Microbio - Review Exam 2

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Biology 225
Study Notes Exam 2 Chapter 8: Microbial Genetics
Genetics – science of heredity Genome: all the DNA in a cell - DNA chains in cell are organized into chromosomes - DNA in nucleus is organized into chromatin (DNA in chromosomes + complex of proteins) DNA chains (strands) pair to form a double helix - each strand in double helix has a sugar-phosphate backbone, with a nitrogenous base attached to each sugar - base pairs form in the center of the double helix by complementary base pairing of nitrogenous bases - Adenine hydrogen bonds to Thymine; Cytosine hydrogen bonds to Guanine The genetic code in a gene is translated to the amino acid sequence in a polypeptide Genotype: the genetic makeup or genetic information that codes for characteristics of an organism Phenotype: the traits specified (expressed) by an organism’s genotype Bacteria generally have a single circular chromosome attached at one or more points to the plasma membrane - The E. coli genome is about 4 million bases (base pairs) & ~ 1 mm in length - Chromosome is packaged to take up only ~ 10% of cell - Genes can be (& have been) roughly mapped to specific regions of the chromosome by conjugation experiments - Genomics – the sequencing & characterization of genomes – can be (& has been) used to determine the sequence of the entire genome DNA Replication: DNA replication is carried out by the enzyme DNA Polymerase, as well as some additional protein factors - the double helix is unwound (hydrogen bonds between complementary bases are broken) in preparation for replication - Replication is unidirectional (5’ to 3’). One strand (the leading strand) is synthesized continuously, while the other strand (the lagging strand which is in the 3’ to 5’ direction) is synthesized discontinuously in short fragments in the 5’ to 3’ direction… the fragments are sealed together by DNA ligase - DNA Polymerase has a proofreading activity to correct replication errors (adding the wrong base). The corrected error rate (after proofreading) is about 1 in 1 billion bases - DNA replication is semiconservative:
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each newly replicated DNA molecule consists of 1 old strand from the original double- stranded DNA molecule, and 1 newly synthesized strand DNA replication is endergonic (requires energy)… the energy required is supplied by the nucleotides - cleavage of phosphates in a nucleotide triphosphate releases energy (high energy phosphate bonds, as in ATP (an RNA nucleotide)

Transcription: Transcription is carried out by a 5’ to 3’ RNA Polymerase, as well as additional protein factors - a strand of mRNA is synthesized by complementary base pairing with the sense strand of the DNA within a gene (producing a short term copy of the gene that the cell can use to synthesize the gene product (polypeptide) for that gene - RNA polymerase binds to the promoter region of the gene to begin transcription - RNA polymerase moves along the DNA within the gene, synthesizing the corresponding strand of RNA, until the terminator region is reached… at which point RNA polymerase & the new mRNA are released - In eukaryotes, the RNA synthesized by RNA polymerase must be modified to produce the mature RNA sent to the ribosome for translation (5’ cap & poly-A tail are added, & exons are spliced together) Translation: Translation occurs at the ribosomes - rRNA along with proteins comprise the structure of the 2 subunits of the ribosome - mRNA moves to the ribosome to begin translation - Ribosome subunits associate immediately prior to translation, and dissociate following translation - Ribosomes generally begin translation at the first AUG (start) codon - The AUG start codon specifies formylmethionine in bacterial cells (elsewhere AUG specifies methionine) & methionine in eukaryotic cells - The ribosome moves along the mRNA in the 5’ to 3’ direction… as soon as the AUG start codon is exposed, another ribosome can bind & start translation of the same mRNA - each sequence of 3 nucleotides (codon) following the start codon in mRNA specifies an amino acid in the polypeptide - most amino acids are coded for by more than one codon (code is degenerate) - tRNA molecules carry amino acids to the ribosome during translation (a tRNA for each amino acid) - anticodon on tRNA binds to codon on mRNA - One of 3 nonsense codons (stop codons; all other codons are sense codons) signals the ribosome to stop translation of the mRNA… following translation, a release factor cleaves the complete polypeptide from the last tRNA and the ribosome, and the polypeptide leaves the ribosome

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- In eukaryotes, transcription occurs in the nucleus, while translation occurs in the
cytoplasm… transcription must be completed & mRNA sent into the cytoplasm before translation can begin - In bacteria, translation can begin before transcription is complete (no nucleus) Regulation of Gene Expression in Bacteria: - some genes whose products are needed constantly by the cell (genes for glycolysis enzymes) are transcribed & translated constitutively (at a fixed rate) in the cell - regulation of gene expression is energy efficient – only produce the gene product when needed - Repression & Induction regulate gene expression at the transcriptional level (regulate mRNA synthesis for a gene) - Repression inhibits gene expression, generally in response to the overabundance of the gene product (usually an enzyme) - Uses a repressor to block binding of RNA Polymerase to the promoter - Induction turns on (upregulates) expression of a gene product (also, usually an enzyme – enzyme induction) - Uses an inducer to block function of the repressor - A common example in bacteria is regulation of β-galactosidase, an enzyme involved in lactose metabolism Operon Model of Gene Expression: - formulated by Jacob & Monod in 1961… based on inducible enzymes for lactose catabolism (lac operon) in E. coli - structural genes code for enzyme gene products - in bacteria, a group of coordinately regulated structural genes with related metabolic functions, plus promoter & operator sites controlling their transcription, is an operon - in an inducible operon (e.g.: lac operon), a regulatory gene codes for a repressor protein - when inducer is absent, the repressor binds to the operator & stops transcription - when inducer is present, it binds to the repressor & transcription is free to proceed - in a repressible operon (e.g.: trp operon), a repressor requires a corepressor to bind to the operator - structural gene transcription is induced by the absence of glucose… cyclic AMP binds to its receptor protein, CRP, which binds to the lac promoter to turn on transcription - glucose effects catabolic repression… when present, cAMP levels are low & CRP cannot bind to the operator to induce transcription Mutation: a change in the base sequence of DNA - Base substitution: a single base is replaced with a different base - If the base substitution results in a codon change & an amino acid substitution, it is called a missense mutation - If the base substitution results in a nonsense codon, it is called a nonsense mutation
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Nucleotide insertions or deletions can result in a frameshift mutation… the reading frame & codons (& amino acids in the polypeptide) change from the site of the mutation Spontaneous mutations occur in the absence of any mutation-causing agent Mutagens are agents that cause DNA mutations

