Microbial life Flashcards
How has ceelular life changed throughout earth’s existence ?
Cellular life starts approx 4bya (3.8 bya), first organisms did not use oxygen but did use sunlight (cyanobacteria) anoxygenic phototrophic bacteria in the anoxic world, cyanobacteria (blue green algae) present 1 billion years later releasing oxygen
2bya were where the modern eukaryotes came about. Then algae diverse then 45 million years ago we had shelly invertebrates
What is the significance of cyanobacteria ?
- Cyanobacteria symbiotic with plants, become chloroplasts
- This stabilises the atmosphere by oxygenating it
How has the tree of life changed with microbes ?
found by rRNA sequencing, detect SNPs, gene loss, gene acquisition
- 3 domains: bacteria (divert first), then archaea diverts then eukaryote 2.8 bya, these all stem from LUCA (3.8 bya) – last universal common ancestor (can be called progenote)
- Archaea has properties of both bacteria and eukarya so is an intermediate and was added later this relationship was found using 16S rRNA sequencing
How are genes selected for sequencing to add to the tree of life ?
- Choosing the gene to sequence: should be conserved, present in all organisms, same function amongst the group of organisms
- Genes in ribosome productions are often used, 16S prokaryotes, 18S in eukaryotes
What is a mesophile ?
favour stable conditions, lots of oxygen and a specific optimum temperature, easy to culture
Where is most of microbial life present ? And what are they a reservoir for ?
Most of microbial life (66%) are on the present on the marine subsurface, 2.5 x 1030 cells on earth, microbial cells are a key reservoir for nutrients in life (carbon (20% of the (planets carbon), nitrogen, phosphorus)
What is required for microbial growth ?
carbon, nitrogen, phosphorus, other macronutrients (S, P, K, Mg), micronutrients (Fe, Mn, Co, Cu, Zn, Mn, Ni)
- Electron acceptors – nitrate, sulfate, iron 3
- Inorganic electron donors (Hydrogen, hydrogen sulfide, iron 2, ammonium, nitrite)
What conditions are required for microbial growth ?
temperature, water potential, pH, oxygen (oxic -> microoxic -> anoxic), light, osmotic conditions
What is the name for microbes that can withstand cold, medium and hot temperatures ?
psychrophile -> mesophile -> thermophile -> hyper thermophile
What is the name for microbes that can withstand low, neutral and high pH ?
acidophil -> neutralophile -> alkaliphile
What is an extremophile ?
survive in environments in challenges with temperature, pH, salinity, pressure etc
How do we know the first microbes were extremophiles ?
Anoxic earth, high temperatures, variable pressure, harmful volcanic gases
How are 66% of all microbes extremophiles ?
- 66% of all microbes are on the marine subsurface, so they are all extremophiles as there is less oxygen, no light, high pressure, low temperature, high salinity (more salt at the bottom of the sea than the top)
What is a facultative microbe ?
prefer one conditions but can survive in both levels of oxygen (high and low). Facultative are the most successful organism.
What makes hydrothermal vents an extreme environment ?
toxic compounds, high pressure and temperature, provide light
Bacteria living in thermal vents are called chemosynthetic bacteria, what does this mean ?
converts heat, methane and sulphur compounds into energy, support other life forms
Microbes are a rich source of …
Carbon source: falling organic matter, carcasses, detritus
- Lots of dissolved minerals
What is the ring of fire ?
The edges of tectonic plates where hydrothermal vents are present, this means these areas are abundant in extremophiles
What are some extreme environments on land ?
