AID Flashcards
What are the 3 domains
- Archaea
- Eucarya
- Bacteria
Different types of eucarya
- Fungi
- Plants
- Animals
- Protists
What are the key reservoirs of biomass and nutrients for all life?
Bacteria and archaea
Why are there so many microorganisms
- Rapid growth rate
- Many chances of speciation through random mutation
- Exchange of genetic material
- Every niche is occupied
What is a phototroph
Gets energy from light
What is a chemotroph
Gets energy from chemicals
What is an organotroph
Uses organic compounds as electron donors
What is a lithotroph
Uses inorganic compounds as electron donors
What is an autotroph
Uses Co2 as a carbon source
What is a hetrotroph
Uses oranic carbon as a carbon source
Two types of primary nutrients
Macronutrients (carbon, nitrogen) and micronutrients (trace metals)
4 different stages of a growth curve
- lag phase (adaptions to new conditions)
- exponential phase
- stationary phase (Limitations in nutrients and a build up of waste products)
- death phase
Different measures of growth in microorganisms
- Cell number (haemocytometry, dilution plating)
- Optical density (turbidmetry)
- Fresh/dry weight
- Protein
- DNA
How to identify microorganisms
- Staining and microscopy
- Growth on selective media/ differential media
- Testing enzyme activity
- Characterising of cell constituents (lipid, cell wall, components)
Modern identification is based on sequencing of conserved genes.
How does selective media identify microorganisms
Allows the growth of some microorganisms
How does differential media identify microorganisms
Based on growth and appearance on that media
Testing enzyme activities towards pathogens
- Culture the organism
- Cell is re-suspended in a buffer
- Test wells are inoculated
- Test strip is used to see enzyme activity by colour change, which is compared to a database
- Used to distinguish between pathogenic strains and benign strains
Surface origin hypothesis
- On the surface there was a primordial soup, organic compounds formed by chemical reactions and electrical activity caused by meteor strikes
- Organic acids came together to form amino acids and nucleotides
- Theory not likely due to hostile conditions on the surface
Subsurface origin
- Life begins in hydro thermal vents where compounds mixed at high temperatures
- Self replicating RNA forms and enzymatic proteins
- DNA forms and leads to the evolution of biochemial pathways
- Divergence of lipid biosynthesis, cell walls and cell type
- Formation of early bacteria and early archaea
What are the landmarks in biological evolution
- Early life is dependent on H₂ and Co₂
- Energy and carbon metabolism diversifying
- Phototrophy using H₂S as am electron donor
- Evolved into oxygenic photosystem using H₂O
Define phylogenics
How related organisms are to each other
Molecular sequences used in phylogenics must be
- Universal
- Contains conserved and variable regions
- Not subject to gene transfer
- Must be truly homogeneous (perform the same function)
Tree of life use comparative ribosomal RNA
Basic evolution of eukaryotes and the 2 theories
- Mitochondrion appeared in a proto eukaryotic cell
- Nucleus formed
- Plants formed when chloroplasts came about
Endosymbiont theory and hydrogen hypothesis
What is the endosymbiont theory
- Mitochondria was formed due to the incorporation of aeorbic chemoorganotrophic bacteria into a host cell
- Chloroplasts formed due to the incorporation of phototropic cyanobacteria into a eukaryotic cell
What is the hydrogen hypothesis
Symbiotic association of an archaeal host using H2 as energy source with an aerobic bacterium that produced hydrogen as a ‘waste’ product.
