Microbiology (1-8) Flashcards
What is the surface origin hypothesis of microbial evolution?
Primordial soup → life began from a series of chemical reactions in a warm pond
→ little evidence
→ hostile environment from high UV, meteor strikes, volcanic activity is very stressful for organisms
What is the subsurface origin hypothesis of microbial evolution?
Life originated at hydrothermal vents on the ocean floor
→ more stable conditions with a constant source of energy (H2 and H2S)
→ enclosed structures allowed for energised reactions of molecules eventually forming lipid bilayers and the first cell type
What is the pathway that lead to the divergence of bacteria and archaea?
- nutrients in hot spring water (NH4+, CN-, HCO3)
- more complex compounds form (RNA + proteins)
- starts using DNA less active than RNA, genetic code
- evolution of biochemical pathways
- divergence of cell walls
- early bacteria + early archaea
Why are certain molecular sequences useful when using phylogenetic methods to determine how organisms are related?
All organisms have DNA, RNA, ribosomes
→ if similarities are found must share common ancestor
→ organism must have the same purpose otherwise they won’t have the same evolutionary pressure
Why are ribosomal RNA genes a universal molecular marker?
They are present in all forms of life
What was the early earth atmosphere?
Anoxic - O2 absent
→ energy generating metabolism was exclusively anaerobic
(methanogenesis CO2 + 4H2 → CH4 + 2H20)
What did the evolution of cyanobacteria lead to?
Cyanobacteria evolved producing O2 as a waste product
→ starts oxidising everything
→ O2 begins to accumulate in the planet, forming oxygenic photosystem
What did the formation of O3 create?
The ozone shield which protects the Earth’s surface from UV radiation
→ becomes hospitable, forming new habitats and evolving diversity
What is the endosymbiont theory of eukaryotic evolution?
Mitochondria arose from the incorporation of aerobic chemo-organotrophic bacteria into the cytoplasm of early eukaryotic host cells
Chloroplasts arose from the incorporation of phototrophic cyanobacteria into eukaryotic host cells
What is the hydrogen hypothesis for eukaryotic evolution?
Eukaryotic cells arose by association of an archaeal host using H2 as energy with an aerobic bacterium that produced H2 as a waste product
→ the merging of cells driven by an initial syntrophic relationship
How are microbes names based on their shape?
Spherical → cocci
Rod → bacili
Spiral → spirilla
Comma → vibrios
Corkscrew → spirochaetes
Why is the traditional definition for a species problematic for microbes?
Asexual reproduction, lateral gene transfer (don’t know if the gene originated in that microbe) and phenotypic/genotypic plasticity
What is the current definition of a microbial species?
A group of strains that show a high degree of similarity and differ considerably from related strain groups with respect to many independent characteristics (pretty vague)
What is polyphasic bacterial taxonomy?
Collective genotypic, phylogenetic and phenotypic methods for determining taxonomic position of microbes
What is the main difference between gram negative and gram positive bacterial cell walls?
Gram -ve → thin peptidoglycan layer surrounded by an outer membrane which contains lipopolysaccharides
Gram +ve → thick peptidoglycan layer, no outer membrane
What are microbial capsules?
Polysaccharide layers, found outside the cell envelope, organised in a tight matrix
→ excludes small particles
→ readily visible by light microscopy
What are microbial slime layers?
Polysaccharide layers, found outside the cell envelope, diffuse unorganised and easily removed
→ does not exclude small particles
→ difficult to visualise
What are the roles of microbial capsules?
- assist in the attachment of microbes to surfaces
- carbon store
- protection against desiccation (drying out)
- may be involved in nutrient capture (acquisition of ions by slime layer)
- exclude things that will damage cell (antimicrobials/disinfectants)
- protection from host immune system, resist phagocytosis
→ immune cells that would recognise as foreign are blocked by capsule
What are the types of capsules in pathogens?
- polysaccharide structures (most common)
- glycolipid ‘capsule’ → extracellular glycolipid of myobacteria
- protein ‘capsule’
- extracellular slime
What are microbial S-layers?
