BMSC 210 Microbial Evolution Lectures (Oct 11 & 16) PDF

BMSC 210 Professor Dillon Introduction and Course Expectations Attendance at lectures highly recommended PDF lecture guide/notes available on Canvas. Please read them. Those who wish to may supplement and review lecture materials with readings indicated in...

BMSC 210 Professor Dillon Introduction and Course Expectations Attendance at lectures highly recommended PDF lecture guide/notes available on Canvas. Please read them. Those who wish to may supplement and review lecture materials with readings indicated in lecture slides. Brock Biology of Microorganisms 16th edition; Madigan, Bender, Buckley, Sattley, Stahl (Pearson) Microbiology, Canadian Edition , Wendy Keenleyside (Pressbooks, Toronto) Try and keep up with the lecture material each week. Recorded classes will be posted in Canvas. I am available after each class and will remain outside the classroom to chat. I like chatting with students. [email protected] (I try to answer questions within 24 hours).  Evolution Changes in the Genetic Blueprint of a Microbe BMSC 210 Lectures 16 and 17 October 11 and 16, 2024 Microbiology: Canadian Edition Chapter 12.5 and 12.6 Brock Biology of Microorganisms Chapter 9 (selected sections Pg 263 - 285) Chapter 13 (pages 403 - 412) 2  Mechanisms of Microbial Evolution Origins of genetics diversity Mutation Selection and genetic drift Gain and loss of function Gene families Horizontal gene transfer – mobilome –detecting horizontal gene flow Evolution of microbial genomes 3  The Evolutionary Process Evolution is a change in allele (alternative gene version) frequencies in a population over time Origins of Genetic Diversity Mutations: random changes in DNA sequence over time drive evolutionary process Most mutations are neutral or deleterious; some are beneficial Several forms including substitutions, deletions, insertions, duplications Recombination breaks and rejoins DNA segments to make new combinations of genetic material can reassort genetic material already present required for integration of acquired DNA can be classified as homologous (requiring short flanking segments of similar sequence) or nonhomologous (does not require high similarity) 4  Mutation and Genetic Exchange in Bacteria Mutation: heritable change in genome can lead to a change in properties of an organism some beneficial, some detrimental, most have no effect Exponential growth in prokaryotes accumulates mutations quickly Bacteria can exchange genes. Bacterial and archaeal genetics can exchange genes by horizontal gene transfer (movement of genes between cells other than reproduction) Horizontal gene transfer (genetic exchange) generates much larger changes Mutation and genetic exchange fuel evolution 5  Mutations and Mutants Genomes of bacterial cells: double-stranded DNA; Viral genomes: double- or single-stranded DNA or RNA Wild-type strain: isolated from nature. “Wild-type” can also refer to just one gene Mutant: A cell or virus derived from wild type that carries a nucleotide sequence (genotype) change. Genotype designated by three lowercase letters followed by capital, italicized (e.g., hisC). Mutations designated his C1, his C2, etc. Observable properties (phenotype) may also be altered. Phenotype designated by capital letter and two lower case letters, then +/- (e.g. His+) MacConkey Differential Media contains Maltose as a carbon source  Isolation of Mutants: Screening vs Selection Selectable mutations confer an advantage Under certain environmental conditions, progeny cells outgrow and replace parent. Example: antibiotic resistance. Relatively easy to detect. Powerful genetic tool Nonselectable mutations do not confer a growth advantage even though they may lead to a phenotypic change Example: colour loss in a pigmented organism. Certain markers require laborious, time-consuming screening (examining large numbers and looking for differences) 7  Some Examples of Mutants Phenotype Nature of change Detection of mutant Auxotroph Loss of enzyme in biosynthetic pathway Inability to grow on medium lacking the nutrient Temperature-sensitive Alteration of an essential protein so it is more Inability to grow at a high temperature that normally supports heat-sensitive growth Cold-sensitive Alteration of an essential protein so it is Inability to grow at a low temperature that normally supports inactivated at low temperature growth Drug-resistant Detoxification of drug or alteration of drug Growth on medium containing a normally inhibitory target or permeability to drug concentration of the drug such as an antibiotic Rough colony Loss or change in lipopolysaccharide layer Granular, irregular colonies instead of smooth, glistening colonies Nonencapsulated Loss or modification of surface capsule Small, rough colonies instead of