Salt Lake, soda lake, deserts soils, hot springs
- Man-made: caves, mines, dams, deserts, global warming, deforestation
- Acid mine drainage: toxic ions and sulfuric acid, catastrophic effect on aquatic environments, some strongly acidophiles archaea can be found
What is a barophile ? halophile ?
withstand high pressure, withstand high salinity
What is a piezophile ?
same as a barophile
What are the types of bacteria morphology we can have ? What are they dependent on ?
coccus (sphere), bacillus (rod), spirochete (spiral), streptococcus (chain), staphylococcus (cluster)
- Shape is based on the environment, more viscous – spiral so they can move
What does pleomorphic mean ?
adopt different shapes depending on the environment
What makes up the bacterial envelope from the outside inwards ?
capsule, cell wall, cytoplasmic membrane
What is the cytoplasmic membrane composed of ?
phospholipid bilayer and proteins
What are the 3 functions of the cytoplasmic membrane?
permeability barrier, protein anchor, energy conservation
How does the cytoplasmic membrane act as a permeability barrier ?
selectively permeable, prevents diffusion in/out, nutrients in, waste out, polar and charged molecules must be transported (transport proteins), accumulate solutes against the concentration gradient
How does the cytoplasmic membrane act as a protein anchor ?
integral (face both inside and outside of the membrane) and peripheral membrane proteins (face one way), transport, energy conservation, adhesion, signalling, chemotaxis
How does the cytoplasmic membrane act as a energy conservation?
protons and hydroxyl ions separated by membrane, create ‘proton motive force’, drives many energy-requiring functions (transport reactions, motility, synthesis of ATP)
How does the cell wall of gram positive and negative bacteria vary ?
Peptidoglycan: many layers in gram positive, gram negative the peptidoglycan is sandwiched in-between the plasma membrane and the lipopolysaccharides (in the outer membrane area) in an area called the periplasmic space
What are the steps of gram staining ?
1) heat-fix the cells (cell in suspension), kill the cells and fix them to the glass
2) add crystal violet
3) iodine treatment
4) decolorization (alcohol, wash the complex ions off the gram-negative bacteria, the alcohol washes away the outer lipopolysaccharides of the gram negative bacteria)
5) counter stain safranin
What is the significance of the crystal violet ?
binds to the peptidoglycan in both grams, it is the primary stain, the stain can get to peptidoglycan in gram negative due to heat fixating and breaking the outer membrane
What is the significance of iodine in gram staining ?
(trapping agent), interacts the crystal violet ion forming a complex, these are trapped in the peptidoglycan strands, more is trapped in gram positive
What is the significance of the safranin dye ?
counter stain safranin (stains both but visible in gram negative only, the gram positive is so deep purple that the pink colour is unobservable, pink – negative, purple – positive
What are most bacteria ?
Gram -ve
Which type of bacteria is more prone to resistance ?
outer membrane gives resistance to detergence, gram negative is more resistant to antibiotics and larger hydrophobic molecules
Why are antibiotics more effective on gram +ve?
the peptidoglycan strands are readily available for forming crosslinks
What is also present in the cell wall of gram -ve ?
protein channels (porins): allow for transport, by passive diffusion (no energy), porins vary in size and shape depending on the molecule they transport
- less variety of porins more selectively permeable more likely to be resistance
What are LPS ?
(lipopolysaccharides): structural integrity, protect cells from toxins like antibiotics, induce a strong immune reaction in animal’s blood, they are called an endotoxin.
- Endotoxin septic shock can be induced
What is the capsule ?
usually polysaccharides, resists desiccation (extreme dryness), virulence factor, attachment to surfaces, can be used as a food source in nutrient low environments
What is loosely packed polysaccharides in the capsule called ?
slime capsule, they dont hold dyes very well
What are the 2 external appendages of bacterial cells?
Pilli & fimbriae, flagella
What are Pilli ?
filamentous protein structure, vary in size and shape, fimbriae: are smaller and assist in surface attachment and movement along a surface, pili facilitate genetic exchange between cells (conjugation)
What is a flagellum ?
o Flagellum: structure that assists in swimming, helical in shape (20 micrometres), long hollow tube, allows locomotion
Attached to the cell membrane, gram negative needs an extra anchor onto the periplasmic membrane space, the hook on the outside rotates allowing for the flagella to move, flagellin (filament) that rotates
What are the different patterns in flagellar ?