Rigid, permeable, paracrystalline outer wall layer composed of protein/glycoprotein
(not associated with pathogens)
→ protects from ion/pH fluctuations, osmotic stress, prevents bursting
→ may protect against host defences
What is peptidoglycan (murein)?
Made up of alternating modified glucose residues:
NAG → N-acetylglucosamine
NAM → N-acetylmuramic acid
Arranged in dimers cross-linked by amino acid side chains - creating amide bonds
→ mesh-like polymer
How is peptidoglycan synthesised?
Chains of linked peptidoglycan subunits are joined by cross-links between peptides
→ often the carboxyl group of terminal D-alanine connected to amino group of diamino pimelic acid (DAPA)
→ peptidoglycan mesh formed is flexible and porous, but strong enough to retain shape - resist turgor pressure, prevent cell lysis
What is the significance of peptidoglycan being formed of D stereoisomer amino acids?
Proteins are always constructed of L-amino acids
→ D-amino acids protect against degradation by proteases
→ way bacteria have evolved to be protected from immune system
What are lysozymes?
‘Antibacterial’ enzymes
→ degrades the beta 1,4-glycosidic bond in peptidoglycan backbone
→ causes cells to be sensitive to changes is osmotic pressure
→ important host defence against bacteria - found in tears, saliva
How does penicillin affect peptidoglycan?
Penicillin inhibits peptidoglycan synthesis
→ transpeptidation is penicillin sensitive
→ halts cell wall synthesis, osmotically sensitive cells lyse
→ more effective against gram +ve bacteria
What is the purpose of teichoic acids in cell walls?
Teichoic acids are found in gram +ve cell walls
→ covalently connected to peptidoglycans or plasma membrane
→ involved in making cell surface negative charged (may help acquire Mg2+ and Ca2+)
How do archaeal cell walls differ from bacterial?
Archaeal cell walls don’t contain peptidoglycan (not sensitive to penicillin - fine because not pathogenic)
→ some methanogens contain pseudomurein - beta 1,3 links instead of beta 1,4
What are cell membrane mainly comprised of?
Phospholipid bilayer
→ hydrophobic tails, hydrophilic heads
→ separates and protects cell from environment
→ hydrophilic/charged substances may attach to the hydrophilic surfaces
→ proteins that traverse the bilayer have hydrophobic regions
What are sterols/hopanoids?
Sterols (eukaryotes), hopanoids (bacteria)
→ rigid planar molecules, stabilise membrane structure
Why are gram -ve cell walls asymmetric?
Due to the insertion of lipopolysaccharide into the external layer of the outer membrane
→ provides defence against cytotoxic chemicals like antibiotics
How is the outer membrane (gram -ve) linked to the cell?
- Braun’s lipoprotein → covalently linked to peptidoglycan and embedded in outer membrane by hydrophobic end
- Adhesion sites → where inner and outer membrane adhere, allow transport of substances to OM and out of cell
How do archaeal membranes differ from bacterial and eukaryotic?
(shows the 2 domains of life are different)
Archaeal membranes branched chain hydrocarbons attached to glycerol by ether links rather than fatty acid-ester links
→ stabilised by isoprene
→ contain double-headed lipids which make a mono-layer - stabilises membrane at extreme temperatures
What are lipopolysaccharides (LPS)?
Large complex molecules containing lipid and carbohydrate
→ lipid A, core polysaccharide, O side chain
When free in host called endotoxin
→ can induce massive immune response (septic shock)
What is the Lipid A component of LPS of gram -ve bacteria made up of?
Two glucosamine residues linked to fatty acids and phosphate
→ anchors LPS into outer membrane
→ remaining LPS projects out of cell surface
→ lipid A most immunogenic as endotoxin
What is the core polysaccharide of LPS of gram -ve bacteria made up of?
Very specific to the species
→ in salmonella: glucose, galactose, heptulose x2, 2-keto-3-deoxyoctonate x3
→ also referred to as R-antigen or R-polysaccharide
What is the O side chain of LPS of gram -ve bacteria?
