Microbes Flashcards
Initial surface of Earth
Molten, atmosphere of methane, CO2, ammonia + nitrogen.
No molecular O2 as reducing conditions.
Water present but only as vapour.
When did life arise?
Stable isotopic analysis - metabolised differently enzymes preferentially fix C12 -> biogenic carbon 3.7-4.1 Bya
Macrofossils, stromatolites from photoautotrophic bacteria -> mucilage secreting, life arose 4 Bya
Microfossils found in 3.5 Byo rocks using SEM in sedimentary rocks (formed from water bodies)
Prebiotic soup hypothesis
Haldane + Oparin 1920s, organic mols in prebiotic oceans formed simpler precursors + energy provided by UV, lightning, volcanic activity.
- supported by Urey + Miller experiment 1950s, RNA nucleobases produced.
Only monomers present though. Clay mineral montmorillonite catalyses large number organic reactions
Deep sea vent hypothesis
Hydrothermal vents have rich + plentiful supply of reduced chemical nutrients -> abiotic production of simple organic compounds.
Mineral structures in vents create potential for compartmentalisation (-> replication processes + lipid bound protocells)
How did life forms arise?
1968 Crick’s RNA world hypothesis - RNA first info containing molecule.
Can self replicate, some enzymatic activity (ribozymes), able to function as both DNA + proteins do.
What are modern phylogenetics based on?
Nucleotide sequence comparisons - sequence data acquired, aligned + used as input to generate phylogenetic tree.
Alignments + evolutionary distance
Alignments: hypotheses of how sequences have diverged since last common ancestor - algorithm used to minimise mismatches + gaps.
- no. of genetic changes used to est. relatedness
- finds most parsimonious alignment
Evolutionary distance can be calculated (3 changes in 12 is 3/12 = 0.25)
Can be increased if take into account how differences arose (multiple mutations)
What makes a good choice of gene sequence to study?
- present in organism of interest
- not laterally transferred
- appropriate level of sequence conservation/divergence
- large enough to contain record of historical info (tRNA too small)
rRNA most useful for phylogenetic reconstruction - has alternating conserved + hypervariable regions
Bacterial metabolic diversity
Chemotrophy has 2 types:
Chemoorganotrophs - oxidise organic mols
Chemolithotrophs - oxidise inorganic mols
Phototrophs convert light -> ATP using chlorophyl
Purple + green bacteria anoxygenic VS cyanobacteria, algae + green plants are oxygenic.
Anaerobic respiration in bacteria uses alternative e acceptors (inorganic N, S, CO2, Fe compounds) - less energy conserved than O2 but still favourable.
e.g. E.coli uses nitrate when O2 not available (facultative anaerobe)
Energy conservation in chemoorganotrophs
- Fermentation - substrate level phosphorylation (ATP directly synthesised from energy rich intermediates, end product often acids/alcohols)
- Respiration - oxidative phosphorylation (ATP produced from proton motive force)
Cocci vs Vibrios
What are other features of bacteria?
Coccus - round cell shapes, 0.5-1um diameter
Vibrios - rod shaped (E. coli, V. cholerae)
Spiral - rigid helix, found in stagnant water
Spirochetes - flexible, helical, very long e.g. syphilis, Lyme disease
Square + flat - e.g. Walsbys square bacterium is phototrophic halophilic Archaeal species.
Star shaped - Stella genus, isolated from soil, water + horse shit, v.large
-> GREAT DIVERSITY
Gram stain
1884, differentiates bacteria due to cell wall structure.
Gram +ve: purple, monoderm so inner mem + thick layer peptidoglycan (20nm)
-> thicker peptidoglycan fixes crystal violet better when iodine added, not washed off as easily by ethanol/acetone
Gram -ve: pink, diderm so inner mem + thin layer peptidoglycan (5-8 nm), outer mem (also has periplasm)
Peptidoglycan
Rigid structure of cell wall - protects from osmotic lysis, remains when boiled, can be destroyed by lysozyme, antibiotics + bacteriophage lysins.
