Chapter 8.pdf

Because learning changes everything. ® Chapter 8 Bacterial Genetics Lecture Outline Nester's Microbiology A Human Perspective, Tenth Edition Denise Anderson, Sarah Salm, Mira Beins © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or...

Because learning changes everything. ® Chapter 8 Bacterial Genetics Lecture Outline Nester's Microbiology A Human Perspective, Tenth Edition Denise Anderson, Sarah Salm, Mira Beins © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC. A Glimpse of History Barbara McClintock (1902 to 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” Matt Meadows/ Photolibrary/Getty Images © McGraw Hill, LLC 2 Introduction Through natural selection, organisms adapt to ever-changing environments Bacteria have two general mechanisms to adjust to new circumstances • Regulation of gene expression • Genetic change • E. coli often used as model system of genetic change • Easy to grow, inexpensive, rapid accumulation of large numbers Dr. Gopal Murti/Science Source © McGraw Hill, LLC 3 Genetic Change in Bacteria 1 Two mechanisms of genetic change in bacteria • Mutation: changes in existing nucleotide sequence • Horizontal gene transfer: movement of DNA from one organism to another Changes are passed to progeny by vertical gene transfer © McGraw Hill, LLC 4 Genetic Change in Bacteria - Figure 8.1 Access the text alternative for slide images. © McGraw Hill, LLC 5 Genetic Change in Bacteria 2 Change in organism’s DNA alters genotype • Sequence of nucleotides in DNA • Bacteria are haploid, so only one copy, no backup Change in genotype often changes observable characteristics, or phenotype • Also influenced by environmental conditions © McGraw Hill, LLC 6 Genetic Change in Bacteria 3 Deletion of gene for tryptophan biosynthesis yields a mutant that only grows if tryptophan is 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 • Trp− cannot make tryptophan • Streptomycin resistance designated Str R © McGraw Hill, LLC 7 Spontaneous Mutations Spontaneous mutations are random genetic changes that result from normal cell processes Occur routinely so every large population contains mutants • A gene mutates spontaneously at an infrequent but characteristic rate • Defined as the probability that a mutation will occur in a gene −12 Mutation rate typically between −4 • 10 and 10 for a given gene • Mutations passed to progeny Occasionally change back to original state: reversion • Also occurs spontaneously at low frequencies Single mutation is rare; two even more unlikely • Physicians may give two antimicrobial medications simultaneously to reduce resistance • Chance that a cell will become resistant to both medications is product of mutation rate for each gene © McGraw Hill, LLC 8 Base Substitution - Figure 8.2 Most common type of mutation • Incorrect nucleotide incorporated during DNA synthesis • Point mutation is change of a single base pair Access the text alternative for slide images. © McGraw Hill, LLC 9 Potential Outcomes of Base Substitutions - Figure 8.3 Base substitution: three possible outcomes • Synonymous mutation: codes for same amino acid as original • Can affect efficiency of translation • Missense mutation: creates codon for different amino acid • Resulting protein often does not function normally • Nonsense mutation: creates a stop codon • Yields shorter, often non-functional protein Access the text alternative for slide images. © McGraw Hill, LLC 10 Potential Outcomes of Base Substitutions Base substitutions more common in aerobic environments • Reactive oxygen species (ROS) produced from O2 • Can oxidize nucleobase guanine • DNA polymerase often mispairs with adenine instead of cytosine © McGraw Hill, LLC 11 Deletion or Addition of Nucleotides - Figure 8.4 Impact depends on number of nucleotides involved Addition or deletion of three pairs changes one codon • Causes one amino acid more or less • Impact depends on location within protein Addition or deletion of one or two pairs yields frameshift mutation • Different set of codons translated • Often results in premature stop codon • Shortened, nonfunctional protein Access the text alternative for slide images. © McGraw Hill, LLC 12 Transposons (Jumping Genes) – Figure 8.5 Transposons (jumping genes): pieces of DNA that can move from one location to another in a cell’s genome; process of transposition • Insertional inactivation: gene into which transposon jumps is inactivated; function disrupted • Most transposons have transcriptional terminators • Block expression of downstream genes in operon Access the text alternative for slide images. © McGraw Hill, LLC 13 Induced Mutations - Figure 8.7 Genetic changes that occur due to an influence outside of the cell • Mutagen: agent that induces the change Chemical Mutagens: Chemicals that modify nucleobases • Change base-pairing properties; increase chance of incorrect nucleotide incorporation • Alkylating agents add alkyl groups onto nucleobases • Nitrosoguanidine adds methyl group to guanine • May base-pair with thymine Access the text alternative for slide images. © McGraw Hill, LLC 14 Base Analogs - Figure 8.8 Resemble nucleobases, but have different hydrogen-bonding properties • Can be incorporated into DNA by DNA polymerase • Wrong nucleotide is incorporated into complementary strand during DNA replication • 5-bromouracil resembles thymine, often base-pairs with guanine • 2-amino purine resembles adenine, often pairs with cytosine Access the text alternative for slide images. © McGraw Hill, LLC 15 Intercalating Agents Increase frameshift mutations • Flat molecules that intercalate (insert) between adjacent bases in DNA strand • Pushes nucleotides apart, produces space • Increases chances of insertions or deletions during replication • Often result In premature stop codon • Transposons can be introduced intentionally to generate mutations • Transposon inserts into cell’s genome • Generally inactivates gene into which it inserts © McGraw Hill, LLC 16 Radiation - Figure 8.9 Radiation can be used as mutagen • Ultraviolet light causes thymine dimers (covalent bonds between adjacent thymines) • Distorts molecule; replication and transcription stall • Mutations result from cell’s SOS repair mechanism • X rays cause single- and double-strand breaks in DNA • Double-strand breaks often lethal • Can alter nucleobases Access the text alternative for slide images. © McGraw Hill, LLC 17 Repair of Damaged DNA Enormous amount of spontaneous and mutagen-induced 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 high probability of breast cancer Mutations are rare because alterations in DNA generally repaired before being passed to progeny © McGraw Hill, LLC 18 Repair of Errors in Nucleotide Incorporation During replication, DNA polymerase sometimes incorporates wrong nucleotide Mutation prevented by repairing before DNA replication • Two mechanisms of repair: “proofreading” by DNA polymerases and mismatch repair • Proofreading by DNA polymerases; checks accuracy • Can back up, remove incorrect nucleotide • Inserts correct nucleotide • Very effective but not perfect © McGraw Hill, LLC 19 Mismatch Repair - Figure 8.10 Mismatch Repair fixes errors missed by DNA polymerase • Enzyme cuts sugar-phosphate backbone of new DNA strand • Another enzyme degrades short region of DNA strand with error • Methylation of DNA indicates template strand • Newly synthesized strand is unmethylated • DNA polymerase, DNA ligase fill in and seal the gap Access the text alternative for slide images. © McGraw Hill, LLC 20 Repair of Damaged Nucleobases - Figure 8.11 • Base excision repair uses DNA glycosylase to remove damaged nucleobase • Another enzyme cuts DNA at this site • A DNA polymerase degrades a short section to remove damage; then synthesizes correct replacement • DNA ligase seals gap Access the text alternative for slide images. © McGraw Hill, LLC 21 Repair of Thymine Dimers - Figure 8.12 Photoreactivation: light repair • Enzyme uses energy from light • Breaks covalent bonds of thymine dimer Nucleotide Excision repair: dark repair • Enzyme removes damaged region • DNA polymerase, DNA ligase fill in and seal the gap Access the text alternative for slide images. © McGraw Hill, LLC 22 SOS Repair SOS repair: last-ditch repair mechanism used when other systems fail • Induced following extensive DNA damage that stalls DNA and RNA polymerases • Several dozen genes in SOS system are expressed • Includes a DNA polymerase that synthesizes even in extensively