Midterm 1 Flashcards
What is the most successful lifeform on Earth? Why?
Microbes; very diverse, populous, and can survive in extreme conditions
What is LUCA?
Last universal common ancestor, was prokaryotic, over 3.5bya, and accumulated as stromatolites
Large organisms consist mostly of:
Carbon
Microbes molecularly consist mostly of:
Nitrogen and Phosphorus
What are the extreme conditions some microbes can tolerate?
Extreme temp (hot or cold), pressure, pH, and salt concentration
What are some pros of microbes?
Important for:
- human microbiome
- food, medicine, and industry,
- biogeochemical cycles
What are some cons of microbes?
Cause:
- food spoilage
- illness
- corrosion
- acid mine drainage
Microbes may cause horrible illness how?
Some may have severe symptoms or antibiotic resistance, in which case, it is hard to treat
Robert Hooke contributions
Discovery of cells
- developed first microscope (30X)
- coined “cell”
- first to observe microbes
Antoni van Leeuwenhoek contributions
Discovery of bacteria
- higher resolution microscope (300X)
- first to observe bacteria
- “wee animalcules”
- thought he saw small humans inside the head of sperm (humunculus)
Louis Pasteur contributions (microbial metabolism)
Microbial metabolism
- many chem. rxns were the metabolic rxns of microbes (alcohol fermentation)
- different microbes carry out different rxns
Healthy vats of beet juice contain _______ and produce ________
Yeast; ethanol
Unhealthy vats of beet juice contain _______ and produce ________
Bacteria (contamination); lactate
Louis Pasteur contributions (spontaneous generation)
Spontaneous generation
- spoiled food is caused by contamination, not spontaneous generation of microbes
- created the swan-necked flask to demonstrate sterile nutrient solutions remain sterile if microbes were prevented from entering the solution (contamination)
Describe how the swan-necked flask is used
- Non-sterile liquid poured into flask
- Neck of flask drawn out into a sideways S shape with a flame
- Liquid sterilized by heating
- a) If flask remains upright, microbes cannot fight gravity to contaminate liquid
- b) If flask is tipped, microbes can contaminate the liquid
The creation of the swan-necked flask disproved what?
Theory of spontaneous generation
Louis Pasteur contributions (sterilization and vaccines)
Sterilization
- aseptic technique
- Pasteurization (lightly boiling)
Vaccines
- attenuated strains (weak form of bacteria) (not a reliable method)
- anthrax and rabies
Robert Koch contributions (germ theory):
Germ theory of disease
- demonstrated link between microbes and infectious diseases
- Koch’s postulates (criteria) used to identify the cause of microbial infections
Describe the criteria of Koch’s postulates
- suspected pathogen must be present in all cases of the disease and absent from healthy animals
- suspected pathogen must be isolated and grown in pure culture
- cells from the pure culture of pathogen must cause disease in a healthy animal
- pathogen must be re-isolated and cultured to show that it is the same as the original
Robert Koch contributions (culture techniques):
Culture techniques
- developed multiple staining techniques
- Walther Hesse (medium) and Richard Petri (dish) developed a mechanism to grow microbes on solid media
- Fannie Hesse suggested agar agar as a medium
Sergei Winogradsky contributions:
Microbial ecology
- specific bacteria are linked to specific biogeochemical transformations (N and S cycles)
- Winogradsky column
- chemolithotrophy and autotrophy
- demonstrated microbial N fixation and nitrification
Chemolithotrophy
Oxidation of inorganic molecules for energy
Autotrophy
Use CO2 for carbon source
Nitrogen fixation
N2 -> NH3
Nitrification
NH3 -> NO2 -> NO3
What is a Winogradsky column?
A column that emulated conditions of the environment by stacking different types of microbes on top of each other
Describe the oxygen gradient of a Winogradsky column
High at the top and low at the bottom due to aerobic photoautotrophs at the top and anaerobic chemolithotrophs at the bottom
Describe the sulfide gradient of a Winogradsky column
Low at the top and high at the bottom due to sulfide-producing bacteria at the bottom
Martinus Beijerinck contributions
Enrichment culture
- developed multiple methods to select for organisms with specific metabolic requirements
- discovered N-fixing bacteria
- first to isolate a virus (“contagious living liquid”)
Why is it important to have multiple methods to select for organisms with specific metabolic requirements?
Essential for studying microbial ecology and nutrient cycling
What is an enrichment culture?
Enriches the growth of specific organisms by providing an environment that promotes their growth
What are the four ways we can study microbial diversity?
- morphological
- metabolic
- genomic (mix of morphological and metabolic)
- evolutionary
Morphological diversity
What they look like and what they’re made of
Metabolic diversity
How they acquire carbon and energy
Genomic diversity
What functions are they capable of
Evolutionary diversity
Where they fit in the tree of life
Ferdinand Cohn contributions
Morphological diversity of bacteria
- coined “bacteria” (little rod/staff)
- organized microbes into groups based on cell shape
What are the four cell shapes Ferdinand Cohn organized bacteria into?
- sphaerobacteria
- microbacteria
- desmobacterium
- spirobacterium
Sphaerobacteria
Sphere-shaped
Microbacteria
Rod-shaped
Desmobacterium
Filamentous (forms chains)
Spirobacterium
Coiled, spring-like
What are some distinguishing features of bacteria used to sort them based on morphology?