Mutagens: - Chemical mutagens: nitrous acid causes base modifications & subsequent base pairing anomalies; nucleoside analogs ( such as 5-bromouracil & AZT) are structurally similar to normal nucleotides, but have altered base pairing properties - Ionizing radiation causes ions & free radicals to form, that react with DNA & cause base substitutions & DNA breaks - UV light causes thymine-dimers - UV damage to DNA can be repaired by repair enzymes that excise & replace the damaged DNA (deficiency in these repair enzymes leads to disorders such as Xeroderma pigmentosum (XP)) Mutation rate: - expressed as 10 to a negative power - mutations generally occur at random locations along chromosome - low spontaneous mutation rates are beneficial for diversity of life Mutant identification: - mutants can be detected & selected by testing for an altered phenotype - replica plating used for negative selection - auxotrophs (require a specific nutrient for growth) can be selected by growing cells in media without the nutrient Identification of Carcinogens - Ames test used for identification of chemical carcinogens - Tests reversion of histidine auxotrophs of Salmonella Genetic Transfer & Recombination: - genetic recombination: the exchange of genes between 2 DNA molecules to form new combinations of genes on a chromosome - crossing over: homologous chromosomes break & rejoin, exchanging information from the breakpoint on (occurs normally during metaphase I of meiosis) - vertical gene transfer: genes are passed from an organism to its offspring - horizontal gene transfer: during conjugation in bacteria, a portion of a cell’s DNA is transferred to a recipient cell - when donor DNA has been integrated into a recipient’s DNA, the resulting cell is a recombinant Bacterial Transformation: genes are transferred from one bacterium (or from the solution) to another bacterium as “naked” DNA
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Bacterial Conjugation: DNA is transferred from one bacterial cell to another by means of a (sex) pilus - F+ cells have a plasmid (F factor) that is transferred to F- cells; when the plasmid is incorporated into the recipient’s chromosome, that cell is a Hfr cell - Hfr cells can transfer portions of chromosomal DNA to F- cells Bacterial Transduction: DNA is transferred from one bacterium to another in a bacteriophage, which is then incorporated into the recipient’s DNA Plasmids: self-replicating extrachromosomal circular molecules of DNA carrying nonessential genes - contain an origin of replication - antibiotic resistance genes can be included in a plasmid to select bacterial cells that have taken up the plasmid - α-complementation uses a portion of the β-galactosidase gene in both the plasmid & the recipient cell… a gene of interest can be inserted in the plasmid within the plasmid’s β-galactosidase gene – results in a nonfunctional β-galactosidase - can be used in blue-white selection to identify recombinant bacteria containing the gene of interest Transposons: small DNA fragments that can “jump” from one region to another region on the same or different chromosome or plasmid - found in chromosomal DNA, plasmids & viral DNA - bacterial transposons may contain genes for enterotoxin or antibiotic resistance, but there are likely no limits on the kinds of genes transposons can have

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Chapter 9: Biotechnology & Recombinant DNA
Biotechnology: the use of microorganisms, cells, or cell components to make a product Genetic Engineering: manufacturing & manipulating genetic material in vitro using recombinant DNA technology - genes from one organism can be inserted into the genome of another organism - transgenic: an organism carrying an inserted foreign in its genome Recombinant DNA Techniques: Vector: a plasmid or virus used to insert foreign DNA into a cell - should be self-replicating Clone: many identical cells originating from one cell Tools of Biotechnology: Selection: - natural selection: survival of fittest - artificial selection: used in biotechnology to select cells with desirable characteristics (the gene of interest) Mutation: alters the nucleotide sequence or chemical properties of nucleotides in a DNA molecule - site-directed mutagenesis: make a specific change in a gene Restriction enzymes: cleave DNA molecules at specific nucleotide sequences - may be sensitive to methylated DNA - may produce blunt ends or sticky ends Other Important Enzymes: DNA Polymerase: makes DNA from a DNA template RNA Polymerase: makes RNA from a DNA template Reverse Transcriptase: makes cDNA from an RNA template Vectors: serve as vehicles for the introduction & replication of desired DNA sequences (genes) in a host cell - must be self-replicating in host cell; need origin of replication - plasmid vectors (circular DNA constructs with required DNA sequences/genes) - viral vectors (from retroviruses, adenoviruses or herpesviruses) Polymerase Chain Reaction (PCR): - amplify specific DNA sequences from a population of DNA molecules (that is, PCR makes multiple copies of a desired DNA fragment enzymatically) - can be used to increase amount of a DNA sequence in a sample to detectable levels - template DNA - product DNA
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primer DNA thermostable DNA Polymerase (from Thermus aquaticus or other thermophilic microbe) thermal cycler: automates cycling process (20-30 cycles)… 3 steps per cycle: o denaturation: double-stranded DNA o annealing: primer binds (anneals) to template DNA (and DNA returns to doublestranded state) o extension: DNA sequence between primers is filled in

Inserting Foreign DNA into cells: Transformation: cells (bacteria) take up DNA from surrounding environment - competent cells: cells treated chemically to enhance uptake of DNA from medium o required for transformation of most organisms Electroporation: uses electrical current to make pores in the plasma membrane; the DNA enters cells through the pores - organisms with cell walls are converted to protoplasts (see below) first Protoplast fusion: protoplasts are cells in which the cell wall has been enzymatically removed - the nuclei of protoplasts can be fused to incorporate new genes/DNA Gene Gun: for plant cells… shoots DNA through cell wall into plant cell Microinjection: for animal cells… DNA injected into nucleus of cell Obtaining DNA: Gene Library: a pool of DNA fragments cloned into vectors corresponding to a specific portion of, or the complete, genome of an organism - uses restriction enzymes to create DNA fragments in a specific size range - eukaryotic genes contain introns & exons, that are spliced to form the mRNA for producing the gene product (polypeptide) - cDNA (complementary DNA) libraries can be constructed using mRNA & reverse transcriptase Selection of a clone: - uses selective media with an antibiotic… the resistance gene for the antibiotic is contained in the cloning vector - alpha complementation: uses a portion of the beta-galactosidase gene in the cloning vector & the complementary portion of the gene in the competent cells Colony hybridization: identify clones (colonies) with a gene of interest - DNA probes used to screen colonies… probe will bind to DNA from colonies with desired gene Making a gene product: - transgenic bacteria
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transgenic yeast transgenic plants transgenic animals/mammalian cells