damaged regions • Has no proofreading ability, so errors made • Results in SOS mutagenesis © McGraw Hill, LLC 23 Mutant Selection Mutations are rare events even when mutagens are used Presents challenges for isolating a desired mutant Two methods: • Direct and Indirect Selection are used to isolate mutants © McGraw Hill, LLC 24 Direct Selection - Figure 8.13 Cells inoculated onto medium that supports growth of mutant but not parent • Antibiotic-resistant mutants grow on medium with the antibiotic, but parents do not Access the text alternative for slide images. © McGraw Hill, LLC 25 Indirect Selection Isolates auxotroph from prototrophic parent strain • More difficult because no medium allows growth of auxotrophs but not prototrophs • Method involves replica plating which indirectly selects auxotrophs • Master plate with mutant and non-mutant cells on nutrient agar is pressed onto velvet • Velvet with adhering cells pressed onto two agar plates—one nutrient agar and one glucose-salts agar in consistent orientation • All cells will form colonies on nutrient agar • Auxotrophs fail to grow on glucose-salts agar • Colonies that are missing on glucose-salts agar allow identification of auxotrophs on master plate © McGraw Hill, LLC 26 Indirect Selection by Replica Plating - Figure 8.14 Access the text alternative for slide images. © McGraw Hill, LLC 27 Penicillin Enrichment - Figure 8.15 Selectively kills prototrophs • Increases auxotrophs before replica plating • Penicillin kills only growing cells • Prototrophs grow in glucosesalts medium; auxotrophs do not • Penicillinase added before cells are plated on nutrient agar to create master plate Access the text alternative for slide images. © McGraw Hill, LLC 28 Screening for Possible Carcinogens Carcinogens cause many cancers; most are mutagens • Animal tests expensive, time-consuming; quicker and cheaper to test mutagenic effect of chemicals in microbiological systems • Mutagens increase low frequency of spontaneous reversions • Ames test measures effect of chemical on reversion rate of histidine-requiring Salmonella auxotroph • Uses direct selection on glucose-salts plate • Only prototrophs that have undergone reversion can grow • If chemical is mutagenic, reversion rate increases relative to control (more colonies grow) • Rat liver extract may be added since non-carcinogenic chemicals may be converted to carcinogens by animal enzymes • Additional tests on mutagenic chemicals to determine if they are also carcinogenic © McGraw Hill, LLC 29 Ames Test to Screen for Mutagens - Figure 8.16 Access the text alternative for slide images. © McGraw Hill, LLC 30 Overview of Horizontal Gene Transfer 1 Microorganisms acquire genes from other cells by horizontal gene transfer (HGT) • Allow organisms to change and adapt • Scientists can use the to intentionally move DNA from one organism to another. • The resulting recombinant cells have properties of each of the original strains © McGraw Hill, LLC 31 Demonstration of Horizontal Gene Transfer - Figure 8.17 Two bacterial strains are used, neither can grow on a glucose-salts medium because of multiple growth factor requirements. • Strain A is His- , Trp- (requires histidine and tryptophan in the medium) • Strain B is Leu−, Thr− (requires leucine and threonine in the medium). Strains are mixed on a glucose-salts agar plate Colonies form only if cells of one strain acquired genes from the other strain. − − Access the text alternative for slide images. © McGraw Hill, LLC 32 Overview of Horizontal Gene Transfer 2 Genes naturally transferred by three mechanisms • Bacterial Transformation: “naked” DNA taken up from the environment • Transduction: bacterial DNA transfer by a virus • Conjugation: DNA transfer during cell-to-cell contact © McGraw Hill, LLC 33 Mechanisms of Horizontal Gene Transfer – Figure 8.18 © McGraw Hill, LLC 34 Homologous Recombination - Figure 8.19 Transferred DNA replicated only if a replicon with origin of replication • Chromosomes, plasmids • Not DNA fragments DNA fragments must be integrated into a replicon to be reproduced Homologous recombination • Donor DNA replaces complementary region of recipient cell’s DNA Access the text alternative for slide images. © McGraw Hill, LLC 