- cell shape
- membrane and wall structures
- motility and flagellar motion
- endospore formation
- swarming
- pigmentation
- cell arrangement (what they look like under a microscope when next to each other)
- growth under various conditions
Describe bacteria that require complex media
Cells that grow on complex media require lots of nutrients to survive, may be dependent on other organisms
Describe the bacteria that are capable of growing on single-carbon media
Cells that grow on single-carbon media do not require lots of nutrients to survive, independent
Chemoorganotrophy
Use organic molecules for energy
Heterotrophy
Use organic molecules for carbon
Phototrophy
Use light for energy
What are some elements of microbial metabolic diversity?
- energy source
- carbon source
- O2 requirements (aerobic, anaerobic, facultative)
- optimal growth conditions (temp, pH, pressure, salt, etc)
- specific nutritional requirements (metal cofactors and vitamins)
- relationship with other organisms
An aerobic microbe:
Relies on O2 for survival
An anaerobic microbe:
Dies in the presence of O2 because it lacks necessary enzymes
A facultative anaerobe:
Can live with or without O2
Planktonic organisms:
Are independent from other organisms
What is the Average Nucleotide Identity (ANI)?
Computational method of comparing two genomes to determine their relation to one another
Describe an example of how a single gene can help group organisms closer together on a phylogeny
If N-fixation genes are present within a group of microbes, then we know they are closely related and are thus within the same clade because they share metabolic diversity
Describe the structure of the cytoplasmic membrane
Made up of a phospholipid bilayer with proteins associated. It is semipermeable and fluid and contains hydrophobic and hydrophilic components
What are the hydrophobic parts of the cytoplasmic membrane? Which way do they point?
Fatty acids; inwards
What are the hydrophilic parts of the cytoplasmic membrane? Which way do they point?
Glycerol, phosphate, and the functional group (ex. ethanolamine); outwards
What kind of linkage does the fatty acid tail have with the glycerol in a phospholipid?
Ester (O=C-O-)
What does semipermeable mean?
The membrane allows nutrients in and wastes out
Which molecules may pass the membrane without transporters?
Nonpolar molecules and small weakly polar molecules (water, ethanol, O2, NH3)
Which molecules cannot pass the membrane without transporters?
Large or strongly polar molecules (ions, sugars, amino acids)
What does unsaturation refer to in the context of fatty acids?
Some double bonds are present in the hydrocarbon chain, creating a bend
How would the membrane be modified in thermophiles?
Would opt for more saturated fatty acid tails to decrease the fluidity of their membranes (increase in van der Waals forces)
How do archaeal membranes differ from bacterial and eukaryotic membranes?
- ether linkages instead of ester
- isoprenes instead of fatty acids
- major lipids are glycerol diethers and diglycerol tetraethers
- side chains are phytanyls instead of fatty acids
- can exist as a bilayer, monolayer, or both
What is the side chain of a glycerol diether?
Phytanyl
What is the side chain of a diglycerol tetraether?
Biphytanyl
In archaeal membranes, a bilayer is formed with:
Glycerol diether and two phytanyl side chains
In archaeal membranes, a monolayer is formed with:
Diglycerol Tetraether and two biphytanyl side chains
Thermophilic Archaea likely uses _____________ for its membrane because __________________
Diglycerol tetraethers; the monolayer increases membrane rigidity
What is the third type of membrane molecule in Archaea?
Crenarchaeol
Why does Archaea use ether instead of ester linkages?
Ether linkages are more thermostable, allowing for a thermophilic lifestyle
What are the three functions of the cytoplasmic membrane?
- permeability barrier
- protein anchor
- energy conservation
How does the cytoplasmic membrane increase energy conservation in prokaryotes?
Since prokaryotes lack the internal membrane system, they must generate proton motive force with their cytoplasmic membrane to create energy
What are the types of membrane proteins?
Peripheral, integral, and transmembrane
How are peripheral proteins associated with the membrane?
Loosely attached usually with hydrogen bonds
What are the major functions of membrane proteins?
- molecule transport
- environmental sensing
- electron transport and respiration
- membrane and cell wall assembly
How do cells accumulate solutes against a concentration gradient?
Use active transport
Provide three active transport mechanisms
- simple transport
- group translocation
- ABC system
Describe simple active transport
Uses secondary active transport to move solutes against their gradients
Describe group translocation
Chemical modification of the transported solute to prevent its accumulation, since it is a different molecule than what just crossed the membrane
Describe the ABC system
Binding proteins outside of the cell bind a specific substance and bring it to the trans-membrane transporter. An ATP-hydrolyzing protein associated with the transporter hydrolyzes ATP to bring the substance across
How is active transport fuelled?
Proton motive force, ATP, NADH, GTP
What are cell envelopes composed of?
- 1-2 cell membranes
- cell wall
- S layers
What are the two types of cell envelopes?
Gram positive (GP) and Gram negative (GN)
Components of a GP envelope
- cytoplasmic membrane
- thick cell wall (multiple layers of peptidoglycan)
Components of a GN envelope
- cytoplasmic membrane
- thin cell wall
- periplasm
- outer membrane
What feature of GP envelopes prevent them from exploding?
Organisms with GP envelopes are usually hypertonic to their environment. To prevent the movement of water into the cytoplasm, a thick cell wall is needed
Describe the structure of a GP envelope
Cell membrane makes up the innermost component, followed by a thick cell wall of peptidoglycan. On top of and within the cell wall are teichoic acids and lipoteichoic acids, respectively. Lipoteichoic acids associate with the cell membrane and the cell wall
Describe the structure of a GN envelope
Cell membrane makes up the innermost component, followed by a thin cell wall of peptidoglycan. This is then followed by an outer membrane covered in lipopolysaccharides. The outer membrane is attached to the cell wall by proteins
What is the periplasm? Where is it found?