Applications of genetic engineering: Therapeutic applications: - large-scale synthesis of pharmaceutical products (e.g.: insulin) using bacteria transformed with insulin genes - subunit vaccines: contain only a portion of a protein from a pathogen - gene therapy

Scientific applications: - Southern blotting: used to locate a gene/DNA sequence in cellular DNA o can be used to screen for mutations - RFLPs (Restriction Fragment Length Polymorphisms): differences in DNA fragments created by restriction enzymes in different individuals - DNA fingerprinting: comparison of a DNA sample with known DNA samples to determine identity (can be used in forensics, to determine disease pathology, etc.) Agricultural applications: - Agrobacterium; Ti plasmid used to engineer plants with desired genes/characteristics - Antisense DNA technology: DNA complementary to mRNA for a troublesome gene used to bind the cellular mRNA & prevent translation Safety Issues & Ethics of Genetic Engineering: - cloning - human genome project

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Chapter 10: Classification of Microorganisms
Systematics (phylogeny): the study of the evolutionary history of a group of organisms - reveals evolutionary or phylogenetic relationships - taxa: taxonomic categories The 3 domains: Archaea: prokaryotes with NO peptidoglycan in their cell wall - often live in extreme environments & have unusual metabolism - methanogens: strict anaerobes that produce methane from CO2 & hydrogen - extreme halophiles: require high salt concentrations for survival - hyperthermophiles: grow in hot, acidic environments Bacteria: prokaryotes with peptidoglycan in their cell walls - includes all pathogenic prokaryotes & many nonpathogenic prokaryotes found in soil & water - also includes photoautotrophic prokaryotes Eukarya: all eukaryotes - includes animals, plants, protists & fungi - endosymbiotic theory: eukaryotic cells evolved from prokaryotic cells living inside one another (mitochondrion resulted from internal bacterium) Classification of Organisms: Scientific Nomenclature: - binomial nomenclature: each organism has 2 names - scientific name: genus & species name - examples: Homo sapiens, Rhizopus nigricans, Streptococcus pneumoniae - eukaryotic species: a group of closely related organisms that interbreed Taxonomic Hierarchy: Domain Kingdom Phylum or Division Class Order Family Genus Species

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Prokaryotes: - divided into 2 domains: Bacteria & Archaea - Kingdom: Prokaryotae or Monera (domain names typically used) - classification based on rRNA similarities - prokaryotic species: a population of cells with similar characteristics - strain: a collection of cells derived from a single cell o a single prokaryotic species may have several strains that differ in certain features Eukaryotes (domain Eukarya): - Kingdom Protista: simple eukaryotes; mostly unicellular Kingdom Fungi: includes unicellular yeasts & multicellular molds & mushrooms o extracellular digestion & absorption of nutrients o cells join to form hyphae o develop from spores or hyphal fragments Kingdom Plantae: includes some algae & all mosses, ferns, conifers & flowering plants o multicellular; carry out photosynthesis Kingdom Animalia: includes sponges, worms, insects & animals with backbones o multicellular; obtain nutrients by ingesting organic matter

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Viruses: acellular; abiotic; obligate intracellular parasites - not classified in 3 domains because they don’t have ribosomes - use anabolic machinery in living host cells to multiply - viral species: population of viruses with similar characteristics occupying a particular ecological niche Methods for Classification: Morphological characteristics: uses differences in cell shape & arrangement, & differences in such structures as endospores & flagella to distinguish organisms Differential staining: - Gram stain - Acid-fast stain - Endospore stain Biochemical tests: enzymatic activities - some examples: o ability to ferment a particular carbohydrate  end products of fermentation o synthesis of a specific enzyme Serology: studies blood serum & immune responses evident in serum - antiserum: antibody solutions used in identification of medically important microbes
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slide agglutination test: tests bacteria for antigens that react with antibody – if positive, agglutination or clumping is observed ELISA: enzyme-linked immunosorbent assay; tests ability of bacteria to react with known antibody placed in wells of microplate Western blotting: used to identify bacterial antigens in a patient’s serum o proteins from serum separated by size, placed on filter & subjected to known antibody

Phage typing: tests which phages a bacterium is susceptible to - phage added to bacteria on plate & observed for evidence of plaques (clearing) due to lysis of bacteria by phage Fatty acid profiles: separates cellular fatty acids from bacteria & compares them to fatty acid profiles of known bacteria Flow Cytometry: fluid is passed through a small opening; scattering of light provides evidence for presence & characteristics of contaminating bacteria - can be used to identify bacteria in a sample without culturing the bacteria DNA base composition: percentage of guanine + cytosine (G + C) in DNA from bacterium compared to known bacterial species DNA Fingerprinting: compares fragments of DNA created by restriction enzyme between 2 bacteria Ribosomal RNA sequencing: used to determine phylogenetic relationships (classification of bacteria) Polymerase Chain Reaction (see Chapter 9) Nucleic Acid hybridization (see Chapter 9) - Southern blotting with DNA probes Classification methods… Dichotomous keys: identification of unknown bacterium based on the answers to a series of questions - questions can involve the morphological characteristics as well as the outcome of various biochemical tests Cladograms: maps that show evolutionary relationships among organisms - branchpoints define features shared by species on that branch - originally based on fossil evidence for vertebrates; confirmed with rRNA sequences - bacterial cladograms based on rRNA sequences (no fossils)