Monotrichious, amphitrichuos, lophotrichous, petrichous
What is the bacterial cytoplasm ?
fluid portion where molecules float, very cramped with interacting molecules
What is the bacterial genome made up of ?
chromosomes and plasmids
What is a bacterial chromosome like ?
closed, circular, aggregate as nucleoid, haploid, contain essential genes (survival), supercoiled (the coiled DNA is coiled further like a phone line), e.coli: 4640 Kbp, 4288 genes
What is a plasmid ?
small circular DNAs, multiple copies, autonomous replication, transmitted between cells, confer specific properties
What might happen if the plasmid contains genes essential to life ?
may become integrated into the nuceloid
What is the bacterial ribosome made of?
2 subunits (30S and 50S), proteins and rRNA
What is polyribosomes in terms of transcription and translation ?
transcription and translation are coupled, different RNA polymerases can bind at once, multiple ribosomes (polyribosomes) can translate at once on the same mRNA
Why do bacteria need to communicate with the chemical world ?
- Compete for occupancy
- Fight starvation or nutrient depletion (search for nutrients)
- Exposure to noxious chemicals (stay away from toxic environments)
What is taxis ? what are some examples of it ?
Taxis: movement toward or away from a stimulus
- Chemotaxis (chemoattractant vs chemorepellent), aerotaxis, phototaxis, magnetotaxis
What is a capillary tube assay and whats the functionality of it ?
Capillary tube assay (put a test tube of a solution into a culture and observe):
- Identify chemotactic cells
- Identify attractant/repellent
What are the 3 types of motility in microbes ?
flagellar locomotion, axial filament (twisted flagellar around the cell, so when it turns the bacteria twists in motion), gliding motility (on surfaces)
What is the relationship between cell length and chemical gradients ?
longer cells can sense more difference in concentration due to either side of the cell being at a different level of concentration.
What is the “biased random walk” ?
movement based on a chemical gradient
How does the random walk in a homogenous mixture work ?
aimless zigzag path and straight runs with random tumbles (change in orientation) – ‘run-and-tumble’
How does the biased random walk work in a heterogenous medium ?
when there is a heterogenous medium. The frequency of tumbles is less frequent in biased random walking but the runs are longer, the downgradient movement is more frequent in tumbles but the time of run is les.
- The longer runs upgradient allows for the general movement to an attractant
How does motility work in peritrichuos bacteria ?
The flagella rotate around the cell body counter clockwise and so every now and again they bundle, the change in where they bundle changes the direction of movement therefore allows for as tumble to take place
Flagellar locomotion is a … signal transduction pathway
chemosensory
How does the chemical signal drive the movement ?
The chemoreceptors on the side that the bacteria wants to move in detect a change then a protein cascade reaction occurs that activates the flagellar motor on the other side of the bacteria allowing for the movement into the direction
What type of reaction allows for flagellar locomotion ?
de/phosphorylation
How does the run-tumble-run work in flagellar locomotion in a homogenous medium ?
The phosphorylation allows for the tumble, the phosphorylated protein caused the motor to move in the opposite direction. The dephosphorylation allows for CCW movement to happen
How does the run-tumble-run work in flagellar locomotion in a heterogenous medium ?
When MCP binds to the chemoreceptors, prevents the CheA phosphorylation, preventing the cascade allowing for a longer run
How does a repellent change the flagellar locomotion pathway ?
A repellent increases the phosphorylation of CheA allowing for more CCW movement and more tumbles.
With a monotrichous flagella, the CW rotation can either lead to a … or a …
CCW rotation, change in direction
In a monotrichious bacteria, what does the change in direction lead to ?
leads to a ‘flick’ due to the kink in the tail
What is Quorum sensing?
Quorum sensing (cooperation): produces and releases chemical signals called autoinducers to increase in concentration as a function of cell density.
- Tells bacteria how many other bacteria there are in order to regulate their own gene expression
What is an example of Quorum sensing ?
o Bioluminescence: requires lots of resources, the luminescent gene is only expressed if it’s worth the cells energy, in other words if there are enough cells around to actually produce a luminescence
Hawaiian bobtail squid – vibrio fischeri stored in the squid
What is the significance of cell density in quorum sensing ?