Found in GN cells. It is the area between the outer side of the inner cell membrane and the inner side of the outer cell membrane and has an important role in metabolism
Which type of envelope is most common in bacteria? Why?
GN. In most cases, having 2 membranes provides greater fitness than just one
Why does Archaea not fall into the Gram reaction categories?
It has different cell walls and membranes, and thus requires a different type of category
Describe the steps of Gram staining
- Flood a heat-fixed smear with crystal violet (dark blue, goes into the cytoplasm)
- Add iodine (complexes with crystal violet within cytoplasm)
- Decolourize with alcohol (dehydrates cell walls. GP walls compact so violet-iodine complex cannot leave, while GN walls are destroyed and the complex does leave)
- Counterstain with safranin (permeates both)
What colour are GP cells after staining?
Purple (mix of dark blue crystal violet and pink safranin)
What colour are GN cells after staining?
Pink/red (only safranin has an effect on colour)
What are the functions of the cell wall?
- maintains cell shape and rigidity
- prevents cell lysis by withstanding osmotic/turgor pressure
What is peptidoglycan?
A rigid polysaccharide layer that provides cell wall strength in bacteria by creating a mesh around the membrane
The glycan component of peptidoglycan contains what components?
Sugar backbone of alternating NAG and NAM (modified glucose) joined by Beta-1,4 linkages
How many amino acids make up the peptide in peptidoglycan?
4
Where does peptidoglycan crosslink with itself?
Two sites on the tetrapeptide and a carboxyl on an adjacent strand
True/False? The peptidoglycan mesh of a bacterial cell wall has strands that run perpendicular to each other
False. They run parallel
GP crosslinks often contain what in their peptidoglycan mesh?
Peptide interbridges
GP cell walls have up to how many layers of peptidoglycan?
20
GN cell walls have up to how many layers of peptidoglycan?
3
What is a peptide interbridge?
A 5-glycine bridge between each peptidoglycan crosslink
What is teichoic acid?
An acidic, negatively charged molecule found in certain GP envelopes that are covalently bound to peptidoglycan. They interact with the immune system as antigens
Difference between teichoic and lipoteichoic acid
Lipoteichoic acid is covalently bound to membrane lipids, while teichoic acid isn’t
What are the functions of teichoic acid?
- maintain porosity of cell wall (spreads out peptidoglycan layers
- anchors cell wall to membrane
- maintains cell shape
- captures essential cations (is negatively charged, so attracts positive ions)
- can be an emergency reservoir of phosphate
Inner leaflet of the outer membrane in GN bacteria
Mostly made up of phospholipids and proteins
Outer leaflet of the outer membrane in GN bacteria
Mostly made up of lipopolysaccharides, proteins, and a few phospholipids
What is a porin?
A protein pore in the outer membrane of a GN envelope that increases permeability of hydrophilic low MW substances and is largely non-specific
What is a lipopolysaccharide?
- used instead of phospholipids on outer leaflet of outer membrane
- facilitates surface recognition, important virulence factors, and accounts for most of the strength of the cell
- polysaccharides covalently bound to lipids
What is Lipid A?
An endotoxin that can cause endotoxic shock in organisms that do not produce it. It is embedded in the membrane and contains over two hydrocarbon tails and a phosphorylated head group
What is the core polysaccharide?
A family and genus specific chain of polysaccharides attached between Lipid A and the O-specific polysaccharide
What is the O-specific polysaccharide?
A species specific chain of polysaccharides that sometimes acts as an antigen
Pseudomurein
Similar to peptidoglycan but found in archaeal cell walls. Uses alternated NAM and TAL saccharides linked with a beta-1,3 linkage
What is Lipid II?
A combination of bactoprenol and a peptidoglycan precursor attached to the phosphate group used to carry the precursor to the outside of the cell. More common in GP because of its thick cell wall
Describe the steps in peptidoglycan synthesis
- Autolysins break the B-1,4 bond between NAM and NAG sugars in pre-existing peptidoglycan chains
- Assembly of Lipid II, which is transported across the membrane via flippase
- Transglycosylases insert the precursors into the broken chain
- Transpeptidation (rebuilds the B-1,4 linkage)
What kind of enzyme is flippase? What are its components?
ABC active transport enzyme. Bactoprenol carries precursor to the enzyme as Lipid II and flippase uses ATP to move the Lipid II outside of the cell
What are the agents that destroy peptidoglycan? How?
- lysozymes (hydrolyzes B-1,4 bond)
- antibiotics (inhibit formation and/or cross-linking of the glycan strands, have a Beta-lactam ring)
How may antibiotic resistance work?
Bacteria may synthesize enzymes that can degrade the antibiotics (Beta-lactamases)
What is biomineralization?
The sequestering of minerals from the environment by a bacteria, which are stored within the cell in inclusion bodies
What are Fts proteins?
Prokaryotic proteins essential for cell division. They form the divisome
What does Fts stand for?
Filamentous temperature-sensitive proteins
What Fts proteins make up the divisome?
FtsZ, ZipA, FtsI, FtsK, FtsA
What is the divisome? How does it work?