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Chapter 11: The Prokaryotes: Domains Bacteria & Archaea
Important Groups: - Gram negative obligately anaerobic rods: Bacteroides, Prevotella, Porphyromonas, Fusobacterium - Intracellular parasites: o Rickettsia: gram-negative rods or coccobacilli; obligate intracellular parasites  require vector (insects or ticks) for transmission o Chlamydia: gram-negative coccoid; obligate intracellular parasites - Bacteria without cell wall: Mycoplasma, Ureaplasma (mycoplasmas) - Acid-fast bacteria: Mycobacterium, (Nocardia is weakly acid-fast) - Gram negative aerobic rods: Pseudomonas, Legionella, Brucella, Bordetella, Bartonella, Francisella, Burkholderia, Rickettsia - Gram negative aerobic cocci: Neisseria, Moraxella, Acinetobacter - Gram negative facultatively anaerobic rods: Escherichia (E. coli), Salmonella, Shigella, Klebsiella, Proteus, Yersinia, (Pasteurella, Haemophilus, Vibrio) - Gram-negative vibrios: Vibrio (comma-shaped or S-shaped) - Gram-negative enteric rods: Escherichia strains (E. coli), Salmonella, Shigella, Klebsiella, Proteus, Enterobacter - Gram-negative rods - respiratory pathogens: Haemophilus, Bordetella, Legionella - Gram-negative zoonotic rods: Yersinia, Brucella, Francisella, Pasteurella - Gram positive anaerobic rods: Clostridium, Propionibacterium, Gardnerella - Gram positive facultatively anaerobic rods: Bacillus, Listeria - Gram positive cocci: Staphylococcus, Streptococcus, Enterococcus - Gram positive endospore-forming rods: Bacillus, Clostridium - Gram positive non-endospore-forming rods: Lactobacillus, Corynebacterium, Listeria, Propionibacterium - Spirilla: Helicobacter, Campylobacter, Spirillum o gram-negative aerobic bacteria with a helical or spiral shape (Campylobacter are curved rods; Helicobacter are spiral/curved rods; Spirillum are helical/spiral) o rigid cell wall & motile by means of ordinary polar flagella (unlike spirochetes) o Helicobacter pylori causes peptic ulcers - Spirochetes: Treponema, Borrelia, Leptospira o thin, flexible, spiral-shaped bacteria that move by means of axial filaments or endoflagella (unlike spirilla, no polar flagella) o most are free living (in mud and sediments), or live in associations with animals (e.g. in the oral cavity or GI tract); a few are pathogens of animals o Treponema pallidum causes syphilis o Borrelia burgdorferi causes Lyme disease - Actinomycetes: Mycobacterium, Nocardia, Corynebacterium, Propionibacterium, Gardnerella - Haemophilus: Haemophilus influenzae are coccobacilli (pleomorphic if no capsule) responsible for meningitis, otitis media, bronchitis & atypical pneumonia o clinical labs use tests for requirement of X factor (heme fraction of hemoglobin) & V factor (NAD+ or NADP+) in growth medium
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In Bergey’s manual, prokaryotes are divided into 2 domains: Archaea & Bacteria - gram-negative bacteria are divided into proteobacteria & nonproteobacteria o proteobacteria are divided into 5 groups: α, β, γ, δ, ε - gram-positive bacteria are divided into low G + C gram-positive bacteria and high G + C gram-positive bacteria Domain Bacteria: Proteobacteria: most gram-negative chemoheterotrophic bacteria - largest taxonomic group of bacteria - few photosynthetic, although thought to be derived from common photosynthetic ancestor… phylogeny based on rRNA similarities - subgroups designated by Greek letters α-proteobacteria: - includes most proteobacteria capable of growth with low nutrient levels - some have prosthecae (stalk- or bud-like protrusions - some agriculturally important (nitrogen fixation) - some plant & human pathogens - Azospirillum: found associated with plant roots – fix nitrogen - Acetobacter & Gluconobacter: aerobic; convert ethanol into acetic acid - Rickettsia: gram-negative rods or cocci o Pathogenic; transmitted to humans by insect bites… cause epidemic typhus, endemic murine typhus & Rocky Mountain spotted fever - Ehrlichia: gram-negative pathogens; live in white blood cells – cause ehrlichiosis - Caulobacter & Hyphomicrobium: prominent prosthecae; found in low-nutrient aquatic environments (lakes); budding - Rhizobium & Agrobacterium: invade plant roots o Rhizobium: agriculturally important; fix nitrogen; symbiotic relationship with plants o Agrobacterium: pathogen in plants; insert bacterial DNA plasmid into plant DNA… cause crown gall Brucella: obligate mammalian parasites; cause brucellosis; can survive phagocytosis Nitrobacter & Nitrosomonas: nitrifying bacteria; chemoautotrophs… use reduced nitrogenous compounds for energy o Nitrosomonas are in ß-proteobacteria

ß-proteobacteria: some overlap with α-proteobacteria - can use hydrogen gas, ammonia & methane for nutrient production; some pathogenic - Thiobacillus: chemoautotrophs; oxidize reduced forms of sulfur - Spirillum: large gram-negative, aerobic, motile bacteria with polar flagella - Sphaerotilus: gram-negative with polar flagella; sheathed bacteria… live in freshwater & sewage in hollow filamentous sheath - Burkholderia: motile with polar flagella; B. cepacia best known… capable of growth in disinfectant
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Bordetella: B. pertussis – pathogen that causes pertussis (whooping cough) Neisseria: aerobic gram-negative cocci; pathogenic species cause gonorrhea & meningococcal meningitis Zoogloea: important in sewage treatment processes

γ-proteobacteria: largest subgroup of proteobacteria - Beggiatoa: gliding motility; uses H2S as energy source - Francisella: Francisella tularensis transmitted by wild animals & arthropods causes tularemia - Pseudomonales o Pseudomonas: motile by polar flagella; many produce water-soluble pigment (blue-green in Pseudomonas aeruginosa)  common in soil & other natural environments  resistant to many antibiotics  can cause opportunistic/nosocomial infections (UTIs, burn/wound infections, septicemia) o Azotobacter & Azomonas: free-living nitrogen-fixing bacteria in soil o Moraxella: coccobacilli; species implicated in conjunctivitis - Legionellales o Legionella: common in streams; can grow in water-supply lines  Legionella pneumophila causes legionellosis; a bacterial pneumonia o Coxiella: intracellular parasites transmitted by arthropods  Coxiella burnetii causes Q fever (a bacterial pneumonia) - Vibrionales o Vibrio: rods that are often curved or comma-shaped  Vibrio cholerae causes cholera, characterized by profuse, watery diarrhea  Vibrio parahaemolyticus causes less severe gastroenteritis - Enterobacteriales (enterics) o inhabit intestinal tracts of animals; most actively ferment sugars o produce bacteriocins that lyse/kill closely related species o Escherichia:  E. coli is common in biological research  E. coli is not usually pathogenic, but can cause UTIs, and enterotoxinproducing strains can cause traveler’s diarrhea and gastroenteritis o Salmonella: almost all members can be pathogenic; divided into serological types or serotypes (strictly not species) by types of antigens on flagella, capsule & cell wall  Salmonella typhi causes typhoid fever, a severe gastroenteritis  other Salmonella members cause less severe gastroenteritis o Shigella: Shigella species cause a severe form of diarrhea called bacillary dysentery (shigellosis), as well as traveler’s diarrhea o Klebsiella: found in soil & water; many are nitrogen-fixing  Klebsiella pneumoniae causes a serious pneumonia o Serratia:  Serratia marcescens produces a red pigment
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can be isolated from hospital environments; may cause nosocomial infections such as UTIs and respiratory tract infections o Proteus: motile by peritrichous flagella; exhibit swarming growth on agar  produce urease enzyme; implicated in many UTIs o Yersinia:  Yersinia pestis causes plague; transmitted by rat flea (respiratory droplets may be involved as well) o Erwinia: primarily plant pathogens (cause plant rot) o Enterobacter: found in animals, water, sewage & soil  Enterobacter aerogenes & Enterobacter cloacae can cause UTIs & nosocomial infections Pasteurellales o Pasteurella: domestic animal pathogen; causes septicemia & cholera in fowl  Pasteurella multocida can be transmitted to humans by cat & dog bites o Haemophilus: requires blood in culture medium (heme fraction called X factor); also requires NAD (V factor)  Haemophilus influenzae causes bacterial pneumonia and bacterial meningitis