larger, smooth colonies Nonmotile Loss of flagella or nonfunctional flagella Compact instead of flat, spreading colonies; lack of motility by microscopy Pigmentless Loss of enzyme in biosynthetic pathway leading Presence of different color or lack of color to loss of one or more pigments Sugar fermentation Loss of enzyme in degradative pathway Lack of color change on agar containing sugar and a p H indicator. Differential Media Virus-resistant Loss of virus receptor Growth in presence of large amounts of virus 8  Molecular Basis of Mutation Spontaneous mutations Occur without external intervention Most result from occasional errors by DNA polymerase during replication Induced mutations Caused environmentally or deliberately Can result from exposure to natural radiation or chemicals that chemically modify DNA Point mutations Change only one base pair Occurs via single base-pair substitution Phenotypic change depends on exact location  Molecular Basis of Mutations Mutations can lead to changes in the protein sequence encoded by the DNA. 10  Missense, Nonsense and Silent Mutations Not all mutations change polypeptides Silent mutations do not affect sequence of encoded polypeptide or phenotype (e.g., UAC to UAU) Almost always third base of codon because of degeneracy Missense mutation changes sequence of amino acids in polypeptide (e.g., UAC to AAC) If at a critical location, e.g., active site, could alter activity Not all missense mutations lead to dysfunction/nonfunction Nonsense mutation (stop codon) Typically results in truncated (incomplete) protein that lacks normal activity  Frameshift Mutations and Insertions/Deletions Frameshift mutations: single base pair deletions or insertions that result in a shift in the reading frame Scrambles entire polypeptide sequence downstream. Insertion or deletion of two base pairs also causes frameshift. Insertion/deletion of three base pairs adds/deletes a codon/an amino acid, which usually is not as bad Insertions/deletions can result in gain/loss of hundreds to thousands of base pairs. Often result in complete loss of gene function Can be lethal. May arise from errors during genetic recombination. Large insertions may be due to transposable elements  Reversions and Mutation Rates Mutation rates depend on frequency of D N A changes and efficiency of D N A repair For most microorganisms, errors in DNA replication occur at a frequency of 10-6 to 10-8 per kb Single genes in same range Eukaryotes have 10x less error rates DNA viruses have 100-1000x greater and RNA viruses even higher due to lack of proof reading and RNA repair mechanisms Reversions (Back Mutations) and Suppressors Reversion: occurs because point mutations are typically reversible Revertant: strain in which original phenotype is restored Same-site revertant: Mutation is at the same site as original mutation (true revertants restore original sequence) Second-site revertant: Mutation is at a different site in the D N A that restores wild-type if functions as suppressor mutation compensating for the original effect mutation somewhere else in the same gene that restores function mutation in another gene that restores function mutation in another gene that results in production of an enzyme to replace a nonfunctional one Identifying Bacterial Mutants – Replica Plating Identification of auxotrophic mutants, like histidine auxotrophs, is done using replica plating. After mutagenesis, colonies that grow on nutritionally complete medium but not on medium lacking histidine are identified as histidine auxotrophs. 14 Identifying the Carcinogenic Potential of New Chemical Compounds – Ames Test 15  Chemical Mutagenesis Mutagens: chemical, physical, or biological agents that increase mutation rates, induce mutations Nucleoside analogues which have different base pairing functions; 5-Bromouracil can base-pair with guanine, causing A T to GC substitutions. 2-Aminopurine can base-pair with cytosine, causing AT to GC substitutions. Mutagens such as nitrous oxide which modify existing DNA bases (cytosine) to produce uracil which pairs with A. Converts CG base pair to a TA base pair Intercalating agents such as ethidium bromide or acridine results in DNA polymerase introducing a deletion or insertion resulting in a frameshift mutation 16  Radiation IONIZING RADIATION may lead to the formation of NONIONIZING RADIATION like ultraviolet light single-stranded and double-stranded breaks in the can lead to the formation of thymine dimers, sugar-phosphate backbone of DNA, as well as to the which can stall replication and transcription and modification of bases introduce frameshift or point mutations. 