Higher organism species definition
They interbreed to produce fertile offspring
Why is it hard to classify bacteria species
- Asexual reproduction
- Lateral gene transfer
- Phenotypic and genotypic plasticity of microorganisms
What is polyphasic bacterial taxonomy
Taxonomy taking in account:
- Phenotypic analysis (Morpholical, metabolic, physiological characteristics)
- Genotypic
- Phylogenetic (evolutionary links)
Gram staining procedure
- Spread culture thin and air dry
- Add crystal violet
- Fix with iodine
- Wash with alcohol
- Counter stain with safrarin
- Positive appears purple, negative appears blue
What does a barophile require
a high pressure
What does a halophile require
very salty conditions
What is a microaerophile require
requires low oxygen concentration
What does a psychrophile require
low tempertures (<15 degrees)
What does a mesophile require
“normal” temperature (15-45 degrees)
What does a thermophile require
high temperature (>50 degrees)
What does a hyperthermophile require
very high temperature (>80 degrees)
Name some colony based characteristics
Colony shape, margin, elevation, opacity, texture, pigmentation, odour
What is molecular analysis FAME (fatty acid methyl ester)
It determines fatty acid profile of membrane lipids by:
- Grow under standard conditions
- Extract lipids and chemically modify to methyl esters
- Analyse products by gas chromatography
Drawbacks of FAME
- Fatty acid profile depends on growth conditions which need to be standardised
- Not all strains can be cultured in these conditions
4 different types of genotyping analysis
- DNA-DNA hybridisation
- DNA profiling
- Multilocus sequence typing (MLST)
- GC base ratios
What is DNA-DNA hybridisation
Genome wide comparison of sequence similarity
What is DNA profiling
Producing DNA fragment patterns for comparative analysis
What is multilocus sequence typing
Sequencing several housekeeping genes and assign different alleles to different strains
What are the outer layers of a gram positive cell (outside to inside)
- Capsule, s-layer
- Cell wall (peptidoglycan)
- Periplasmic space
- Cytoplasmic membrane
What are the outer layers of a gram negative cell (outside to inside)
- Capsule, s-layer
- Outer membrane
- Periplasmic space with a layer of peptidoglycan
- Cytoplasmic membrane
What is a capsule and what are its roles
- It is a polysaccharide layer outside of the cell wall
- Prevents cell dehydration
- Involved in the attachment to surfaces
- Involved in the capture of nutrients
- Acts as carbon store
What is the s-layer
- A layer of protein and glycoprotein external to the cell wall
- Protects against ion and pH fluctuations as well as osmotic stress
What is peptidoglycan (PG) made of
- Alternating residues of NAG and NAM which are cross linked by amino acid side chains
- Has D-amino acids which protects against degradation by proteases
- It retains the stain in gram ve+ cells
What is a lysozyme and what is its significance
- An antibacterial enzyme
- Degrades 1-4 glycosidic bonds in pepidoglycan back bone
- Loss of PG makes the cell sensitive to changes in osmotic changes
- Important om host defence against bacteria
What are teichoic acids
- Only found on gram positive cells
- Ribitol or glycerol polymers joined by phosphate groups
- Covalently bound to peptidoglycan
- Gives the cell wall a negative charge
- May help the cell to obtain Mg2+ and Ca2+
roles of sterols and hopanoids
- Hopanoids in bacteria, sterols in eukaryotes
- They are rigid molecules that stabilise the membrane structure
The 2 ways the outer-membrane is linked
- Braun’s lipoportein
lipoproteins covalently linked to peptidoglycan and embedded in the outer membrane - Adhesion sites
Where inner and outer-membrane adheres
What are archaeal cell walls
- Typically has no outer membrane
- No peptidoglycan
- Instead has a polysaccharide called pseudomurein which is similar to peptidoglycan
- If the archaeal cell doesn’t have pseudomurein it will contain other polysaccharides
What is the lipopolysaccharide (LPS) composed of?
- Lipid A
- Core polysaccharide
- O side chain
Also called an endotoxin when it is free in the host
Significance of the O-antigen
- Variable region responsible for antigenic make up of bacteria
- Different O serotypes link to different diseases
- Responsible for species specific attachment
- Smooth and rough variants
Function of the different components in the LPS
- Lipid A stabilises the outmembrane
- Core polysaccharide is negatively charged, reducing permeability of hydrophobic substances
- Protects against host defences
- O antigen is used as a key diagnostic tool due to its variability
Function of endotoxin and when is it produced
- Produced by pathogens during cell division or by lysis of bacteria
- It primes the immune system against a pathogen (Immunogenic)
How are endotoxins tested for
By an LAL assay, where blood cells from Limulus polymephus clot when they come in contact with an endotoxin
Important properties of endotoxins
- Heat stable
- Toxic in nanogram amounts
- Triggers release of cytokines and activates transcription factors
- Results in inflammation, fever, vasodilation
What are porins
- Highly conserved transmembrane proteins that form water filled channels
- Made out of 3 identical subunits
- Most are nonspecific channels but some are specific
- Very stable due to a salt bridge
Function of the periplasm
- Nutrient acquisition
- Energy conservation, electron transport proteins
- Peptidoglycan synthesis
- Binding proteins that deliver compounds to ABC transporters
2 pathways that transport protein into the periplasm
Sec pathway and TAT pathway
What is the sec pathway
- Exports nascent polypeptide through cytoplasmic membrane using a translocase
- Folding of protein occurs after translocation
What is the TAT pathway
-Exports fully folded enzymes across the cytoplasmic membrane
What is a flagella
Long thin extracecllular helical structures that aid in motility. Proton motive force causes conformational changes in the motor.
Structure of the flagella
- Cap
- Filament (made out of flagellin)
- Has a base known as the hook
- Ring structure (transfer of protons through the ring structure drives the motor)
rings and hooks are attached to the membrane
Synthesis of flagella
- MS and C rings form
- Motor proteins form
- P and L rings form, the hook and cap
- Filament grows through the flow of flagellin through the hook
What are the different types of flagella
- Single flagella
- Flagella at opposite poles
- Multiple flagella at opposite poles
- Have flagella anywhere on the cell
What are the different motility patterns
Run and tumble. It is a fairly random process, but bacteria dont move in random directions.