Density: high cell density leads to group behaviours, autoinducers diffuse freely across the cell envelope. Low cell density leads to individual behaviours
What is an autoinducer ?
- Autoinducers: AHL (acylated homoserine lactone), diffuse allowing for a cascade reaction leading to genes being switched on for group behaviour. This occurs in gram negative bacteria
o Each specie has its preferred autoinducer, each specie has their own ‘language’ of AHL, they can feel international language (other species AHL) and can sense how prevalent their specie is in the population by comparing their intraspecies AHL and the interspecies AHL
What are some quorum sensing controlled behaviours ?
bioluminescence, biofilm formation, virulence factor production, swimming motility.
What is quorum quenching ?
one species can interfere with communication of another specie, allowing for them to fight off other species
How is quorum sensing being used to prevent cholera ?
Preventing cholera infection using engineered gut flora, introduction of engineered commensal strain, intercellular signalling blocks virulence of the cholera bacteria, the quorum sensing inhibits the virulence genes. The AHL tricks the cholera into thinking there is too high cell density to produce the virulence factors.
How do bacteria change their external environment?
- Through their metabolic:
o Consume nutrients, release waste products - Through secretion of proteins and chemicals:
o For nutrition, for competition, for habitat, for motility
What are metabolic outputs and inputs ?
Metabolic outputs: external accumulation of secreted metabolic products and by-products
Metabolic inputs: consumption of nutrients and compounds from their environment
What is bioremediation ?
uses microorganisms to clean up contaminated soils and groundwater, bacteria utilise contaminants as source of foods and energy, can break down hazardous substances into less toxic or non-toxic substances
- Example: metal decontamination: anaerobic respiration, metal gets reduced
What is simple transport ?
cross a transmembrane protein, driven by the energy in proton motive force e.g., sodium ions, potassium ions, lactose
What is group translocation?
multiple proteins involved, chemical modification of the substance, driven by energy-rich organic compounds (phosphoenolpyruvate, PEP), glucose, fructose, mannose
What is ABC transport ?
ATP-binding cassete, periplasmic binding proteins involved, energy comes from ATP, sugars, amino acids, inorganics
How does the gram -ve cell secrete a signal peptide ?
signal peptide: 15-20 amino acids at N terminal (cleaved) for secreted and membrane proteins
- Through cytoplasmic membrane translocases to insert or export proteins (sec pathway)
- Gram negative:
o 2-steps: translocases through inner and outer membranes
o 1-step: proteins exported directly outside
What is a type III secretion system ?
pathogenic bacteria use injectosomes to deliver toxins directly into host cells. Injectosome is embedded into the plasma membrane with an extracellular of a needle similar to a flagellum. Evolutionarily using the same body for a different function
- Can synthetically add genes for the injectosome into the bacteria producing a SIEC (synthetic injector E. coli)
What is a VI secretion system ?
Type VI secretion system: a firing mechanism. A tube contains an arrow, the tube then contracts ejecting the arrow into the target cell
- Used for bacterial competition for a niche
What is an example of secretion of hydrolase ?
Secretion of hydrolases: break down complex molecules into smaller compounds
- Cellulose; cytophaga is a genus of gram-negative soil bacteria (release of a cellulosome which degrades cellulose cell walls)
What is an exo-enzyme and where is it used ?
Exo-enzymes: enzymes that work extracellular
- Used in biological washing powder, a mix of enzymes is used so it works on a wider range of stains. Enzymes are harvested and purified by industrial bacteria, the enzyme is then locked in a structure to avoid them being allergenic
What do bacterial cells secrete in order to compete ?
- Bacterial antibiotics: aminoglycosides, macrolides, tetracyclines, most inhibit protein synthesis (target ribosomes), resistance to own antibiotics
- Bacteriocins: narrow spectrum of activity, kill strains of the same or closely related species, bind to receptors on sensitive cells and penetrate through plasma membrane, encoded on plasmid with immunity genes e.g., colicins from E. coli.