Purple & Green Photosynthetic Bacteria: scattered in many taxonomic subgroups - green nonsulfur bacteria - green sulfur bacteria - purple nonsulfur bacteria - purple sulfur bacteria ∂-proteobacteria: some bacterial predators; some sulfur-reducing bacteria - Bdellovibrio: attacks other gram - bacteria - Desulfovibrionales o Desulfovibrio: obligately anaerobic sulfur-reducing bacteria - Myxococcales o Myxococcus: move by gliding (slime trail); digest other bacteria for nutrients ε-proteobacteria: gram-negative rods; helical or vibrioid - Campylobacter: microaerophilic vibrios (name means curved rod) o Campylobacter jejuni is a cause of foodborne intestinal disease - Helicobacter: microaerophilic curved rods with multiple flagella - Helicobacter pylori is a common cause of gastric ulcer Nonproteobacteria Gram-Negative Bacteria: phylogeny based on rRNA - Cyanobacteria: once called blue-green algae due to color o Carry out oxygen-producing photosynthesis; many fix nitrogen o Unicellular, colonial & filamentous forms - Chlamydiales: intracellular parasites (cultivated in cells, animals, embryonated eggs) o Chlamydia: coccoid bacteria; spread by interpersonal contact or airborne  Chlamydia trachomatis: causes blindness, nongonococcal urethritis (NGU)  Chlamydia psittaci causes psittacosis (ornithosis)  Chlamydia pneumoniae causes mild pneumonia
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o Spirochetes  Treponema: Treponema pallidum causes syphilis  Borrelia: bacteria transmitted by ticks or lice; cause relapsing fever • Borrelia burgdorferi causes Lyme disease  Leptospira: cause leptospirosis; spread though contaminated water from urine of infected animals Bacterioides: anaerobic bacteria; members of genus Prevotella in oral cavity o Bacterioides: inhabit human intestinal tract & gingival crevice  infect through puncture wounds or surgery; cause peritonitis Fusobacteria: pleomorphic but often fusiform (spindle-shaped) o Fusobacterium: may be responsible for dental abscesses Sphingobacteria: chemoheterotrophic bacteria; hydrolyze plant oils (commercial use) o Cytophagia: degrade cellulose in soil; important in sewage treatment

Gram-Positive Bacteria: divided into 2 groups, based on ratio of G+C (Guanine + cytosine nucleotides relative to total nucleotides in chromosome) Low G + C Gram-Positive Bacteria - Mycoplasmatales: do not form cell walls o have high sterol content in the plasma membrane o aerobes or facultative anaerobes; highly pleomorphic o Mycoplasma - Can be grown on artificial media with sterols, but cell culture methods are often used o Mycoplasma pneumoniae - primary atypical pneumonia o Ureaplasma urealyticum - nongonococcal urethritis - Clostridiales o Clostridium - obligate anaerobes that form endospores o Clostridium tetani – causes tetanus o Clostridium botulinum – causes botulism o Clostridium perfringens – causes gas gangrene, foodborne diarrhea o Clostridium difficile - antibiotic-associated pseudomembranous colitis - Epulopiscium – large, originally thought be a protozoan - Veillonella: normal flora of the mouth (dental plaque), colon, vagina o anaerobic cocci that occur in pairs or short chains; non-motile, non-endospore forming o opportunistic pathogen – abscesses of sinuses, tonsils and brain - Lactobacillales o Lactobacillus - normal flora of oral cavity, vagina and intestinal tract  aerotolerant rods that produce lactic acid through fermentation - Streptococcus – aerotolerant cocci that grow in chains o catalase-negative; some produce exotoxins that destroy phagocytes & host tissues o α-hemolytic streptococci produce α-hemolysin, which reduces hemoglobin (red) to biliverdin (green) o β-hemolytic strep produce a hemolysin (streptolysin-O or S) that completely lyses hemoglobin, producing a clear zone around colonies o γ-hemolytic strep are actually nonhemolytic; do not produce a hemolysin
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o S. pyogenes - Group A, β-hemolytic strep; causes pharyngitis, impetigo, scarlet fever, and rheumatic fever o S. mutans - γ-hemolytic; causes dental caries o S. pneumoniae - α-hemolytic; causes pneumonia, bacteremia, meningitis, otitis and sinusitis Staphylococcus: aerobes or facultative anaerobes that grow in grapelike clusters, under high osmotic pressure/low moisture conditions o Staphylococcus epidermidis -normal flora of the skin o Staphylococcus aureus - pyogenic infections such as endocarditis and osteomyelitis; food poisoning, and toxic shock syndrome o Staphylococcus saprophyticus - urinary tract infections Listeria o Listeria monocytogenes - meningitis and sepsis in newborns and immunosuppressed adults; can cause stillbirth or serious damage to the developing fetus; contaminates foods, survives phagocytosis, grows at refrigeration temperature. High G + C gram-positive bacteria – phylum Actinobacteria Mycobacterium - aerobic, non-endospore forming rods; fungus-like in that they occasionally exhibit filamentous growth o mycolic acids in outer layer from waxy, water-resistant layer, resistant to desiccation and many antimicrobial drugs o Mycobacterium tuberculosis: causes tuberculosis (TB) o Mycobacterium leprae: causes leprosy Corynebacterium – pleomorphic, varies with age of cells o Corynebacterium diphtheriae: causes diphtheria Propionibacterium – forms propionic acid, species used for fermentation of Swiss cheese. o Propionibacterium acnes: causes acne Gardnerella o Gardnerella vaginalis – common cause of vaginitis, gram-variable and pleomorphic. Actinomycetes – filamentous, soil bacteria o Frankia – forms nitrogen-fixing nodules in alder tree roots o Streptomyces –strict aerobes, produce asexual spores (conidiospores); source of most commercial antibiotics o Actinomyces – facultative anaerobes, inhabit mouth and throat of humans and animals  Actinomyces israelii – causes actinomycosis, tissue destroying disease of the head, neck or lungs. o Nocardia – aerobic, produce filaments that fragment into short rods  Nocardia asteroides – may cause pulmonary infections or mycetoma (localized destructive infection of feet or hands)