Structure:
- alternating amino sugars (glycan chain), NAG & NAM
- peptide chains covalently linked to glycan chain (strong to due to many cross links)
- NAG/NAM chains conserved, peptides more variable
Cell walls of Gram +ve bacteria
- homogenous architecture
- single thicker layer (20-80nm)
- has PG and teichoic acid (TA) -> wall TA & lipoTA
Teichoic acid is polymer of glycerol (3C) or ribitol (5C) phosphate
- WTA covalently bonded to NAM, LTA embedded in mem via lipid component
Bacterial division
Divide through binary fission once cell doubled in length.
- division stimulated by FtsZ ring (attracts proteins) + forms division plane
- Cell doubles length, DNA + ribosomes portioned, septum forms, DNA attached to mem.
Can be measured by mean doubling time i.e. in E. coli its 20 mins.
Growth of bacterial cells
MreB - allows rod shaped cells to grow longer
It is an actin homologue, forms helical, filamentous structure (scaffold for PG synthesis machinery)
- autolysin breaks B1-4 linkage between NAM & NAG
- transglycosylase adds PG unit
Cocci lack MreB so PG synthesis occurs at septum
Bacterial growth curve
Lag phase - cells innoculated into fresh medium, adapting (no increase in numbers.
Exponential (log) phase - cells double each unit of time, logarithmic plotting (straight line) allows calculation of mean doubling time
Stationary phase - pop growth decreases (turbid culture), nutrient limitation + toxic waste means division stops e.g. lactic acid.
Death phase - cells may die at constant rate.
Methods of measuring bacterial growth
1) turbidity (optical density) using spectometry - not exact
2) calculate viable cell count -> 1 cell forms whole colony on solid medium
optimum temperature can vary from 4C to 113C
Psychophiles & mesophiles
low Topt, found in oceans (<15C).
- unsaturated f. acids so semi-fluid membranes at low temps so still function
- altered proteins, more alpha helices give more flexibility
- anti-freeze mols bind ice crystals used in fish, not found in prokaryotes
Mesophiles - midrange Topt (14-45C)
Thermophiles
High Topt, soil (45-80C)
- saturates f. acids so semi fluid membranes at high temps (stable)
- altered proteins have heat resistant folding conformations
Hyperthermophiles
Very high Topt, hot springs + deep sea vents (80-100C)
- no f. acids in membranes (phytane is C40 hydrocarbon covalently joined head to tail), lipid monolayer not bilayer
Sterilisation
Heat can be used - cells die at constant rate as they lose viability, pop death exponential
D value is time required to kill 90% cells (1 log ycle)
Steam sterilisation by autoclaving at high temps (112C) for 20 mins at high pressures (138kPa)
Gram -ve cell wall & LPS structure
Outer mem made of lipopolysaccharide (LPS): lipid A (hydrophobic), core oligosaccharide, O antigen + trimeric porin proteins.
Braun’s lipoprotein embedded in inner leaflet of outer mem, attached to PG layer.
Thin PG layer in periplasmic space.
LPS:
- Lipid A has beta1-6 linked disaccharide w/ P groups at 1C & 4C, hydrophobic f. acids instead of sugars at 2C & 3C.
- core oligosaccharide has KDO, 7C heptoses, glucose + galactose sugars
- O antigen v. structurally variable
Lipid A is endotoxin + can be recognised by TLR4 arm of immune system -> inflammatory response
Archaeal cell envelopes
Extremophiles:
monoderm from monolayer of tetra-ether lipids, impermeable to H+ so thermoacidophiles have stable pH, S layer has membrane anchoring protein.
Mesophiles:
Bilayer of diether lipids - pseudomurein layer + S layer.
-> Pseudomurein has NAM replaced w/ NAT, NAG/NAT connected by beta 1-3 glycosidic linkages (resistant to lysozyme), peptide bridges only have L-amino acids.
-> S layers universal in archaea, covered by paracrystalline structure, made of many copies of single glycoprotein.
Flagella
Subunit of filament of flagellin - helical structure, moves by rotation in CW or CCW directions, energy provided by ion powered motor.