What is biocontainment ?
Biocontaiment: toxin-antitoxin system, use bacteriocins for biological containment
What do bacterial cells secrete in order to live in a habitat ?
o Surface niches: save energy, greater access to nutrients, protection from predation
o Adhesion through: cell surface proteins, pili, capsule
o Biofilms: most common form of bacterial growth, secretion of polysaccharide slime and cell debris to form a mixed environment (yeast (eukarya) may grow here too)
What are some benefits of a biofilm ?
protection from shear stress, exclusion of molecules (antimicrobials)
What are some chemical benefits to the biofilm ?
absorption of nutrients (high local concentration), micro-niches with optimal parameters, effective concentration of QS (quorum sensing) molecules
What are some biological benefits to biofilms ?
local release of extracellular enzymes, local production of inter-species effectors (bacteriocins), processes relying on cell-cell contact
Describe the biofilm life-cycle ?
- Attachment (swimming cells with a flagella, finding a niche through chemotaxis) colonisation (express biofilm genes like slime production), (differentiate to sessile cells by losing the flagella) growth maturation (some cells revert back to being planktonic) Dispersal (bursts releasing the planktonic bacteria)
What is the importance of biofilms ?
- Bacterial resistance: biofilms increase resistance of pathogens to immune system and antibiotics; resistance may depend on their place in the biofilm
o They may go into a slow growing state (quiescent) in the biofilm to reduce the antibiotics effectivity, as antibiotics target fast growing cells. - Medical implant biofouling
- Industrial biofouling (vector for bioinvasion, accumulation of microorganisms on submerged structures like a ships hull)
o Cooling systems: growth of biofilms in pipes
o Waste water treatment: biofilm carriers (K1: bacteria form biofilms on them allowing them to be removed from the treated water)
o Biofilm engineering: adhering proteins can be put under blue light for imaging, detecting sessile bacteria not planktonic
What is the difference between planktonic and sessile ?
Planktonic: free-swimming phase
Sessile: multicellular life phase
What does secretion for motility result in ?
swarming
What is swarming ?
example of bacterial surface motion (different from swimming in bulk liquid)
What does swarming depend on ?
- Collective behaviour characterised by: high cell density, secretion of surfactants (decreases surface tension), migration in packs within a thin liquid layer, super-diffusive cell motility (move like a liquid)
What are the cells like in a swarming colony ?
- In swarming colony, bacteria cells are:
o Unattached, motile through flagellar locomotion (crowded environment promotes forward motion)
o Metabolically active
o Altered physiologically
o Swarms differ from biofilms (biofilms = mass of metabolically quiescent (dormant) cells attached firmly to a surface)
What are some advantages to swarming ?
- Proteus mirabilis: 50x more flagella when in a cluster
- Confers advantages to the group as a whole colonise new niches faster, nutrient shared within a group, better survival (against lethal antibiotic concentrations) predation mechanisms (myxococcus xanthus)
What is a compartment ?
an area isolated from the rest of the cell in order to carry out an independent function.
an area isolated from the rest of the cell in order to carry out an independent function.
What is a differentiated cell?
cells become different to other cells, within a multicellular organism
What is the difference between a compartment and a differentiated cell ?
compartment: Physical or functional region in tissues or cell, Segregates or organizes biological activities, transient
differentiated: Individual specialized cell, Performs a specific, specialized function, fixed identity
What is the role of compartmentalisation and differentiation ?
- A new compartment can add a new process in a cell to help it survive
- Some functions need to be done in specific isolated environments
- Differentiation can be essential for cell survival to new conditions or as part of a cell cycle
What are some characteristics of magentotactic bacteria ?
o Generally -ve (2 membranes)
o Geomagnetically sensitive
o Motile
o Widespread worldwide distribution
o Aquatic or in sediment (free living)
o Microaerophilic or/and anaerobe (requires little to no oxygen)
o Can be spirillum, coccus, rod-shaped, multicellular
What is key about taxis ?
no effect on the speed of movement just the direction
What is a magnetosome ?