35 Bacterial Transformation Also referred to as DNA-mediated transformation Transformation involves uptake of naked DNA • DNA not within a cell or virus • Originates from cells that have burst or secreted it • Addition of DNase prevents transformation Recipient cell must be competent • Most take up DNA regardless of origin; some accept DNA only from closely related bacteria • Some species are always competent; others become so under certain conditions © McGraw Hill, LLC 36 Bacterial Transformation – Figure 8.20 • A double-stranded donor DNA encoding streptomycin resistance (StrR) binds to receptor on surface of the competent cell • One strand enters the cell; nucleases degrade other strand • New DNA integrates into chromosome by homologous recombination • One daughter cell will inherit donor DNA • Transformed cell grows on a medium containing streptomycin • Other donor genes are possible Access the text alternative for slide images. © McGraw Hill, LLC 37 Focus Your Perspective 8.1 Transformation demonstrated by Griffith in 1920 • Streptococcus pneumoniae (“pneumococcus”) only pathogenic when encapsulated • Living non-encapsulated cells were transformed by DNA from heat-killed encapsulated cells and produced living encapsulated cells Access the text alternative for slide images. © McGraw Hill, LLC 38 Transduction Transduction: transfer of bacterial genes by bacteriophages (phages) • Phages infect bacterial cells • Attaches to cell and injects its nucleic acid • Phage enzymes cut bacterial DNA into small pieces • Bacterial cell enzymes produce phage nucleic acid and a phage coat – components of new phage particles • Phage particles are released from bacterial cell • Generalized transduction results when a fragment of bacterial DNA enters the phage protein coat • Produces a transducing particle © McGraw Hill, LLC 39 Transduction - Figure 8.21 Transducing particle may attach to another bacterial cell and inject the DNA it contains • New DNA may be integrated into chromosome Access the text alternative for slide images. © McGraw Hill, LLC 40 Conjugation - Figure 8.22 Conjugation: DNA transfer between bacterial cells • Requires contact between donor, recipient cells • Conjugative plasmids direct their own transfer • Replicons; do not have to integrate into chromosome • F plasmid (fertility) of E. coli most studied • Encodes F pilus (sex pilus) • F+ cells contain F plasmid; F− cells do not • Other plasmids encode resistance to some antibiotics • Spread resistance easily Dennis Kunkel/SPL/Science Source © McGraw Hill, LLC 41 Plasmid Transfer - Figure 8.23 • F pilus binds to receptor on recipient cell wall • F pilus contracts, pulling cells together • Enzyme cuts plasmid at origin of transfer • Single DNA strand is transferred • Complementary strands synthesized • Both cells are now F+ Access the text alternative for slide images. © McGraw Hill, LLC 42 Chromosome Transfer - Figure 8.24 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′ plasmid is replicon; transferred to F− cells • Carries bacterial DNA into new cells Access the text alternative for slide images. © McGraw Hill, LLC 43 Conjugation - Figure 8.25 Chromosome transfer • Hfr cell produces F pilus • Integrated F plasmid begins transfer of genes on one side of origin of transfer of plasmid • Part of chromosome transferred to recipient cell; chromosome usually breaks before complete transfer • Recipient cell remains F− since incomplete F plasmid transferred Access the text alternative for slide images. © McGraw Hill, LLC 44 Genome Variability - Figure 8.26 Much variation in the genes of different strains of a single species • Less than 50% of E. coli genes found in all strains • Led to the concept of a pan-genome • Consists of three sets of genes • Core genome is common to all strains • the accessory genome is present in more than one but not all strains • unique genes are found in only one strain of the species). Mobile genetic elements can move from one DNA molecule to another Includes plasmids, transposons, genomic islands, phage DNA © McGraw Hill, LLC 45 Mobile Genetic Elements (MGEs) Plasmids common in microbial world • Usually circular double stranded DNA with origin of replication • Generally encode nonessential