Variable region responsible for the antigenic make-up of bacteria
→ difference in O antigen defines receptor
→ different O serotypes linked to disease
→ lipid A and core polysaccharide straight, O side chain flexible and bent
What is the function of LPS?
- lipid A stabilises outer membrane structure
- core polysaccharide is charged contributing to the negative charge on surface
→ helps protect cell + attracts nutrients - charged hydrophilic external layer reduces permeability of hydrophobic substances
→ preventing entry of bile salts, antibiotics - protects against host defences
- toxic to animals - toxicity linked to LPS lipid A (endotoxin/0
- key diagnostic tool
What are endotoxins?
Released by pathogens during cell division or by lysis
→ can act to prime immune system against pathogen if detected
→ LPS in the blood can cause septic shock syndrome
→ immunogenic even in absence of living cells
tests for endotoxins: rabbit pyrogen test, limulus amoebocyte lysate (LAL) assay
What are the important properties of endotoxins?
- heat stable
- toxic in nanogram amounts
- interacts with innate immune system cells
→ can trigger the release pf cytokines in a cascade
→ activates transcription factors like interferon-B and TNF - can result in; inflammation, fever, vasodilation, thrombosis, depletion of platelets/clotting factors (leading to haemorrhage), shock
What are porins?
Transmembrane proteins that act as channels for entrance and exit of solutes
→ makes the outer membrane relatively permeable to small molecules
→ most are non specific, some are selective
→ high thermal stability
→ resistant to protease and detergent degradation
What is the structure of porins?
16-stranded antiparallel B-barrel
→ exceptionally stable; extra stability from salt bridges between N- and C-termini
→ hourglass shape with central constriction
→ charges inside define size of solute that can traverse
What is the periplasmic space?
Compounds diffuse through porins into periplasm
→ periplasm in the space between the outer and cytoplasmic membrane
→ gel like consistency due to abundant proteins
What is the enzyme activity in periplasm?
- nutrient acquisition - hydrolytic enzymes like alkaline phosphate
- energy conservation - electron transport proteins )produce energy)
- some peptidoglycan synthesis enzymes
- periplasmic binding proteins - deliver specific compounds to transporters in cytoplasmic membrane
- chemoreceptors - involved in chemotaxis, sensing what enters
What are flagella?
Long thin extracellular helical structures made of protein subunits that aid in motility
→ connected to a motor that spins them clock- or anti- clockwise allowing bacteria to swim
→ rotates at very high speeds
→ biological motors
What is the structure of flagellum?
Complex ring structures anchored into the membrane and call wall/outer membrane
→ rings (lL, P, S-M, & C) and hooks are rigid and attached
→ filament id made of a single protein called flagellin
→ flagellum shaft is easily removed by vigorous shaking
→ different antigenic properties of shaft, tip of shaft and hook
→ the motor is driven due to transfer of protons through the ring structure (from periplasm to cytoplasm)
How are flagellum synthesised?
Growth occurs at the tip
→ subunits made in cytoplasm are exported to periplasm and sent up the flagella
→ constant growth to repair damage caused by movement
How does the flagella motor work?
Rotary motion provided by the basal structures
→ powered by proton motive force
→ requires lots of energy (one rotation requires translocation of 1000 protons)
→ changes in charge lead to conformational change of MotA, causing movement relative to MotB, second conformational change with loss of charge
How are gram +ve flagella different to gram -ve?
- no L and P rings
- anchor in the membrane layer is more complex
- mot proteins surround inner ring and movement of these relative to each other provides the force
How do bacteria move?
Via flagella; motility pattern alternate between ‘run’ and ‘tumble’
→ run - motor rotates anti-clockwise, flagellar filaments form bundle and propel cell
→ tumble - quick reversal of motor to clockwise rotation, produces twisting force that transforms flagella
→ separates filaments act in uncoordinated ways to generate forces that change orientation of cell - toward favourable conditions
What are the types of tactic responses bacteria make?