- Confer a permanent magnetic dipole moment to the bacteria
- Synthesised/biomineralized by the bacteria
- Contains magnetitie (Fe3O4) and greigite (Fe3S4) in a lipid bilayer membranous vesicle
- Big variation in number of magnetosomes per cell, crystal size, shape and arrangement
- Can be arranged into chains, dispersed aggregates, clumps or clusters
How long does magnetosome formation take ?
15 minutes
What are the steps in magnetosome formation?
1) Cytoplasmic membrane invagination (cytoplasmic membrane invaginates, the periplasmic membrane does not)
2) Protein sorting (proteins in the membrane attract the metals required to form the magnetosome)
3) Chain formation and assembly to actin-like magnetosome filaments
4) Iron transport, redox control
5) Biomineralization/nucleation
6) Control of the size and shape of the crystal
What is the role of magentotaxis ?
Magnetotactic bacteria live in places where there is oxygen and chemical vertical gradients (water and sediments) they need little or no oxygen and specific nutrients
- Main role: to ensure that cells find and maintain their optimal location for survival and growth
- Magentotaxis and chemotaxis work together to reduce a 3D problem into a 1D problem
- Might also have a role in iron storage and elimination of reactive oxygen species
- Important in the geochemical cycling of iron, sulphur, nitrogen and carbon
What is a carboxysome ?
- Found in all cyanobacteria and some proteobacteria
- Microcompartments
- Have defined subcellular localisations in a cell
- Polyhedral bodies of about 120nm of diameter
- Solid, granular interior with a 3-4nm thick, selectively permeable protein shell
- Site of carbon dioxide fixation
What is the size order of magentosome, carboxysome and heterocyst?
(small) Magnetosomes carboxysomes heterocysts (large)
What are the 2 reactions in a carboxysome ?
- CA = carbonic anhydrase
o Transforms HCO3- into CO2 (bicarbonate ion into carbon dioxide) - RuBisCO = ribulose-1,5-bisphosphate carboxylase/oxyeganse
o RuBisCO uses D-RuBP (ribulose-1,5-bisphosphate) and carbon dioxide to make 3-PGA (3-phosphoglycerate), which fixes carbon dioxide into a sugar
o Oxygen is a problem as it is also a substrate for RuBisCO but the reaction with oxygen does not result in the production of sugar (waste).
What are the role of carboxysomes ?
- Site of carbon dioxide fixation
- Optimise the action of RuBisCO by concentrating the quantity of carbon dioxide and decreasing the quanitiy of oxygen around the enzyme
- Organisms containing carboxysomes have an essential role in maintain the global carbon balance.
What is a cyanobacteria ?
- Almost ubiquitous (wet and dry environments, on and below the surface, symbiotic interactions with plants and animals)
- Important primary production at the basis of food chains
- Important role in cycling of carbon and nitrogen in the biosphere
- Perform oxygenic photosynthesis
- Essential role in the evolution of life on earth (responsible of the great oxygenation event)
What are the advantages of being multicellular ?
- Defence against predation
- Sharing information – cooperation
- Reduce competition
- Permit more complicated activities than an individual cell
- More efficient acquisition and utilisation of nutrients division of labour cell differentiation (heterocyst)
Characteristics of filamentous cyanobacteria ?
- Multicellular organisms (one of the oldest on Earth)
- Form chains of cells (up to hundred cells)
- Gram-negative type of envelope
- Exchange between cells thanks to the continuous periplasm and septal junctions (small pores/channels between cells)
What is a heterocyst ?
- 2 out of 5 taxonomic orders of cyanobacteria can differentiate into heterocyst
- Differentiated cells (cannot divide)
- Sensitive to oxygen (oxygen concentration needs to be low)
- Only made by filamentous cyanobacteria under nitrogen deprivation in medium
- Specialised in the fixation of atmospheric nitrogen (N2)
- Vegetative cells fix carbon and heterocyst fix nitrogen; then they exchange their molecules
What are some characteristics in heterocysts ?