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Domain Archaea: extreme halophiles, extreme thermophiles, and methanogens

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Chapter 12: The Eukaryotes: Fungi, Algae, Protozoa & Helminths
Fungi: - Pros: decompose dead plants using extracellular enzymes like cellulases - important for food chains/webs o mycorrhizae – symbiosis with plants (live on roots), aids absorption of minerals & water from soil o used as food (mushrooms), in baking/brewing (yeasts), & as source of antibiotics (penicillin) - Cons: Fungal infections (hospital-acquired & from weakened immunity) o Fungal diseases of plants Mycology: study of fungi Characteristics of fungi: - fungi are chemoheterotrophs (require preformed organic compounds for energy & carbon) - fungi are generally aerobic or facultatively anaerobic; very few anaerobic fungi - Vegetative structures: fungal colonies… cells involved in catabolism & growth Molds & fleshy fungi: thallus (body) composed of hyphae (long filaments of cells) - septate hyphae have septa (cross-walls) dividing hyphae into uninucleate units - coenocytic hyphae have no septa… appear as long continuous cells - hyphae fragments can grow fully into new hyphae - vegetative hyphae obtains nutrients… reproduction occurs in reproductive or aerial hyphae (project above media surface) that often bear spores - mycelium: visible mass of hyphae Yeasts: nonfilamentous unicellular fungi; spherical or oval shape - budding yeast (Saccharomyces): divide unevenly by formation of new cell from small bud o pseudohypha: undetached buds that form short chain of cells - fission yeast (Schizosaccharomyces): divide evenly - yeast are facultative anaerobes… carry out aerobic respiration when oxygen is present & alcohol fermentation in the absence of oxygen Dimorphic fungi: can grow as either a mold or yeast - temperature-dependent in some pathogenic fungi (yeastlike at 37˚C, moldlike at 25˚C); CO2 concentration-dependent in others Life cycle: - asexual reproduction in filamentous fungi is possible by fragmentation of hyphae - spores formed by aerial hyphae o asexual spores: form fungus identical to parent
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conidium: unicellular or multicellular spore not enclosed in sac; produced in chain at end of conidiophore • arthrospore: formed by hyphae fragmentation • blastoconidia: buds from parent cell chlamydospore: thick-walled spore from enlarged hyphal segment sporangiospore: formed within sac (sporangium) at end of sporangiophore (found in Rhizopus)

o sexual spores: result from fusion of nuclei from opposite mating strains of the same species (not identical to either parent)  3 phases of sexual reproduction: plasmogamy (donor nucleus enters recipient cell); karyogamy (+ & - nuclei fuse); meiosis (diploid nucleus forms haploid nuclei)  sexual spores used in classification Nutritional adaptations of Fungi: - low pH (~ pH=5) environment best for growth - molds generally aerobic; yeast generally facultative anaerobes - fungi generally more resistant to high osmotic pressure - can grow in low moisture environment - reduced requirement for nitrogen - can metabolize complex carbohydrates (lignin, cellulose) Medically Important Phyla of Fungi: Teleomorphs: produce both asexual & sexual spores Zygomycota: conjugation fungi; saprophytic molds with coenocytic hyphae - example: Rhizopus nigricans (black bread mold) - asexual spores are sporangiospores - sexual spores are zygospores (large, thick-walled spore) Ascomycota: sac fungi; molds with septate hyphae, some yeasts - asexual spores are conidia - sexual spores are ascospores (spores produced in saclike structure called ascus) Basidiomycota: club fungi; fungi with septate hyphae that produce mushrooms - asexual spores are usually basidiospores (formed externally on base pedestal called basidium), sometimes conidiospores Anamorphs: only produce asexual spores Deuteromycota: undefined fungi; rRNA sequencing now being used to classify fungi in this phylum - example: Penicillium - most deuteromycetes may be anamorph phases of Ascomycota Fungal Diseases: Mycoses: fungal infection… 5 groups:
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systemic mycoses: deep infections, not localized – can affect many organs/tissues subcutaneous mycoses: fungal infections beneath skin; caused by saprophytes in soil (spores enter wound) cutaneous mycoses (dermatomycoses) o dermatophytes: infect epidermis, hair & nails  secrete keratinase – degrades keratin superficial mycoses: localized infections along hair shafts & in epidermis opportunistic pathogens: normally harmless fungi can become pathogenic in weakened or immunocompromised host o many fungi can be fatal in AIDS patients o yeast infection (candidiasis): caused by Candida albicans, frequent in newborns, AIDS patients & those under antibiotic treatment

Economic effects of fungi: - Aspergillus niger used to produce citric acid for foods - The yeast Saccharomyces cerevisiae used to produce bread, beer & wine… & also to make many proteins - Trichoderma used to produce the enzyme cellulase - Taxomyces produces taxol (anticancer drug) - Various fungi can be used in pest control On the other hand… - Molds responsible for food spoilage - Fungal blights have destroyed entire crops (potato blight), & prevent chestnut trees from growing in certain regions - Dutch elm disease has devastated the US elm population Lichens: mutualistic symbiotic relationship between a green algae (or cyanobacterium) & a fungus (usually an ascomycete) - crustose, foliose & fruticose forms - the fungi use carbohydrates from algae photosynthesis for food, & the algae in turn is protected from drying out (by the fungal cortex or covering) & can attach to substrates it otherwise could not, such as wood & rocks (by attachment of fungal hyphae or holdfast) - used to make clothing dyes Algae: unicellular, filamentous & multicellular forms - eukaryotic photoautotrophs without plant tissues - multicellular algae: body or thallus consisting of holdfasts (anchor to rock), stipes & blades - supported by water or pneumatocysts (gas-filled bladders) - sexual & asexual reproduction; alternation of generations in some - Brown Algae (kelp): macroscopic; used to produce algin (used as thickener) - Red Algae: branched thalli; used to produce agar & carageenan (thickener) - Green Algae: believed to be ancestors of terrestrial plants; unicellular, multicellular & filamentous forms
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Diatoms: unicellular or filamentous algae; cell wall with pectin & silica; responsible for domoic acid intoxication Dinoflagellates : unicellular algae (plankton) or free-floating; some produce neurotoxins o produce saxitoxins responsible for paralytic shellfish poisoning o large marine concentrations of some species produce red tides periodic/seasonal planktonic algae increases called algal blooms algae believed to produce most atmospheric oxygen