17  Chemical and Physical Mutagens and Their Modes of Action Agent Action Result Blank Blank Base analogs 5-Bromouracil Incorporated like T; occasional faulty pairing with G A T yields G C and occasionally G C yields A T AT → GC GC → AT 2-Aminopurine Incorporated like A; faulty pairing with C A T yields G C and occasionally G C yields A T Blank AT → GC Blank GC → AT Chemicals that react with D NA A T yields G C and G C yields A T Nitrous acid H N O sub 2 Deaminates A and C (HNO2 ) AT → GC and GC → AT G C yields A T Hydroxylamine N H sub 2 O H Reacts with C Alkylating agents (NH2OH) Blank GC → AT Blank Monofunctional (for example, ethyl methanesulfonate) Puts methyl on G; faulty pairing with T G C yields A T GC → AT Bifunctional (for example, mitomycin, nitrogen mustards, Cross-links DNA strands; faulty region excised by D nase Both point mutations and deletions nitrosoguanidine) Blank Blank Intercalating agents Acridines, ethidium bromide Inserts between two base pairs Microinsertions and microdeletions Blank Blank Radiation Ultraviolet (U V) Pyrimidine dimer formation Repair may lead to error or deletion Ionizing radiation (for example, X-rays) Free-radical attack on DNA, breaking chain Repair may lead to error or deletion  Mutations can be Corrected – DNA REPAIR 1. Proofreading by DNA polymerase 2. Mismatch repair –errors introduced during replication are corrected 3. Repair of thymidine dimers by a) nucleotide excision repair, or b) photoreactivation 19  Microbial Evolution in Gene Families: Duplications, and Deletions Duplication and deletion has major evolutionary role: Governs genome size; gene content, removing nonessential genes and expanding function; classify genes based on shared ancestry for comparison Homologous genes (homologs): Genes that all descended from single ancestral gene. Tend to have similar nucleotide sequences Orthologs: Homologous genes sharing same function Paralogs: Single ancestral gene diverges to many different functions in many different organisms Gene families: groups of gene homologs Gene duplications thought to drive evolution of gene families and organisms. Duplication creates redundant copy. Second copy can mutate without loss of function. 20 21 Mechanisms of Microbial Evolution: Gene Duplication Orthologs, paralogs and homologs 22 RubisCO Family of Genes (fix CO2 during photosynthesis) 23  Do Gene Duplications Increase Microbial Fitness? A duplication mutation creates a redundant copy of a gene sequence in the genome. The original gene copy retains its function, making it possible for the second copy to accumulate mutations freely without a loss of gene function in the cell. In this way, evolution can “experiment” with one copy of the gene. Deletion mutations that remove biosynthetic genes might have little effect on the fitness of an intracellular pathogen if those biosynthetic functions are provided by the host 24  Gene Deletions in Microbial Genomes Play an important role in microbial genome dynamics In prokaryotic cells, nonessential/nonfunctional materials deleted over time Deletions occur much more often than insertions/duplications Maintains small size of microbial genomes Selection counters effect of deletions, preserving genes that benefit fitness Deletions drive tiny genomes in obligate intracellular symbionts and intracellular pathogens – Metabolites available in host cytoplasm – Deletions removing biosynthetic genes might have little effect on fitness – Also eliminate redundant functions (e.g., insect endosymbionts, mitochondria, chloroplasts) 25  The Evolutionary Process Selection Evolutionary selection is defined by fitness (ability of an organism to produce progeny and contribute to genetic makeup of future generations) Most mutations are neutral (no effect) and accumulate over time Deleterious mutations decrease fitness and are removed by natural selection over time Beneficial mutations increase fitness and are favored by natural selection (e.g., antibiotic resistance during therapy) Mutations occur by chance; environment selects for advantageous mutations Genetic drift: random process that can cause gene frequencies to change over time, resulting in evolution in the absence of natural selection. Some populations have more offspring by chance. Most powerful in small populations and those experiencing frequent “bottleneck” events (severe reduction in population size followed by regrowth from remaining cells, such as pathogens) Evolutionary selection is defined by fitness (ability of an organism to produce progeny and contribute to genetic makeup of future generations) – Most mutations are neutral (no effect) and accumulate over time – Deleterious mutations decrease fitness and are removed by natural selection over time – Beneficial mutations increase fitness and are favored by natural selection (e.g., antibiotic resistance during therapy) – Mutations occur by chance; environment selects for advantageous mutations 27 28 Genetic Drift Genetic drift is an