- Number of microcompartments are lost from heterocyst (including carboxysomes)
- Additional cell envelope = 2 layers outside the outer membrane (less permeable to gas)
- Necks form at poles in contact with vegetative cells (narrow cytoplasm and septum) and the additional cell envelope is thicker = reservoir of organic nitrogen
- Honeycomb form near poles in contact with vegetative cells (contorted intracellular membranes used in respiration)
Reactions in a heterocyst ?
- Nitrogenase
o N2 into NH4+ (nitrogen into ammonium) - Glutamine synthetase:
o NH4+ + glutamate = glutamine
Where does the glutamate and sugar come from in a heterocyst ?
Glutamate and sugar (energy) come from vegetative cells
Glutamine is exported to vegetative cells
What are spores ?
- Spores are metabolically dormant, stress resistant and poised for germination when conditions are right
- Spores have specialised, multi-layered surface structures and unique composition (low core water content, high levels of stable spore proteins, compacted chromosomal DNA).
- Spores contain proteins important for stress resistance, anabolism and cell signalling
- Spores germinate with heterogeneity (from hours to days even in the same population)
What is sporulation ?
Formation of spores, Sporulation is carried out by organisms across the tree of life (bacteria, protozoa, plants, fungi, mushrooms, plant pathogens) = most are fungi
What is the role of spores?
- Spores survive in response to adverse growth conditions (can survive heat, desiccation, UV, outer space, DNA damaging agents, hydrolytic enzymes, ionising radiation, pH and mechanical disruption
- Spores are used to disperse the organism to new (more hospitable) environments
- Spores can be used as a reproductive strategy (sexual or asexual) or for cellular propagation
What is a vegetative organism ?
asexual
What is phase contrast microscopy ?
Phase Contrast Microscopy is a technique used in light microscopy to visualize transparent, unstained specimens, such as living cells, in high contrast. This method takes advantage of differences in the refractive index and thickness of different parts of a specimen to produce contrast in the final image.
What are the 3 types of endospores in Bacillus subtilis ?
Terminal spores (form at the pole), subterminal spores (near the pole but not at the pole), central spores
What is B.subtilis ?
rod-shaped, gram positive firmicutes (phylum of bacteria), found in soil and the gastrointestinal tract of ruminants (grazing mammals) and humans.
- Numerous survival mechanisms: motility, uptake of exogenous DNA, biofilm formation, destruction of surrounding cells/cannibalism and sporulation.
What is important in sporulation of B.subtilis ?
- Happens when there is a lack of nutrients and a high cell density
- Only occurs in a subpopulation
- Complex and irreversible
- Highly regulated developmental pathway
- Very demanding in energy
- Last resort for cell survival
- Spores resistant to heat, desiccation, treatment with detergents, pH and mechanical disruptions
- Forms subterminal spores
What are the layers in a B.subtilis spore, outside to inside ?
Outer Coat
Inner Coat
Outer Membrane
Cortex
Inner Membrane
Core
Compacted Chromosomal DNA
What do mature spores look like in B.subtilis ?
White blobs at one terminal
What do immature, developing spores look like in B.subtilis ?
grey blobs at one pole
What is the formation process of a spore in B.subtilis ?
activation (lack of nutrients, high cell density)
asymmetric cell division, produces a forespore at one pore
one cell engulfs the spore, forming a coat
late sporulation: outer membrane and inner membrane form
Mother cell lysis
germination
How does the genome change during sporulation of B.subtilis ?
2 chromosomes, chromosome condensation, polar septum formation, engulfment, cortex and coat assembly, spore maturation, further chromosome condensation, mother cell lysis
How does 2 chromosomes go to 30% of the genes in a forespore ?
There must be 2 full chromosomes for sporulation to start, origins tethered at opposite poles, 30% of the DNA is in the forespore, activate translocation of the rest of the chromosome in the forespore
How long does sporulation take in B.subtilis ?