The cell division apparatus used in prokaryotes. It breaks up and replaces the cell wall, pinching around the ring formed by FtsZ
What is the function of FtsZ?
Forms a ring around the center for the cell and has structural homology to tubulin in eukaryotes
What is the function of ZipA?
Anchor that connects FtsZ ring to cytoplasmic membrane
What is the function of FtsI?
Peptidoglycan biosynthesis protein
What is the function of FtsK?
Assists in chromosome separation
What is the function of FtsA?
ATPase
What is the function of MinCD?
Inhibits the formation of the FtsZ ring
What is the function of MinE?
Oscillates from pole to pole, sweeping MinCD aside to the poles. Clears MinCD from the middle of the cell so FtsZ can form a ring
When in the cell cycle does MinE work?
During M phase
What are the two proteins that are determinants for prokaryote cell morphology
Crescentin and MreB
What is the default shape of prokaryotes?
Spherical (coccus)
What is the role of crescentin?
Organizes into filaments ~10nm wide that localize on the concave surface of curved cells (curves cells inwards)
What is the role of MreB?
Forms simple cytoskeleton with patch-like filaments around inside of cell just below cytoplasmic membrane in bacteria and some Archaea. Makes bacteria elongate
What kind of bacteria lack MreB?
Cocci (spherical)
What is the major shape-determining factor in prokaryotes?
MreB
What direction does the cell grow relative to the formation of MreB polymers?
Perpendicular
What are S-layers?
Cell wall component in Archaea, that consists of proteins or a glycoprotein monolayer. Acts as structural support, molecular sieves, and form a pseudo-periplasmic space
What characteristics of S-layers allow them to carry out their roles?
Rigid (structural support) and semi-permeable (molecular sieve)
What pattern do S-layers form when observing from a bird’s eye view?
Hexagonal
What are some names for the capsule? What does the name depend on?
Exopolysaccharide, glycocalyx, slime layer; depends on species of bacteria (GP or GN)
What is the capsule homologous to in Archaea?
S-layer
What are the functions of the capsule?
Nonspecific attachment to surfaces, biofilm formation, protection from grazing, immune systems, antibiotics, and desiccation
What is the flagellum?
A protein tail on the outside of the bacterial cell that is used for mobility
What are the types of bacterial flagella
Peritrichous, monotrichous, lophotrichous
What are peritrichous flagella?
They appear in any direction on the bacteria (looks like a starfish or something)
What are monotrichous flagella?
Only one flagellum
What are lophotrichous flagella?
Multiple flagella coming from the same origin (looks like the bacteria is having a bad hair day)
What are pili and fimbriae?
Very thin and fragile filamentous protein structures ~2-10nm wide that extend from the bacteria that have different roles
What is the function of fimbriae
Enable organisms to stick to surfaces or form pellicles (thin sheets of cells on a liquid surface), attach specifically
What is the function of pili?
Have roles in conjugation (attachment to other bacteria for genetic exchange) and twitching mobility (“grapple” to attachment point and pull themselves towards the point)
Compare fimbriae and pili
Pili are typically longer, thicker, and fewer
What are inclusion bodies?
The generalized name for sequestered minerals found in prokaryotes
What do PHA’s store?
Lipids
What is a PHB?
A lipid family within PHA’s
What does glycogen store?
Carbohydrates for energy
What do phosphate granules store?
Inorganic PO4 linked together
What do sulfur globules store?
Elemental sulfur
Where are sulfur globules stored? What does this imply?
In the periplasm; they are found in GN cells
Biomineralization of barium, strontium, and magnesium is used to store what?
Carbonate
Why do PHB’s take up most of the volume in a bacterial cell?
Carbon is very important as a source of energy and as a building block, so there needs to be lots of it
Why are sulfur globules located in the periplasm?
The periplasm is the main site for metabolism, and some lithotrophic bacteria use sulfur for metabolism
What are the two types of protein-bound compartments?
Chlorosomes and carboxysomes
What are chlorosomes?
Light-harvesting complex located around the perimeter of the cytoplasm that allow bacteria to grow at low light intensities. They use Bchl (bacterial chlorophyll)
What are carboxysomes?
They house Calvin cycle enzymes (RubisCO-) for CO2 fixation
What are gas vesicles?
Buoyancy regulators in some aquatic planktonic bacteria
How do bacteria use gas vesicles?
They increase buoyancy in low light conditions to get closer to the surface vice versa
What are magnetosomes?
A collection of magnetic iron oxides that allow cell to undergo magnetotaxis. They help the cell differentiate between up and down
What is magnetotaxis?
Migration along magnetic field lines
What are endospores?
Highly differentiated cells resistant to heat, harsh chemicals, and radiation responsible for the dormant stage in the bacterial life cycle
Why are endospores ideal for bacterial dispersal?
They can withstand harsh conditions what would otherwise kill the bacteria
True/False? Endospores are found in both GP and GN cells
False. Only GP
When are endospores formed?
When growth ceases due to lack of an essential nutrient such as carbon or nitrogen
How long can endospores stay dormant? How long does it take for them to germinate into a bacterial cell?
Can be hundreds or thousands of years; convert rapidly
How is it possible for some bacteria to have internal membranes? What kinds of bacteria have them?
Invaginations of the cytoplasmic membrane to increase surface area; phototrophic, nitrifying, and methanotrophic bacteria
Why do bacteria need invaginations in their cell membranes?