unpredictable change in the gene pool, and it usually limits 29 diversity because some alleles become either eliminated or expressed too much. When a small group of individuals breaks away from a larger population and creates its own population in a separate location, rare alleles could be overrepresented in this newly "founded" population. If this new population is isolated and interbreeds, then the resulting population could have a high frequency of certain traits. The bottleneck effect occurs when a random event, such as a natural disaster, unselectively reduces the size of a population. The resulting population is much less genetically diverse than the original population. Some alleles may become entirely eliminated and some may become overrepresented 30 Survival of the Fittest and Natural Selection in a Population of Phototrophic Purple Bacteria Rhodobacter: anoxygenic phototrophic purple bacterium. In anaerobic culture, photopigments are synthesized. In light, pigments lead to ATP synthesis; in dark, no benefit. In constant darkness, loss of function mutants with reduced levels of photopigments have an advantage due to energy savings and rapidly take over due to fitness. In the light, pigment loss is not advantageous, and mutants are lost. 31  The Evolution of Interdependence in Microbial Communities In nature, microorganisms grow in communities, so deletion preventing production of essential nutrient may not be lethal if nutrient is available elsewhere. Can increase fitness but promote interdependency. Explains need for one or more growth factors in pure culture. 32 Long-Term Evolution of Escherichia coli – Gain of Function Gain of Function Long-term evolution experiment (LTEE) started in 1988 and has tracked 12 parallel lines over 60,000+ generations Culturing in minimal glucose medium represents an adaptive environment in which E. coli can evolve over time A marker that colors cells red or white enables fitness measurement of evolved strains relative to ancestor by competition Dramatic increase in fitness over first 500 generations, then slowed down New ability to use citrate found in only one of 12 lines. Gain of Function. Evolutionary pressures can shift major properties very quickly 33  How Do Organisms Whose Reproduction is Asexual Create Genetic Diversity? Horizontal Gene Transfer Impacts microbial evolution Allows transfer of DNA between distant branches of evolutionary tree At least three mechanisms: transformation, transduction, and conjugation Recombination in which donor DNA passed to recipient and becomes part of recipient genome Differs from sexual recombination: unidirectional, asymmetrical (small amount of DNA), not constrained by species boundaries 3 fates: degradation, replication by itself, recombination with host genome 34  Vertical Versus Horizontal Gene Transfer Vertical gene transfer Horizontal gene transfer Chromosome Mobile Elements: Genome Plasmids, Phage, replication Transposons and and cell Insertion division sequences Horizontal: transfer of genetic information between organisms Vertical: inheritance from parental organism(s) 35  Consequences of horizontal gene transfer Homologous recombination – process that results in genetic exchange between homologous DNA from two sources Gene conversion (homologous recombination results in replacement of recipient copy with donor copy). If no fitness benefit, deleted over time; If a fitness benefit, will persist Effective means for acquiring/evolving new functions/traits 36 Transformation  Transformation Transformation: Genetic transfer process by which free (naked) D N A is incorporated into a recipient cell and brings about genetic change Typical transformable size ~10 genes; 10,000 nucleotides competentence: a cell that can take up D N A and be transformed; genetically determined.In some bacteria, competence linked to pili In Gram-negative Bacteria, proteins within pilus recognize and bind extracellular DNA, pilus retraction pulls DNA into cell Some Gram-negative bacteria require uptake sequences in their DNA to be transformed (N. gonorrhoeae) In Gram-positive bacteria, pili or other proteins can In V. cholerae, The pilus binds extracellular DNA and pilus bring DNA into the cell retraction brings extracellular DNA through the outer NOT all bacteria are competent. Some must be membrane to the competence (Com) system associated with made artificially competent (e.g. E. coli) the cytoplasmic membrane Transformation  Conjugation Conjugation (mating): Horizontal gene transfer that requires cell-to-cell contact Plasmid-encoded Occurs between closely related or distantly related cells Donor cell: contains conjugative plasmid Recipient cell: does not contain plasmid Other genetic elements (e.g., other plasmids or host chromosome) may be mobilized (transferred during conjugation) Conjugation: F Factor Typical conjugation of the F plasmid from an F+ cell to an F− cell is brought about by the conjugation pilus bringing the two cells into contact. A single strand of the F plasmid is transferred to the F− cell, 42 which is then made double stranded.  