4 hours since last sporulation event
What is Streptomyces coelicolor ?
- Gram positive actinobacteria (high GC content)
- Filamentous multicellular
- Found in soil and decaying vegetation
- Break down organic material in the soil and produce a characteristic “earthy” smell
- Produce more than half of all known antibiotics, metabolites and other bioactive natural products (when partial nutrient limitation)
- Sporulate in solid medium
What does the vegetative growth look like in S.coelicolor ?
- Coherent mycelium of branching filaments penetrating the substrate b
- Apical tip growth or branching
- Long multinucleated mass of cytoplasm irregularly separated by cross-wall
How does sporulation happen in S.coelicolor ?
- When growth becomes limited on a solid medium
- Formation of a fluffy white hydrophobic aerial mycelium (not branched)
- An aerial tip contains more than 50 copies of the genome
- Then, multiple cell divisions string of unigenomic compartments regular chain of spores (no endospores) that become grey
- Finally, spores are released thanks to cell wall hydrolase
What do the spores look like in S.coelicolor ?
average size of 1 micrometre, have a thick protective cell wall, permit spreading to environment, permit long term survival in detrimental conditions, resistant to heat, desiccation, detergents, high osmolarity, cell wall damage by lysozyme or vancomycin (glycopeptide antibiotic)
What happens to peptidoglycan synthesis in S.coelicolor sporulation?
- Switch in peptidoglycan synthesis machinery
o Vegetative cell = new peptidoglycan at tips and branches
o Sporulation = peptidoglycan synthesis at the septum and to form the spore wall - Tight coordination between DNA segregation and septum formation
How does the spore in S.coelicolor know its time to germinate ?
- The spore then sense nutrients, induction of production of stress proteins, signalling cascade and checkpoints to restart developmental gene expression, 1 or 2 germ tubes grow and branch
What is Myxococcus xanthus ?
- Gram negative delta (fruit-body-forming) proteobacteria (predominant phylum in gram negatives)
- Large bacterial genome – 7,500 genes
- Complex life cycle with a temporal multicellularity and sporulation (on a solid surface)
- Great abundance in a wide range of soil and water habitats
- Dangerous predator
What are the Predation methods of M. xanthus?
- Production of exoenzymes and antibacterial molecules
- Two gliding mobility engines
- Production of molecules to lubricate the environment
- Cells bind each other in a swarm or to surface to move as a whole (cooperative behaviour)
- Cells ripple to stay in the same region for longer to scavenge lysed cells and for faster penetration in the prey colony
Why does M. xanthus need multicellular behaviour ?
- Very high cell density and starvation on solid medium
- To be very good at sensing their environment, to recognise and interact with their kin
- Numerous signalling and regulatory machineries
- Complex set of pathways that lead to, organise and maintain multicellularity
- Communication, coordination and cooperation between individuals
How does M. xanthus recognise its kin ?
- Cells in a fruiting body have a high genetic similarity
- Kin recognition is carried out by a cell surface receptor
- Kin cells that come into contact fuse their outer membrane and exchange part of their membrane, periplasm and associated proteins
- Membrane exchange is critical for M. xanthus social motility and development
- If it is not the right receptor - no kin recognition no fusion - no development
Why does M. xanthus need to recognise its kin ?
- Allows communication and synchronised behaviours
- A way to maintain a table population (homeostasis)
- A way to control selfish behvaiours
How do bacteria keep their kin close ?
- Keep the kin nearby by using a viscous, dense population
What does the fruiting body of M.xanthus look look ?
- A large fraction of cells in the fruiting body undergoes programme cell lysis (to provide nutrients?)
- Cells outside the fruiting body are peripheral rods
- A subset of the population in the fruiting body differentiates in spores = cells inside the fruiting body
- The full cell differentiates with 2 entire genomic copies per spore (no endospore)
What are some characteristics of spores in M. xanthus ?
Spores are highly resistant to heat, deisiccation, cell wall/ membrane perturbants