What can motile molecules move towards
- Oxygen
- Nutrients, away from toxins
- Move along magnetism line
- Light
Role of methylated MCP
It is “bacterial memory” and remember previous concentrations of attractant and repellent
- High conc. attractant: Methylated MCP results in short runs and tumbling to stay in a “good” environment
- High conc. repellant:Methylated MCP results in longer runs and less tumbling to leave “bad” environment
What is gliding motility
Bacteria glides across a surface by the lateral movement of outermembrane protein. Does not need flagella or pili
What is twitch motility
Type 4 pili extend from the surface than retracts itself, pulling itself along. Powered by ATP hydrolysis. Pili is different to flagella.
What are gas vesicles
- Protein vesicles containing gas, which confers buoyancy to the cell
- Found in planktonic bacteria and some archaea
- Cells can float towards light or oxygenated water
What are fimbriae/pili
- Surface appendages involved in bacteria adhesion to surfaces
- Allows pathogens to attach to tissues
- Aid resistance to phagocytosis
- Are antigenic
Type 1 and type 4 pili
- Type 1: Thinner and shorter than flagella, hundreds to thousands per cell
- Type 4: Had an adhesive tip that binds to a glycolipid or glycoprotein. Pili pulls bacerium closer to the host cell.
Structure of type 1 pili
- FimH is the tip adhesin
- FimF and FimG link FimH to FimA
- FimA is the fimbriae
- FimC is a chaperone
- FimD is an usher protein (catalyses FimA polymerisation at the base)
What is the F pilus
- Pilus connecting two bacteria cells
- Involved in the transfer of genetic material, transfers DNA in the form of a plasmid
Stages in sex pilus conjugation
- Best known F pilus
- Conjugation:
1. F plasmid cell attaches itself to a cell without the F plasmid
2. Pilus retracts and fuse
3. Exhange of F plasmid
4. Both have the F plasmid
What are bacterial endospores
- Dormant stage in bacterial life cycle (metabolically inactive)
- Forms when the cell becomes vegetatively stressed
- They are extremely resistant to heat, desiccation and radiation
- Germinate when conditions become favourable
- Only found in gram ve+
What triggers sporulation
- Formation of endospores
- Complex series of differentiation events, controlled by many genes
- Triggered by nutrient depletion
Stages of sporulation
- Vegetative cell is under stress
- DNA is organised along cell axis
- Genome copy is enclosed in a forespore septum, forespore is formed
- Cell membrane engulfs forspore in a second membrane
- Spore is dehydrated making it heat and chemical resistant
- Exosporium layer are produced
- Spore matures with complete cortical layers
- Cell lyses releasing spore
Germination of endospore
- Uptake of water and amino acids trigger germination
- Re-hydration and a loss of resistance
- Cell is released and grows in the normal way
- Gram negative when released but develops and becomes positive again.
How was spontaneous generation disproved
Pasteur sterilised contents of the flask and showed the flask remained sterile as long as it was untouched
What is kosh’s postulates
That a specific microorganism causes a specific disease
- Pathogen is present in disease and absent in healthy animals
- Suspected diseased organisms are grown in pure culture
- Cells from the pure culture of the suspected diseased organism should cause disease in a healthy animal
- The new infected organism is isolated and shown to be the same as the original
Suggested benefit of the human microbiome
- Shields body tissue against invasion of “bad bugs”
- Production of vitamins from bacteria
Hard to determine what microbes cause disease and what are effected by disease
Factors that determine virulence
- Adhesion and entry into cells
- Antiphagocytic activity and immune system invasion
- Production of toxins
Why is MRSA and VRSA a threat
They have limited or no treatable antibiotics left
What are emerging “new” bacterial pathogens
“New”- not previously known as pathogens
What are the 4 major classes of pathogens
- Extracellular bacteria, parasites and fungi
- Intracellular bacteria and fungi
- Intracellular viruses
- Extracellular parasitic worms
What are the challenges faced by the immune system
- Has to protect against a variety of pathogens
- Pathogens and mutate and recombine, so the immune system must be adaptive
- Re-exposure (memory)
- Rapid division (rapid response)
- Has to distinguish between self and non-self
- Has to be tissue specific
What is antigenic drift
An accumulation of mutations within genes that code for antigen
What is antigenic shift
When two or more viruses combine to form a new subtype by having a mixture of antigens from the combined viruses
What is autoimmunity
- Immune system attacks healthy tissues and cells
- Autoimmunity results from impaired regulation of powerful immune response
What are 2 immune responses
- Innate (rapid, non specific, no memory and encoded into the germ line, dependent on family history)
- Adaptive (Highly specific, slow to adapt , has memory, somatic recombination)
What is cell mediated immunity
Defence provided by specialised cells in the blood and tissues. Relies on phagocytic cells and natural killer cells.