Arrangements:
Monotrichous (1 at single pole), lophotrichous (multiple at single pole), amphitrichous (1 at both poles), peritrichous (multiple flagella all around cell surface)
Flagella semi rigid + rotate at 300 revs per second. Hook is flexible, acts as universal joint (20nm wide filaments)
- filaments grow from tip + need to constantly elongate as they break
Taxis mechanism
Taxis is enabled by switching flagella rotation direction.
- gradient of desired chemicals induces CCW -> CW rotation
Runs up gradients elongated, runs down gradient shortened -> random bias walk.
What is a virus?
Very small (20nm-1um), infectious, obligate intracellular parasite that is NOT living.
Replication dependent on infection, cannot generate ATP, no ribosomes, some have single stranded genomes.
Pandoravirus largest w/ 2,000 kBP dsDNA vs polivirus (30nm) w/ 7kBP ssRNA
Genetic material in viruses
ss or ds RNA or DNA. + sense (polio) can make proteins using mRNA, - sense cannot synthesise proteins so must be converted to + sense 1st
mRNA must be formed:
- Retrovirus RNA genome replicates using reverse transcriptase, via DNA intermediate
- Hepadnavirus DNA genome replicates via RNA intermediate
Virion structure + different cellular states
Nucleic acid core surrounded by protein capsid (monomer like parvovirus or polymer)
Can be enveloped by membrane (influenza + CV) or naked (adenovirus)
Extracellular state - metabolically inert, stable structure that protects genome
Intracellular state - glycoproteins bind host cell, capsid breaks down, genome inserted, host machinery redirectd to produce virions late in infection.
Influenza virus
Orthomyxovirus, enveloped ss RNA virus.
Helical nucleocapsid - 15 types of haemagglutinin (H) & 9 of neuraminidase (N)
-> ensures annual variability
Symmetry of viruses
Rod viruses have helical, spherical viruses have icosahedral.
Helical - e.g. TMV has +ss RNA genome
Icosahedral - e.g. parvovirus, 20 triangular faces -> efficient packing + 12 pentons
*bacteriophages have icosahedral head w/ tail fibres + sheath (injects DNA), have lysozyme for cell entry, breaks PG in cell wall
One step growth curve + its features
Cell monolayer infected w/ 10 viruses per cell
Eclipse - coat + n. acid are separated , cannot detect virus
Latent period - cannot detect virus whatsoever as replication + synthesis only starts
End of assembly + release, number of viruses detcted increases.
No of virions = burst size (< 1,000)n
Chytridiomycota
Aquatic, mainly asexual but some sexual spores flagellated.
- most primitive
Large thallus where rhizoids emerge + have motile zoospores
-> present in cattle rumen, causes fatal disease in amphibians
Zygomycota
Terrestrial, can cause food spoilage.
Asexual, non-motile spores germinate + produce new mycelium.
Conjugated - large sexual zygospore w/ thick coat to stop desiccation.
Undergo meiosis + produce haploid spores.
Aseptate so coenocytic.
Ascomycota
Sac fungi, largest + most diverse group inc. Penicillin + A. niger + fungi w/ fruiting bodies.
Aerial borne chains of asexual conidiospores.
8 sexual ascospores in asci borne/ascocarp. Many plant pathogens + spoil food.
Basidiomycota
Most advanced, from visible mushrooms + toadstools.
Usually has 4 sexual basidiospores (+or-) borne on basidium club structure.
No asexual reproduction.
Basidia line gills/pores of mushroom. Has many mycorrhizal associations w/ trees.
-> basidiocarp is above surface, mycelium underneath forms network of associations.
3 basic fungi cell structures
Yeasts (unicellular) - grow by budding division, new organism attached as it grows, asexual reproduction (genetically identical to parent)
Filamentous (mycelium + hyphae network) - produce conidia (asexual spores) from conidiophore) which germinate into hyphae (has apical growth), release enzymes from hyphae + take up nutrients.
Dimorphic - can be either ^ depending on temps e.g. sporothrix.
Types of fungal hyphae
Septate - hyphae divided into separate cells by perforated septa, tiny holes in septa allow flow of nutrients between cells.
Coenocytic - no septa, large cells w/ multiple nuclei e.g. zygomycota.
