavy metals (mercury, arsenic) • Compounds found in hospital environments • Often two parts • R genes • RTF (resistance transfer factor) – Codes for conjugation • Often broad host range • Normal microbiota can transfer to pathogens Copyright © 2016 McGraw-Hill Education. Permission required for reproduction or display. Use fig. 8.26 in 8th edition 8.9. The Mobile Gene Pool Transposons provide mechanism for moving DNA • Can move into other replicons in same cell • Simplest is insertion sequence (IS) • Encodes only transposase enzyme, inverted repeats • Composite transposons include one or more genes • Integrate via non-homologous recombination 8.9. The Mobile Gene Pool Transposons yielded vancomycin resistant Staphylococcus aureus strain • Patient infected with S. aureus • Susceptible to vancomycin • Also had vancomycin resistant strain of Enterococcus faecalis • Transferred transposoncontaining plasmid to S. aureus • Transposon jumped to plasmid in S. aureus 8.9. The Mobile Gene Pool Bacteria can conjugate with plants • Natural genetic engineering • Agrobacterium tumefaciens causes crown gall • Different properties, produces opine, plant hormones • Piece of tumor-inducing (Ti) plasmid called T-DNA (transferred DNA) is transferred to plant • Incorporated into plant chromosome via nonhomologous recombination 8.9. The Mobile Gene Pool Genomic islands: large DNA segments in genome • Originated in other species • Nucleobase composition very different from genome • G-C base pair ratio characteristic for each species • May provide different characteristics • • • • Utilization of energy sources Acid tolerance Development of symbiosis Ability to cause disease – Pathogenicity islands