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 organism¹s 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 (5¹ to 3¹). One strand (the leading strand) is synthesized continuously, while the other strand (the lagging strand which is in the 3¹ to 5¹ direction) is synthesized discontinuously in short fragments in the 5¹ to 3¹ 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:
- 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 5¹ to 3¹ 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 (5¹ 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 5¹ to 3¹ 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
- 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 b-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
- 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
- Ames test used for identification of chemical carcinogens
- Tests reversion of histidine auxotrophs of Salmonella
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
- 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 double-stranded 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
- 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
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
Taxonomic Hierarchy:
Domain
Kingdom
Class
Order
Family
Genus
Species
Prokaryotes:
- 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
Viruses: acellular; abiotic; obligate intracellular parasites
- not classified in 3 domains because they don¹t 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
- 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 patient¹s 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
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
- 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)
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
In Bergey¹s manual, prokaryotes are divided into 2 domains: Archaea & Bacteria
- gram-negative bacteria are divided into proteobacteria & nonproteobacteria
o proteobacteria are divided into 5 groups: a, b, g, d, e
- 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
a-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 a-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
- 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
g-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 enterotoxin-producing strains can cause traveler¹s 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 traveler¹s 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
§ 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
e-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
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
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)
Domain Archaea: extreme halophiles, extreme thermophiles, and methanogens
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 37C, moldlike at 25C); 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
§ 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:
- 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
- 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
Euglenozoa: photoautotrophs; move by flagella; lack sexual reproduction
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 cell¹s 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
- 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 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
§ 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
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 l)
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
Biosynthesis of Viral Nucleic Acid:
- DNA viruses: viral DNA synthesized in nucleus using enzymes from ³early² genes & capsid synthesized in cytoplasm using mRNA from ³late² 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 as template 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
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