Fungal cell walls have glucans, chitin + mannoproteins -> give shape + rigidity.
Fungal hyphae growth
Occurs at apical region, rapid extension, movement of material from older regions -> tip.
Apical growth gives penetrating power + powered by actin polymerisation + cytoplasmic expansion forces.
Growth is polarised.
Spitzenkorper is a cluster of small mem. bound vesicles of different sizes - embedded in meshwork of actin filaments.
Opportunistic pathogens
Normally commensal (don’t cause disease)
P. aeruginosa - G -ve (plants + soil) infects burns patients, colonises lung of cystic fibrosis patients.
S. epidermis - G -ve (skin) colonises intravenous catheters + grow as biofilm, multiply & form community on antibiotic catheter
N. meningitidis - G -ve causes bacterial meningitis, commensal of nasopharynx but can infect. Transmitted through respiratory droplets
-> meningococcal septicaemia is systemic infection, can cause brain damage
Highly virulent pathogens
M. tuberculosis - develops over years, G +ve rods, replicate in alveolar macrophages in lung -> granuloma formation (white patches), spread by respiratory droplets.
T. pallidum - syphilis infection in stages, flexible helical structure. Primary lesion after 2 weeks, secondary stage 10 weeks infection spreads, latent phase (years) 40% get tertiary syphilis -> insanity + death.
Robert Koch’s postulates + its problems
- Organism found in lesion
- Grow organism outside body in lab.
- Organism must reproduce the disease
- Re-isolate from test animal
Problems: cant grow cultures on lab media (leprosy or viruses), some are human specific so ethical issues, no suitable animal model (gonorrheoa)
Measures of virulence
Minimum infectious dose - smallest number of bacteria needed to cause disease.
10 in S. pneumoniae vs 10,000 in V. cholerae
Lethal dose - dose to kill 50% animals/cells in given time, can quantify relative toxicity
Virulence determinants
nj
- capsule of poly-D-glutamic acid-mucoid (-ve charge) inhibits phagocytosis
- toxins suppress immune cell responses, later lethal levels induce toxic shock + death
Strain becomes attenuated if capsule or toxins are lost
Bacterial disease processes
Enter host + adhere by specific mechanisms e.g. E. coli attaches via Type I peritrichous fimbriae (subunits have adhesin that attach mannose receptors)
Pathogens can also adhere via non-fimbrial adhesins. e.g. S. pyogenes binds M protein.
Some bacteria penetrate tissues.
-> systemic infection of L. monocytogenes causes food poisoning (unpasteurised dairy or unwashed lettuce)
Microbiota
Collection of micro-organisms which live in our bodies in mutualistic relationship - commensals.
Lungs + brain only sterile parts of body.
Variation of abundances of bacterial phyla at different body parts
Skin - restricted -> S. epidermis + S. aureus (MRSA), some transient bacteria like E. coli.
Nasal cavity - restricted -> opportunistic pathogens N. meningitidus + S. penumoniae.
Oral cavity - v. high biodiversity -> 300 bacterial species in dental plaque (Streptococcus + Actinomyces)
-> dental plaque formed by bacteria attaching to salivary pellicles, corn cob formations in mature plaque.
Stomach has pH2 , but H. pylori G -ve motile + able to colonise mucosa in stomach -> gastritis.
Small intestine has pH4-5, low bacterial biomass vs colon pH 7 huge bacterial biomass
In colon: facultative aerobes (E. coli + E. faecalis) use up O2, vast majority are obligate anaerobes.
Gut microbiota
Acquired on passage through birth canal.
Exclusive breast feeding selects for specific bifidobacteria + transition to solid food marks stable gut bacteria
In over 60s - ratio shifts in bacteroides to firmicutes + decrease in bifidobacteria.
-> crucial for health/digestion, strenghten immune system, protects surface from pathogen, gut-brain axis (stress, depression, obesity, autism)
Chemical byproducts of microbiota
Organic acids (short chain fatty acids) vital for health - 95% used as energy store for cells lining colon.
- generated by complex + resistant fibres.
- glycosidase reactions release glucose
Gut microbiota provides 10% calories from food. Convert complex carbs to short chain fatty acids.