The increase in surface area increases the number of membrane-bound enzymes possible, which increases the cell’s metabolism and other important functions (like nitrification)
What is nitrification?
The lithotrophic oxidation of ammonia to nitrate
How many bacteria does nitrification need? What are their names?
2; ammonia oxidizers and nitrite oxidizers
What is the purpose of ammonia oxidizers?
Oxidize ammonia into nitrite (using two steps)
What is the purpose of nitrite oxidizers?
Oxidize nitrite into nitrate
What is an annamoxosome?
A special nitrogen fixation “organelle” that is really just invaginated cytoplasmic membrane
Define habitat
Parts of an ecosystem suited to a particular group of populations (ex. a meadow within a forest)
What are habitats limited by?
- resources (carbon, macronutrients, micronutrients, electron acceptors and donors
- conditions (temp, water availability, pH, O2, light, and salinity content)
Compare the ecosystem range for prokaryotes vs. eukaryotes
Prokaryotes have a much broader range
What two terms are used to describe diversity?
Species richness and species abundance
Species richness
The total number of different species present
Species abundance
The population size of each species within an ecosystem
What is a guild?
A subdivision of a community based on metabolic similarities between microbe species
Microenvironment
A small part of a local environment encountered by a given species where physiochemical conditions are subject to rapid change both spatially and temporally
Compare growth rates in a natural environment versus a lab
Much slower in natural environment
What are soils composed of (include percentages)
- inorganic mineral matter (40%)
- organic matter (5%)
- air and water (50%)
- living organisms (5%)
What three factors limit microbial activity in surface soils?
- water availability
- energy source (organic matter) availability
- inorganic nutrient availability
What is a rhizosphere?
The area around plant roots where plants secrete sugars and other compounds. Rich in organic matter and microbial life
What allows for microbial diversity in a small sample?
Microenvironments
Trend of O2 concentration in a small ball of soil
[O2] decreases the further you go inwards
How are bacteria identified in environmental samples?
SSU rRNA sequences
Describe the steps of SSU rRNA collection
- extract DNA from environmental sample
- amplify 16S gene by PCR
- run on a gel to make sure you have the right band size
- sequence with next-gen sequencing
- generate a tree with known sequences
What is an operational taxonomic unit (OTU)?
A 16S rRNA gene sequence that differs from all other sequences by >3% (presumed to represent a species)
Which four families of bacteria are the most common in a soil sample?
- Proteobacteria
- Acidobacteria
- Bacteroidetes
- Actinobacteria
Why do we only have 13k named species of bacteria?
The 1min-1tril other ones have either not been discovered or cannot be cultured
Why are so many bacteria hard to cultivate?
Many have highly specialized growth requirements
Compare microbial diversity in oceans vs. soil. Why?
Much more in soil, less in oceans. Soil is warmer and has more nutrients and lower salinity
The microbial activity in oceans is a major factor in what?
Carbon cycle (about 25% of the earth’s primary production)
Describe the distribution of chlorophyll (and thus, microbes) in the ocean
Almost none in the open ocean but very high around the coasts and lakes due to the open ocean being devoid of light, warmth, and nutrients
Which four families of bacteria are the most common in an oceanic sample?
- Proteobacteria
- Bacteroidetes
- Marinimicrobia
- Actinobacteria
What is Prochlorococcus?
A cyanobacteria that accounts for most of the primary production in open oceans
- >40% of the biomass of marine phototrophs
- 50% of the net primary production of the ocean
- 25% of the net primary production of the world
What is Pelagibacter?
The most abundant marine organoheterotroph. It is an oligotroph and uses proteorhodopsin to use light to drive ATP synthesis when nutrient levels are low (cannot sustain life just on light)
What is an oligotroph?
An organism that grows best at very low nutrient concentrations
Why was Pelagibacter hard to culture until we learnt more about it?
Because it’s an oligotroph, it wouldn’t grow on rich media, which is usually used to culture new bacteria when we don’t know anything about their nutrient requirements
Describe the deep water Horizon oil spill
It was the largest marine oil spill ever, where an oil plume was released at great depths. Following this event, there was a bloom of hydrocarbon-degrading bacteria, which reduces the environmental impact of the spill. These types of bacteria are common at deep levels as they do not rely on light for food
What is the most common type of microorganism between 1-6km deep in the ocean? Why?
Extremophiles (chemolithotrophs); they must deal with low temp, high pressure, low nutrients, and the absence of light
Planktonic bacteria
Free-floating bacteria
Biofilms
Produced by bacteria that attach to a surface. They are assemblages of cells adhered to a surface and enclosed in a polysaccharide adhesive matrix excreted by the cells
Why are most bacteria attached and not planktonic?
Easier for them to gain nutrients
Describe the steps to biofilm formation
- attachment of cell to surface and expression of biofilm-specific genes
- genes encode proteins that initiate matrix formation
- quorum sensing for development and maintenance of biofilm
What is quorum sensing?
Allows bacteria to sense and respond to population density. They detect a high level of nutrients when density is high
Under a microscope, what do biofilms resemble?
Stromatolites
What are the functions of biofilms?
- self defense
- trap nutrients
- prevent detachment of cells in a flowing system
- facilitates cross-feeding and symbiosis
How does the biofilm help with self defense?
Resists phagocytosis and penetration of toxins because they are so thick and viscous
How do biofilms negatively impact our lives?
- implicated in several medical and dental conditions
- slow the flow of liquids through pipelines
- very few effective antibiotics are available because of how strong the polysaccharide matrix is
How are biofilms in pipelines usually dealt with?