Protozoa: unicellular, eukaryotic chemoheterotrophs - feeding & growing stage (trophozoite) feeds on bacteria & particulate nutrients - reproduce asexually by fission, budding or schizogamy (multiple fissions) - ciliates (Paramecium) reproduce sexually by conjugation (fusion of micronucleus from one ciliate & macronucleus from another) - some protozoa produce gametes that fuse to form a zygote - can form a protective capsule called a cyst… phylum Apicomplexa forms an oocyst that can reproduce asexually - some have a protective covering called pellicle Archaezoa: eukaryotes with no mitochondria; spindle-shaped with 2 or more flagella - Trichomonas vaginalis: infects vagina & male urinary tract Microsporidia: lack mitochondria & microtubules; cause diarrhea in AIDS patients Rhizopoda: amoeba - Entamoeba histolytica causes amoebic dysentery Apicomplexa: nonmotile in mature form; obligate intracellular parasites - Plasmodium reproduces in Anopheles mosquito & is transferred to human blood cells by mosquito bite; lyse blood cells… causes malaria Ciliophora: move by means of cilia Euglenozoa: photoautotrophs; move by flagella; lack sexual reproduction Slime Molds: - cellular slime molds resemble amoebas; ingest bacteria by phagocytosis - plasmodial slime molds are a multinucleate mass of protoplasm… engulf bacteria & debris as they move Helminths: - parasitic flatworms in phylum Platyhelminthes - parasitic roundworms in phylum Nematoda - multicellular animals; some are human parasites - adult stage found in definitive host - larval stages require intermediate host
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can be monoecious (male & female sex organs in same animal) or dioecious (separate male & female organisms) Platyhelminthes: dorsoventrally flattened; parasitic flatworms may lack digestive tract o Adult trematodes (flukes) have oral & ventral sucker to attach to host o A cestode (tapeworm) consists of a scolex (head) & proglottids (segments) o intermediate host consumes eggs (from feces/soil); larvae encyst in muscle; when definitive gost consumes meat; larvae mature & migrate to other tissues (GI tract) o humans as definitive host:  beef tapeworm (Taenia saginata); cattle are intermediate host  pork tapeworm (Taenia solium); humans can also be intermediate host o humans as intermediate host:  tiny tapeworm Echinococcus granulosus; dogs & coyotes are definitive hosts o diagnosis based on presence of mature proglottids in feces Nematoda: roundworms with a complete digestive system o most species are dioecious o males are smaller than females & often have a hooked tail with spicules o free-living (soil & water) and parasitic species o can be divided into those in which egg is infective & those in which larva is infective o egg infective for humans:  pinworm Enterobius vermicularis  Ascaris lumbricoides: large roundworm that lives in the digestive tract of humans & domestic animals • eggs excreted in feces into soil & ingested by another host o larvae infective for humans  hookworm (Necator americanus): live in small intestine of humans; eggs are excreted in feces/soil; larvae enter host by penetration of skin  Trichinella spiralis: causes trichinosis in humans through ingestion of encysted larvae in undefcooked pork or game animals (bears)

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Arthropods as Vectors - arthropods (Phylum Arthropoda) are animals characterized by segmented bodies, hard external skeletons & jointed legs

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include arachnids (spiders, mites, ticks), crustacea (crabs, crayfish) & insects (bees, flies, lice) arthropods that carry disease are called vectors control or eradication of vectors is best approach to elimination of diseases they carry

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Chapter 13: Viruses, Viroids & Prions
Viruses: - are obligatory intracellular parasites - contain a single type of nucleic acid – either DNA or RNA - contain a protein coat surrounding the nucleic acid (& some have an envelope composed of lipids, proteins & carbohydrates surrounding the protein coat) - multiply in living cells using the cell’s metabolic machinery (do not have their own enzymes for protein synthesis or ATP generation) - cause synthesis of structures that can transfer viral nucleic acids to other cells - viral size: ranges from 20-14,000 nm Viral Host Range: viruses exist that infect invertebrates, vertebrates, plants, protists, fungi & bacteria - viruses that infect bacteria are called bacteriophages or phages - the host range depends on the presence on the surface of the host cell of viral receptors for a given virus Viral Structure: virion – infectious viral particle - Nucleic Acid: either DNA or RNA o Can be either single-stranded or double-stranded; size varies - Capsid & Envelope: o the capsid is the protein coat of the virus surrounding the nucleic acid o the capsid is composed of protein subunits called capsomeres o in some viruses, the capsid is enclosed by an envelope consisting of proteins, lipids & carbohydrates  some envelopes are covered by carbohydrate-protein complexes called spikes that may aid in attachment to the host cell  in nonenveloped viruses, the capsid protects the nucleic acid from nucleases & promotes attachment to host cell General morphology: - Helical viruses: helical capsid; o examples: rabies & Ebola viruses - Polyhedral viruses: polyhedral (many-sided) capsid; usually icosahedral (20 sides) o examples: adenovirus, poliovirus - Enveloped viruses: capsid enclosed by envelope o examples: Influenzavirus (helical enveloped), herpes simplex virus (icosahedral enveloped) - Complex viruses: complicated structure o example: bacteriophage Viral taxonomy: - viruses grouped into families based on: nucleic acid type, replication strategy, & morphology - order names end in –ales - family names end in –viridae
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genus names end in –virus species names use common names (e.g.: human immunodeficiency virus) o subspecies use a number (e.g.: HIV-1)