information, but may allow survival in particular environment • Variable, few to many genes • Low-copy-number (few per cell) to high-copy-number (up to 500) • Most have narrow host range; some replicate in many different species • Conjugative plasmids carry all genetic information for transfer • Mobilizable plasmid requires conjugative plasmid for transfer • Some plasmids can be transferred to various unrelated species – even plants © McGraw Hill, LLC 46 Table 8.3 Some Plasmid-Encoded Traits Trait Organisms in Which Trait is Found Antibiotic resistance Many Antibiotic synthesis Streptomyces species Gas vacuole production Halobacterium species Increased virulence Yersinia and Shigella species Insect toxin synthesis Bacillus thuringiensis Nitrogen fixation Rhizobium species Oil degradation Pseudomonas species Pilus synthesis E. coli, Pseudomonas species Toxin production Bacillus anthracis Tumor formation in plants Agrobacterium species (see Focus Your Perspective 8.2) © McGraw Hill, LLC 47 Focus Your Perspective 8.2 Bacteria can conjugate with plants • Agrobacterium tumefaciens causes crown gall • Cells multiply without hormones and produce opine • Piece of tumor-inducing (Ti) plasmid called T-DNA (transferred DNA) is incorporated into plant chromosome • Tumor formation genes can be replaced with beneficial genes and incorporated into plant genome Access the text alternative for slide images. © McGraw Hill, LLC 48 Resistance or R Plasmids - Figure 8.27 • • • • Encode resistance to antimicrobial medications Many are conjugative plasmids with broad host range Resistance to many medications can spread quickly Normal microbiota with R plasmids may transfer them to pathogens Access the text alternative for slide images. © McGraw Hill, LLC 49 Transposons - Figure 8.28 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 Access the text alternative for slide images. © McGraw Hill, LLC 50 Genomic Islands Genomic islands: large DNA segments in genome that originated in other species • Nucleotide composition very different from genome • G-C base pair ratio characteristic for each species • Characteristics encoded by genomic islands include • Use of specific energy sources • Acid tolerance • Ability to cause disease • Pathogenicity islands © McGraw Hill, LLC 51 Focus on a Case 8.1 Transposons yielded vancomycin-resistant Staphylococcus aureus strain • Patient infected with S. aureus that was susceptible to vancomycin • Normal microbiota included vancomycin-resistant strain of Enterococcus faecalis • Resistance encoded in transposon of plasmid transferred to S. aureus • Transposon jumped to plasmid in S. aureus Access the text alternative for slide images. © McGraw Hill, LLC 52 Phage DNA Certain types of phages can insert their DNA into the host cell chromosome • Phage DNA becomes part of the host cell’s genome • Will be replicated and passed on to progeny cells • Such phage DNA is called a prophage. © McGraw Hill, LLC 53 Bacterial Defenses Against Invading DNA Some bacteria recognize and destroy foreign DNA that enters the cell • Systems likely evolved as defenses against phages • Important in biotechnology because they allow scientists to cut DNA at precise nucleotide sequences • Once a cut has been made, the nucleotide sequence at that site can be manipulated © McGraw Hill, LLC 54 Restriction-Modification Systems - Figure 8.29 Bacteria use to degrade foreign DNA from phage infection • Restriction enzyme cuts DNA at specific sequence • Modification enzyme protects cell’s own DNA by adding methyl groups Access the text alternative for slide images. © McGraw Hill, LLC 55 CRISPR Systems CRISPR systems include small segments of phage DNA that recognize the specific DNA if it invades the cell again • First invasion: complex of Cas proteins cuts DNA into short fragments • Inserted into chromosome at CRISPR array; integrated DNA fragment called a spacer • Subsequent invasion: transcription of CRISPR array generates crRNAs that direct DNA-cutting enzymes to invading DNA © McGraw Hill, LLC 56 CRISPR System - Figure 8.30 Access the text alternative for slide images. © McGraw Hill, LLC 57 Because learning changes everything. www.mheducation.com © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC. ®