Just by scraping them off
What is a microbial mat?
A thick biofilm built by phototrophic and/or chemolithotrophic bacteria and often occur in systems with low predation and grazing (extreme ecosystems)
Provide an example of a phototrophic mat
Stromatolites (they have existed for billions of years!)
True/False? Extremophiles are usually resistant to every environmental stress
False. Usually only a couple of extreme environments are paired at a time per species
What is the suffix given to organisms that can grow in different ranges of environmental stress parameters?
-phile
What is the suffix given to organisms that can survive (can’t grow, but don’t die) in different ranges of environmental stress parameters?
-tolerant
What are the Cardinal temperatures and the enzyme activity at each?
Minimum: low
Optimum: peak
Maximum: none
What happens at the minimum temperature?
Membrane gelling, transport processes so slow that growth cannot occur
What happens at the optimum temperature?
Enzymatic rxns occurring at peak capacity
What happens at the maximum temperature?
Protein denaturation, collapse of the membrane, and thermal lysis
What is a Psychrophile and what is its optimum temperature range?
A microorganism that lives in cold environments; < 15C
What is a Mesophile and what is its optimum temperature range?
A microorganism that lives in “normal” temperatures; 15 - 45C
What is a Thermophile and what is its optimum temperature range?
A microorganism that lives in hot temperatures; 45 - 80C
What is a Hyperthermophile and what is its optimum temperature range?
A microorganism that lives in extremely hot temperatures; > 80C
Where do Thermophiles live? Hyperthermophiles?
Geothermal systems, decaying matter, the deep biosphere; deep within the earth
Which organisms make up the majority of Hyperthermophiles?
Archaea
What is the maximum temperature a eukaryote can survive? What does this make this particular species?
62C; Thermophile
What can be observed in hot springs above 73C vs at the edges, where it’s below 73C? Explain why
Above, there is no photosynthetic bacteria, and below, there is. This is because photosynthesis stops at 73C, as the necessary enzymes for this process do not work above this temperature
Thermophiles and Hyperthermophiles usually adopt what kind of metabolic strategy near hydrothermal vents? What does this mean?
Chemolithotrophs; they use inorganic materials from the vents to use as electron donors and acceptors in the ETC
Why do some eukaryotic organisms live near hydrothermal vents despite not being chemolithotrophs?
The water around the base is cooler, so they are able to survive and feed off of the thermophiles and hyperthermophiles. They are usually coated in the sediment ejected from the vents
What are the problems for microbial life at high temperatures? What are the solutions?
- protein denaturation
- DNA and RNA denaturation
- membranes become too fluid (leaky)
Solutions: - stronger bonds to stabilize proteins
- increase DNA and RNA stability
- decrease membrane fluidity
What are some features of thermophilic microorganisms that provide thermal stability to proteins?
- amino acid substitutions in enzymes (not in the active site) that provide more heat tolerant folds
- increased number of ionic bonds between basic and acidic amino acids resists unfolding in the cytoplasm
- hydrophobic interiors
- production of solutes helps to stabilize proteins
- smaller, more spherical proteins with less quaternary structure
How is DNA stabilized in thermophiles?
Positive supercoiling by reverse gyrase requires more energy to unwind, which protects the DNA
How is RNA stabilized in thermophiles?
A higher GC content to maintain secondary folding structures
How is membrane rigidity increased in thermophiles?
Increased saturation, longer tails, and tetraethers in the case of Archaea
What are the problems for microbial life at low temperatures? What are the solutions?
- proteins too rigid and slow rxn rates
- membranes too viscous
- DNA and RNA is too rigid
Solutions
- reduced amino acid interactions that stabilize tertiary structures
- increased membrane fluidity
- decreased GC content
What is the pH range all microbes can grow in within their optimal pH?
2-3 pH units
Neutrophiles
Grow optimally within a range of 5.5 - 7.9. May be acidotolerant or alkalitolerant
Alkaliphiles
Grow optimally above a pH of 8. May be neutrotolerant
Acidophiles
Grow optimally below a pH of 5.5. May be neutrotolerant
What problems do pH extremophiles face?
Their cytoplasm must be near a neutral pH, so the membrane must be very impermeable to protons. Because of this, maintaining a proton motive force is difficult
How do acidophiles solve their problem with maintaining their PMF?
Because the environment is more acidic than their cytoplasm, proton leakage into the cell through ATPase is uncontrolled. To maintain a neutral pH, protons are pumped back out
How do alkaliphiles solve their problem with maintaining their PMF?
Since they are more acidic than their environment, they cannot create a PMF. Instead, they use sodium ions to generate a SMF (sodium motive force) in the same fashion. Protons are pumped in from the environment at the same time
True/False? pH extremophiles still rely on the ETC despite using other methods to make energy
False. It isn’t entirely useful, as acidophiles do not need to generate a PMF, and alkaliphiles use an SMF instead
Halophiles
Love salt
Xerophiles
Thrive in dry conditions
Osmophiles
Like halophiles, but use other solutes instead of salt (like sugar). Usually yeasts
Non-halophile optimum salinity
0%. Steep drop in growth if above 0
Halophile optimum salinity
1-15%
Extreme halophile salinity
> 15%
What is a halotolerant organism
An organism that can survive in saline conditions, but cannot grow
What is water activity?