- aerotaxis - movement towards oxygen
- chemotaxis - movement towards nutrients also away from toxins
- magnetotaxis - movement along lines of magnetism
- phototaxis - movement towards light
How do bacteria move by chemotaxis?
Bacteria sense changes in nutrient concentration in environment
→ methyl-accepting chemotaxis proteins (MCP) detect and interact with cytoplasmic proteins
→ che proteins interact with rings of motor regulating direction, dictates run or tumble by switch of rotation
→ CheA-p phosphorylates CheY - CheY-p binds to flagellar motor conformational change causes bacterium to tumble
attractants decrease CheY-p → less switching, longer runs
repellents increase CheY-p → more tumbling
What are gas vesicles?
Protein vesicles that contain gas in planktonic bacteria
→ confer buoyancy, allows cells to float up to oxygenated water or towards light
→ can be involved in vertical migration in aquatic systems q
What are fimbriae/pili?
Surface appendages, mediate adhesion
→ don’t spin like flagella
→ involved in host-pathogen interactions, colonisation, biofilm formation, adhesion, resistance to phagocytosis, antigenic
→ specialised pili involved in genetic exchange between bacteria
How do fimbriae/pili aid in adhesion?
They allow the cell to break mucus layer and attach to epithelial cell and invade
What are type I fimbriae?
Thin surface polymer
→ made up of 500-3000 subunits of the protein FimA, stacked in a helical cylinder
→ important virulence factor in a range of pathogens (like E.coli, salmonella)
What is the tip fibrillum of type I fimbriae made up of?
FimH → tip adhesion, binds to surface of target
FimF & FimG → link FimH adhesin onto the fimbriae
What catalyses FimA polymerisation in type I fimbriae?
FimC → chaperone protein
FimD → usher protein
What is P-pili?
PAP: Pylonephritis-Associated-Pili
→ critical virulence factor of uropathogenic E. coli which can cause cystitis and pyelonephritis (UTI that reaches kindeys)
→ similar in structure and assembley to type I fimbriae - PapG is the tip adhesin,
What are type IV pili?
Widely distributed in gram -ve
→ longer than fimbriae, only a few pili per cell, typically at both cell poles, most are not hollow unlike flagella
→ twitching motility - allows movement by attaching
What are the roles of type IV pili?
- host cell adhesion
- biofilm formation
- twitching motility - crawl along surface
- enable enteropathogenic E. coli to form microcolonies on tissue monolayers
→ often aggregate laterally to form bundles - lack virulence
What determines species specificity of pathogens?
LPS (O antigen) and fimbriae (K antigen)
e.g E. coli → CFA (colonising factor agent) fimbriae are present on E. coli pathogenic to humans but K99 and K88 are fimbiral antigens in animal pathogens
What is the difference in adhesion between CFA (colonising factor antigen) and no CFA strains of enterotoxigenic E. coli (ETEC)?
ETEC strains have specific interactions with mucosal epithelium depending on CFA
have CFA → pathogenic, adhere to e.g. small intestine and urinary tract, express pathogenicity by producing toxins
no CFA → non pathogenic, adhere to colon, routinely secreted in faeces
What is a F pilus used for?
Involved in transfer of genetic information (from cell that contains F plasmid to one that doesn’t)
→ unlike other pili/fimbriae have a central 2nm wide channel
→ found on many Gram -ve bacteria
→ all plasmid encoded
What is conjugation?
Transfer of genetic information via F pilus
→ F+ donor attaches to F- recipient via surface protein on recipient
→ retraction - cells brought close together
→ exchange - replication and transfer of plasmid
→ results in 2 F+ cells
What are bacterial endospores?
Dormant stage in bacterial life cycle
→ simple, tough version of bacterial cell
→ form inside of a bacterial cell - when a vegetative cell becomes stressed
→ survival mechanism - resistant to heat, desiccation and radiation
→ some viable for 100,000 years
→ only Gram +ve produce endospores
How can you visualise endospores?
Staining with malachite green
→ heat/steam required for dye penetration
→ after washing only spores will remain with the primary dye (malachite green)
What is sporulation?