Viral Families by Genome & important viruses - single-stranded DNA, nonenveloped o Parvoviridae (human parvovirus B19) - double-stranded DNA, nonenveloped o Adenoviridae (Mastadenovirus) o Papovaviridae (Papillomavirus (human papillomavirus or HPV)) - double-stranded DNA, enveloped o Poxviridae (Orthopoxvirus (vaccinia virus, smallpox virus)) o Herpesviridae  Simplexvirus (herpes simplex virus HHV-1 & HHV-2)  Varicellovirus (varicella zoster virus HHV-3)  Lymphocryptovirus (Epstein-Barr virus (EBV) HHV-4)  Cytomegalovirus (CMV, HHV-5)  Roseolovirus (HHV-6) o Hepadnaviridae (Hepadnavirus (hepatitis B virus (HBV))) - + strand single-stranded RNA, nonenveloped o Picornaviridae (Enterovirus, Rhinovirus, hepatitis A virus (HAV)) o Calciviridae (hepatitis E virus (HEV), Norovirus) - + strand single-stranded RNA, enveloped o Togaviridae (Rubivirus (rubella virus)) o Flaviviridae (Flavivirus, hepatitis C virus (HCV)) o Coronaviridae (Coronavirus) - - strand single-stranded RNA o Rhabdoviridae (Lyssavirus (rabies virus)) o Filoviridae (Ebola virus) o Paramyxoviridae (Paramyxovirus (measles virus)) o Deltaviridae (hepatitis D virus) - multiple strand RNA (- strand) o Orthomyxoviridae (Influenzavirus A, B & C) o Bunyaviridae (Hantavirus) o Retroviridae (Lentivirus (HIV)) - double-stranded RNA, nonenveloped o Reoviridae (Reovirus, Rotavirus) Viral Isolation: - bacteriophage growth visualized on agar plates as plaques (clearings where bacteria have been lysed) - can be counted – plaque-forming units - animal viruses are more difficult… can use: o living animals o embryonated eggs o cell cultures
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cause cytopathic effect – can be counted similarly to plaques primary cell lines – cells isolated from tissue; short life span • diploid cell lines – isolated from embryos; about 100 generations possible  continuous cell lines: immortal; infinite generations – usually isolated from cancerous tissue Viral Identification: can use serological methods (Western blotting), RFLPs, PCR

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Viral Multiplication: (bacteriophage model) - Lytic cycle: (T-even bacteriophages) o Attachment: phage attaches to host cell (wall) o Penetration: phage penetrates host cell (using tail core) & injects its DNA o Biosynthesis: host cell used to synthesize phage proteins o Maturation: synthesized viral components packaged into virions o Release: host cell lysed (phage lysozyme breaks down cell wall) & new virions released - burst time: time from phage attachment to release - burst size: number of new phage particles released from a single infection - Lysogenic cycle: (bacteriophage λ) o Lysogenic phages may use a lytic cycle, but can also integrate their DNA into the host cell chromosome – the inserted phage DNA is known as a prophage o If the integrated phage DNA is excised (removed from host DNA by UV light; chromosome break), it can enter the lytic cycle o During lysogeny, the phage remains latent (inactive), & the host cell is known as a lysogenic cell o Lysogenic cells are immune to reinfection with the same phage o Phage conversion: lysogenic cell may acquire new properties (from phage gene products) o Specialized transduction: excised phage DNA can carry adjacent bacterial genes which will be packaged into phage capsids – recombinant phage Multiplication of animal viruses: - Stages: o Attachment: virus attaches to plasma membrane receptors of host cell using surface attachment sites (capsid fibers, spikes,…) on virus o Penetration: virus taken into cell by endocytosis or by fusion with the plasma membrane (enveloped viruses) o Biosynthesis: synthesis of capsid & viral nucleic acid (see below) o Uncoating: viral nucleic acid separated from capsid (by enzymes, etc.) o Maturation & Release: enveloped viruses released by budding from host cell plasma membrane (membrane may become viral envelope) – host cell may survive; nonenveloped viruses released through ruptures in plasma membrane – host cell usually dies

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Biosynthesis of Viral Nucleic Acid: - DNA viruses: viral DNA synthesized in nucleus using enzymes from “early” genes & capsid synthesized in cytoplasm using mRNA from “late” genes – capsid proteins migrate to nucleus for packaging o ssDNA viruses: family Parvoviridae (parvovirus) o dsDNA viruses: families Herpesviridae (herpesvirus), Papovaviridae (papillomavirus) & Poxviridae (smallpox & cowpox viruses) - RNA viruses: o ssRNA viruses:  + strand ssRNA viruses use + strand as mRNA for viral proteins, as template for synthesizing – strand (use RNA-dependent RNA polymerase make more + strand) & as viral genome • example: picornaviruses (poliovirus)  - strand ssRNA viruses use – strand to make + strand & as viral genome • example: rhabdoviruses (rabiesvirus) o dsRNA viruses: RNA-dependent RNA polymerase copies the – strand to make + strand (mRNA)  example: reoviruses o multiple strand RNA viruses: reverse transcriptase uses RNA as template to synthesize; DNA is integrated into host cell chromosome (provirus) & used as template for viral mRNA synthesis  example: retroviruses (HIV) Viruses & Cancer: - oncogenes: mutation in these genes makes the cell more susceptible to oncogenic transformation - transformation: tumor cells acquire properties unlike normal cells (loss of contact inhibition, virally transformed cells may express viral antigens) Viral Infections: - latent viral infection: viral activity may be reactivated by immunosuppression or other conditions o e.g.: infections by HSV-1 (cold sores, fever blisters) & HSV-2 (genital herpes) - persistent viral infection (slow viral infection): disease caused by a viral infection that progresses slowly over a long time period; usually fatal o e.g.: subacute sclerosing panencephalitis caused by measles virus Prions: proteinaceous infectious particles - suspected cause of nine types of neurological diseases (mad cow disease (BSE), kuru, Creutzfeldt-Jacob disease (CJD)…) - diseases called spongiform encephalopathies – large vacuoles develop in brain - sheep scrapie: infectivity of infected sheep brain reduced with proteases - human prion protein (PrP) gene found… located on chromosome 20 - normal PrP is PrPC (cellular); abnormal PrP is PrPSc (scrapie); injection of PrPSc in normal animal brains causes disease (and refolding of existing PrPC to PrPSc) - fragments of abnormal prion protein accumulate in plaques in infected brain
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Plant Viruses & Viroids: - cell wall reduces plant susceptibility to viral infection, yet plant viruses (bean mosaic virus, wound tumor virus) cause many plant diseases - viroids: short pieces of naked RNA

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