A measure of the vapour pressure (or free energy) of water, where aw = 1 for pure water at atmospheric pressure. Lower values are caused by dryness and solutes
If the cytoplasm has a greater [solute], what is the relative water activity and what does this cause?
aw(in) < aw(out); water moves into the cell
What happens to a cell if its aw is lower than its environment’s?
Water moves into the cell, causing positive pressure within the cell. This does not kill GP cells because they have a strong cell wall
What would happen to a normal freshwater bacterium if it was placed in a saline environment?
aw(in) > aw(out), so water moves out of the cell, killing it
How do halophiles survive being in high salt concentrations despite having a less concentrated cytoplasm?
- salt-in by accumulating KCl (requires salt-adapted enzymes)
- synthesize compatible (organic) solutes that do not damage enzymes
What domain uses the salt-in strategy?
Archaea (and some Bacteria)
What domain uses compatible solutes?
Bacteria and Eukarya
What molecules are used as compatible solutes? How are they in terms of usefulness?
Glycine betaine and ectoine; very useful, as they both have a minimum aw of 0.75, which helps to prevent cell dehydration
Explain the significance of bacteriorhodopsin
It allows halophilic bacteria to be phototrophic without being photosynthetic. It uses light energy to generate PMF
Aerobes. Where are they located?
Require O2 for respiration. The surface
Microaerophiles. Where are they located?
Require a low [O2]. Buried in soil
Facultative aerobes/anaerobes
Will respire if O2 is available, but can survive without it. Uses fermentation in its absence
Aerotolerant anaerobes
Don’t use O2 but can survive in its presence
Obligate anaerobes. Where are they found?
Don’t use O2, nor can they tolerate it due to a lack of necessary enzymes. Located deep in soil
What is Thioglycolate broth?
A complex broth that distinguishes microbes based on their oxygen requirements. Resazurin within the broth reacts with O2 (turns pink, the rest is yellow) at the surface, which prevents O2 from penetrating deeper into the broth. Turbidity in different layers shows where the bacteria localize
Why is oxygen toxic?
Although molecular oxygen has low toxicity, its byproducts (ROS: O2-, H2O2, OH radical) cause damage to surrounding molecules by ripping electrons off of them. Organisms lacking enzymes that deal with ROS are not able to survive in O2
What is the function of catalase? Which enzyme is it functionally similar to and how do they differ?
It removes hydrogen peroxide. Similar to peroxidase, although catalase combines H2O2 molecules, peroxidase reduces it with NADH. They both create water
What is the function of superoxide dismutase? Which enzyme is it functionally similar to and how do they differ?
It removes superoxide anions. Similar to superoxide reductase, although dismutase combines superoxide molecules, while reductase reduces them. They both produce H2O2
True/False? The catalysis of superoxide requires two enzymatic steps
True. First, superoxide dismutase or reductase convert it into hydrogen peroxide, then catalase or peroxidase convert that into water
What are the types of motility in microorganisms?
- gas vesicles
- swimming
- gliding
- twitching
Gas vesicles provide motility in which dimension?
Vertical
True/False? All microorganisms have some mode of motility
False
What are flagella?
A helical “tail” found on rod-shaped or curved bacteria made of flagellin
Describe the structure of a flagellum, from outside to inside
- filament made of flagellin (hollow)
- hook
- rod ringed in L and P rings
- MS ring enclosed in stator and C ring
What two parts of the flagellum make up the rotor?
MS ring and rod
Function of the hook
Attaches the filament to the rod
Functions of the L and P rings
Sleeve the rod in the lipopolysaccharide and peptidoglycan sides of the outer membrane, respectively
What is the catalytic component of a flagellum? What does it do?
The stator; uses PMF generated in periplasm to spin the MS ring
Function of the C ring
Anchors the flagellum into the inner membrane
What can the working parts of a flagellum be compared to?
ATP synthase (both spin using PMF)
What kind of bacteria usually have flagella?
Rod-shaped GN bacteria (periplasm needed to generate PMF)
Describe the steps for the formation of a flagellum
- MS/C rings formed
- Stator formed
- Rod and P ring
- L ring
- Early hook
- Late hook and cap
- Filaments grow between hook and cap, coming through the hollow tube and assembling at the tip
Peritrichous bacteria use ______________ flagellar rotation to run forwards
Counterclockwise
Peritrichous bacteria use ______________ flagellar rotation to tumble and change direction
Clockwise
What happens to the flagella not in use when the bacteria uses CCW rotation vs. CW rotation?
CCW: trail behind bacteria and group together around flagellum in use
CW: fly apart
What do peritrichous bacteria look like while running?
Lophotrichous bacteria because their flagella trail behind them
How do peritrichous bacteria choose the direction they’re going?
They don’t, its random. They change the direction they’re going with CW rotation, and go in the direction they end up in. This usually results in them staying in the same area
Why is movement for bacteria so difficult?
The friction of water is massive compared to their momentum (low Reynolds number), so when they stop driving their flagellum, they stop dead in place
How do bacteria avoid being washed away from good environments?
They attach to a surface either with a pilus (twitching) or adhesion proteins (gliding)
Describe movement via twitching
A pilus is extended from the bacteria, attached to the surface, and retracted like a grappling hook
Describe movement via gliding
Adhesion proteins move along a helical track on the outside of the bacterium, sticking to the surface when in contact with it. They get moved up and down the length of the bacteria so new ones don’t need to be synthesized
What are the 6 forms of taxis?