Hfr and F’ Cells The F plasmid can occasionally integrate into the bacterial chromosome, producing an Hfr (High frequency of recombination) cell. Imprecise excision of the F plasmid from the chromosome of an Hfr cell may lead to the production of an F’ plasmid that carries chromosomal DNA adjacent to the integration site. This F’ plasmid can be transferred to an F− cell by conjugation. 43 Conjugation: Hfr Conjugation Transfer of the Chromosome in an Hfr Isolate to an F- Recipient 45  Transduction Transfer of DNA from one cell to another by a bacteriophage 1. Generalized transduction: DNA from any portion of the host genome is packaged inside the virion Donor genes cannot replicate independently Will be lost without recombination 2. Specialized transduction: DNA from a specific region of the host chromosome is integrated directly into the virus genome, typically replacing some viral genes Homologous recombination may occur or may be integrated during lysogeny occurs in many Bacteria and at least one Archaea examples: multiple-antibiotic-resistance genes in Salmonella, Shiga-like toxins in Escherichia coli, virulence factors in Vibrio cholerae, photosynthetic genes in cyanobacteria Generalized Transduction Generalized Transduction  Specialized Transduction Lysogeny Extremely efficient transfer of genetic material Selective and transfers only small part of bacterial chromosome Phage genome is integrated at specific site (e.g., Lambda in E. coli: next to galactose utilization genes) Viral replication is under control of bacterial host chromosome Upon induction, viral D N A separates via process that reverses integration Sometimes the phage excises incorrectly and takes adjacent host genes along with it, which can be transferred to another cell (specialized transduction) Limit to amount of host D N A that can replace phage D N A, but helper phage can assist Transduction: Specialized Transduction  Transduction – Phage Conversion Alteration of the phenotype of a host cell by lysogenization Prophage from normal, nondefective temperate infection becomes immune to further infection by same phage Selective value for host because of resistance to further similar infection Many natural lysogens found, suggesting this is an essential process for survival e.g., Pathogenic vs nonpathogenic Vibrio cholera and toxin production Mobile Elements (the Mobilome) Promote Genome Evolution 52 Transposon Mutagenesis 53  Mobile DNA: Transposable Elements Insertion Sequences and Transposons Insertion sequences (ISs) Simplest transposable element ~1000 nucleotides long Inverted repeats are 10 to 50 base pairs Only gene encodes transposase Found in chromosomes and plasmids of Bacteria and Archaea and in some bacteriophages Transposons Larger than I S but have same essential components (inverted repeats and transposase) Transposase recognizes inverted repeats and moves any D N A between them between sites Genes inside vary widely (e.g., various antibiotic resistance genes)  Detecting Horizontal Gene Flow presence of genes typically found only in distantly related species signals genes originated from horizontal transfer presence of a DNA with GC content or codon bias that differs significantly from remainder of genome example: Thermotoga maritima, with 400 genes (20 percent) archaeal origin Phylogenetic difference must be large to be readily detectable (e.g., Chlamydia trachomatis contains histone H1-like proteins). Horizontally transferred genes typically do not encode core metabolic functions. 55  The Evolution of Microbial Genomes Chromosomal Islands Comparative genomic analyses show entire genetic pathways (chromosomal/genomic islands) can be acquired via horizontal gene transfer Often flanked by inverted repeats, implying transposition Base composition/codon bias differs significantly compared with genome Chromosomal islands often belong to pan genome Some carry integrase PAI - pathogenicity islands Encode virulence factors (E. coli, S. aureus) In environmental microorganisms can encode pollutant degradation pathways 56  Chromosomal islands Plasmids Transposon Pathogenicity Island. Integrated Core Genome Insertion may not be present in all strains phage DNA 57 Pan and Core Genome 58 The Core and Pan Genome Concept – 3 Genomes from E. coli E. coli strain K-12 and two pathogenic strains only share 39 percent of genes; vary by million base pairs, contain several genes through horizontal gene transfer On average 4721 genes (4068– 5379) Core genome: 1976 genes, less than half the total 59 Pan Genome 60