Complex series of cellular differentiation stages forming an endospore
→ in response to extreme stress
→ each stage controlled by different gene (over 200 genes involved - evolutionarily important)
→ process take 8h to complete
→environmental trigger stops production of normal cell growth proteins and switches to sporulation genes
→ trigger is probably nutrient depletion
What are the stages of sporulation?
- vegetative cell under stress
- DNA organised along cell axis
- genome copy enclosed in forespore septum
- cell membrane engulfs forespore in second membrane
- cortex between membrane accumulates calcium and dipicolinic acid, becomes dehydrated to <10%
→ resistance to chemicals and heat increases - complex exosporium layers produced
- spore matures with complete cortical layers
- original cell lyses releasing spore
What is the structure of endospores?
Many new layers, outermost exposporium - thin layer of spore-specific proteins
→ not essential for survival but maybe dispersal
Inside core containing; genome, cytoplasm, ribosomes - metabolically inactive
Small acid soluble proteins maintain genome integrity → prevent formation of pyrimidine dimers in DNA (cause DNA damage)
How are endospores resistant?
Dehydration (due to peptidoglycan cortex) of endospores prevents denaturation
→ can be resistant up to 150C
→ autoclaving destroys spores (conditions not seen in nature)
Where is death infectious disease more common?
Low income countries
→ in the USA leading cause of death in 1900 - infectious disease
in 2000 - heart disease and cancer
→ in 2016 in Africa 50% of all deaths due to infections
What are some barriers to infection?
Systems designed to stop infection
→ lysozymes in tears and other secretions - dissolves cell walls
→ skin (physical barrier) produces antimicrobial fatty acids, normal flora inhibits pathogen colonisation
→ rapid pH changes inhibit microbial growth
→ flushing urinary tract prevents colonisation
→ mucus, cilia lining move microorganisms out of the body
→ blood proteins inhibit microbial growth
What body sites are densely populated with bacteria?
Colon (GI tract contains the most), oral cavity, vagina
What is the human microbiome?
The ecological community of commensal, symbiotic and pathogenic microorganisms that share our body space
What are the suggested health benefits of the human microbiome?
Shield body tissues against invasion of pathogens
Production of vitamins by bacteria
What is virulence determined by?
- adhesion to and entry into cells
- antiphagocytic activity, immune system evasion
- production of toxins
→ conventional virulence factors; bacterial toxins, adhesins, cell surface carbohydrates and capsules, LPS
Why is MSRA a great concern for public health?
MRSA → Methicllin Resistant Staphylococcus aureus
Limited options of antibiotic therapy left due to multiple antibiotic resistance - last choice in vancomycin
Now VRSA isolates have been isolated → untreatable by any known antibiotic
What are the 5 major types of antibiotics?
- beta-lactams → interrupt cell wall formation (penicillins and cephalosporins)
- macrolides → inhibit protein synthesis (erthyomycin)
- fluroquinolones → inhibit DNA gyrase (ciprofloxacin)
- tetracyclins → inhibit protein synthesis (tetracycline)
- aminoglycosides → inhibit protein synthesis (kanamycin) `
What are some mechanisms of antimicrobial resistance?
- reduces permeability → penicillins
- inactivation → penicillins, aminoglycosides
- target alteration → macrolides
- new resistance pathway → sulfonamides
- efflux, tetrayclines, erthyromycin
What was the first vaccination using a weakened pathogen?
In 1879 Louis Pasteur used weakened Pasteurella multocdia (chicken cholera) to infect chicken - did not cause disease
→ tested on sheep, no vaccinated sheep died/suffered
→ evidence that weakened vaccination worked
What do tetanus toxins do?
Tetanospasmin - released by cell lysis, spreads through blood and lymph
→ targets CNS, binds to peripheral nerve terminals
Halts the release of glycine and GABA neurotransmitters
→ normally check nerve impulses
→ absence leads to muscular spasms
Why doesn’t the tetanus vaccine produce herd immunity?
The vaccine targets the toxin not the organism
→ recovery from tetanus does not confer immunity