What is humoural immunity
Soluble phase defence by secreted proteins in bodily fluids. Relies on barriers and chemical warfare.
3 lines of innate immune system defence
- Barriers: skin, tight epithelial cells, stomach acid, mucus layers
- Cell-intrinsic responses: Pathogen induced phagocytosis and degradation of ds RNA
- Specialised proteins and cells: professional phagocytosis (e.g.neutrophils and macrophages), natural killer cells, complement system
What is the mucus layer and its function
- Made of secreted mucins and other glycoproteins
- Protects against microbial, mechanical and chemical assaults
- It is slippery so it is hard for pathogens to attach
- Contains defensins
What are defensins
- Small positively charged antimicrobial peptides which can kill or inactivate bacteria, fungi, parasites and envelope viruses.
- There are multiple defensins which have different targets
The mechanism of defensins
Hydrophobic domains enter into the core of the lipid membrane of the pathogen destabilising it, leading to cell lysis.
Why do defensins lyse pathogens but not our own epithelial surfaces
They are more active on membranes that dont contain cholesterol
What are PAMPs
Pathogen associated molecular patterns are molecules which the innate immune system recognises as pathogens.
Various classes of PAMPs
- N-formylmethionine (fmet), which is used for bacterial initiation
- Peptidoglycans from cell walls
- Bacterial flagella
- LPS from gram ve-
- Mannans, glycans and chitin from fungi
What are PAMPs recognised by
Soluble receptors in the blood (complement system) and by cellular receptors (Toll like receptors)
How does complement activation target pathogens for lysis
- Lectin pathway (pathway that binds to a sugar) or alternative pathway by pathogen surfaces cause cleaving of proenzymes resulting in a amplified proteolytic cascade to c3
- c3 cleaves c3a and c3b
- c3b binds to the pathogen membrane while c3a stimulate inflammation and attracts phagocytes and lymphocytes
- Pathogen bound c3b causes a cascade of reactions
- c9 is inserted into the membrane
- c9 pore breaches the membrane, membrane attack complex is formed
- Pathogen lysis
Toll like receptors
- Binds to pathogenic fungi and sends signals to the nucleus that result in the expression of anti-fungal defensins and inflammation
- They are found in epithelial cells, macrophages, dendritic cells and neutrophils
What are the 3 major classes of phagocytes
- Neutrophils
- Eosinophil
- Macrophage
Properties of neutrophils and what are they recruited by
- Short lived cells
- Abundant in blood
- Not present in normal healthy tissue
recruited by:
- Macrophages
- PAMPs
- Peptide fragments of cleaved components
What are granulocytes and which two phagocytes are they
- Neutrophils and eosinophils
- There cytoplasm is granular
What do macrophages remove
- Larger and longer lived than neutrophils
- Removes senscent (old), dead and damaged cells in many tissues
- Can digest microorganisms
What are eosinophils
- They work in gangs and collectively kill large parasites
- They also mediate allergic inflammatory responses.
What are the cell surface receptors on phagocytes
- TLRs
- Receptors of antibodies produced by the adaptive immune system
- Receptors for complement c3 protein
What does an active phagocyte release and induces
- Releases cytokines to attract more white blood cells
- Induces the phagocyte to engulf the pathogen into a phagosome
How is the pathogen killed once in the phagosome
- Granules fuse with the phagosome releasing lysozymes and acid hydrolases in attempt to digest the cell wall.
- Also release defensins which destabilises the membrane
- Respiratory burst allows NADPH oxidase complexes to priduce highly toxic compounds
What happens when neutrophils die
They will eject their DNA in a sticky web that traps bacteria preventing their escape from the killing frenzy
How do some pathogens survive phagocytosis
- Addition of sialic acid to capsule components avoids complement attack
- Some bacteria survive and replicate in neutrophils
- Some bacteria can neutralise actin polymerisation and therefore phagocytosis
- Some bacteria can survive in macrophages
How does inflammation aid in the killing of pathogens
- Blood vessels dilate leading to swelling and accumulation of components of the complement cascade.
- Macropages also secrete cytokines inducing chemokines that attract cytokines
How does the innate immune system recognise viruses
- Recognise CpG motifs in viral DNA
- Recognition of viral ds RNA that is an intermediate in the life cycle of viruses
What are interferons
- Cytokines that interfere with viral infection
- Induced by ds RNA
- Work in an autocrine nature, induces change in infected cells
- Works in a paracrine nature, induces change in neighbouring cells