- phototaxis
- aerotaxis
- thermotaxis
- pH taxis
- magnetotaxis
- chemotaxis
Phototaxis
Movement towards certain wavelengths of light
Aerotaxis
Movement along an O2 gradient until the preferred concentration is reached
Thermotaxis
Response to temperature or a temperature gradient
Magnetotaxis
Directed movement along the geomagnetic lines of force which allows magnetotactic bacteria to seek microaerophilic environments for growth
Chemotaxis
Directed movement in response to chemicals known as chemoeffectors (attractant or repellant)
How are external signals sensed by prokaryotes?
- transmitted directly to a target
- sensed and signal transmitted to regulatory machinery (signal transduction)
What are the most important signal transduction systems?
Two-component regulatory systems
What are the two components used in two-component regulatory systems?
- sensor kinase
- response regulator
What is a sensor kinase and where is it found?
Detects an environmental signal and autophosphorylates. Transmits signal via phosphorylation cascade; cytoplasmic membrane
What is a response regulator and where is it found?
Usually a DNA-binding protein that regulates transcription; cytoplasm
What is the purpose of a feedback loop in the two-component system?
Terminates the signal when no longer needed
Describe the general function of a two-component regulatory system
- A ligand binds the sensor kinase
- A histidine residue on the SK gets phosphorylated
- Response regulator also gets phosphorylated and regulates transcription
- Phosphatase removes the phosphate, stopping response regulator function
Why is taxis complicated when considering the two-component regulatory pathway?
Bacterium are too small to sense concentration gradients, so they have to move around until they can sense a difference (temporal, not spacial)
What is a biased random walk?
Allows directed movement by lengthening the time the bacteria spends running until different conditions are sensed
Describe the steps for the mechanism of chemotaxis in E. coli in the presence and absence of attractant
Presence
1. binding of attractant to MCP shuts off pathway (MCP demethylated)
2. no binding of CheY allows for CCW rotation of the flagellum, and running occurs
Absence
1. no binding of attractant to MCP causes pathway to turn on (methylated)
2. CheA autophosphorylates, which then phosphorylates CheY
3. CheY binds to flagellar switch and causes a tumble
4. CheZ dephosphorylates CheY, which may become phosphorylated again (repeat step 3)
5. CheB is phosphorylated by CheA (still phosphorylated), which demethylates MCP, deactivating it
6. To restart the MCP, CheR remethylates MCP
Describe the steps for the mechanism of chemotaxis in E. coli in the presence and absence of repellant
Presence
1. binding of repellant to MCP causes pathway to turn on (methylated)
2. CheA autophosphorylates, which then phosphorylates CheY
3. CheY binds to flagellar switch and causes a tumble
4. CheZ dephosphorylates CheY, which may become phosphorylated again (repeat step 3)
5. CheB is phosphorylated by CheA (still phosphorylated), which demethylates MCP, deactivating it
6. To restart the MCP, CheR remethylates MCP
Absence
1. no binding of repellant to MCP shuts off pathway (MCP demethylated)
2. no binding of CheY allows for CCW rotation of the flagellum, and running occurs
In the MCP pathway, what protein acts as the sensor kinase?
CheA
In the MCP pathway, what protein acts as the response regulator
CheY and CheB (secondary)
Where are MCPs usually located?
Usually at the poles of the cells, in the direction of travel when running
What is the function of CheZ in the MCP pathway?
Acts as an off switch for CheY-P
What is the function for CheR in the MCP pathway?
Acts as an on switch for MCP
What is the function for CheB in the MCP pathway?
Acts as an off switch for MCP
Describe the run cycle for the MCP pathway
Response regulators (CheY and CheB) are inactive (non-phosphorylated), so the flagella is rotating CCW. CheR gradually methylates MCPs, making tumbles more likely ***
Describe the the tumble cycle for the MCP pathway
MCP signals CheA to autophosphorylate, which then activates CheY and CheB. CheY interacts with Fli proteins to reverse the flagellar motor (CW)
Describe the reset cycle for the MCP pathway
CheB-P demethylates MCPs, causing them to become less active and stop phosphorylating CheY. CheZ dephosphorylates CheY-P, inactivating it. The cell runs (CCW), and CheB-P gradually returns to inactive CheB. CheR gradually remethylates the MCPs
What happens if the concentration of the chemoeffector does not change around a peritrichous cell?
A constant amount is bound to the MCPs and the run-tumble cycle repeats at roughly regular intervals
What is defined media?
The exact chemical composition of the media is known
What is complex media?
It’s composed of digests of microbial animal, or plant products in an unknown amount and proportion
What is selective medium?
It contains compounds that selectively inhibit growth of some microbes but not others (ex. contains antibiotic)
What is differential medium?
It distinguishes organisms with specific metabolic capabilities. Usually contains an indicator
What are the four phases of a typical growth curve?
Lag, log (growth), stationary, and decline
Why do bacteria have a lag phase?
They are acclimating to their environment and preparing for division. Switch from dormant to active form
When is the endospore formed within the growth curve?
Stationary phase to prepare for the upcoming decline
What is the formula for exponential growth? What transformation is applied to linearize it?
Nt = N0(2^n); log transformation
logNt = logN0 + nlog2
What is the formula for the number of generations (n)?
n = t/g where t = the time given and g = generation time
What are the methods of measuring bacterial growth?
- flow methods
- microscopic counts
- viable plate counts
- MPN
Describe Coulter counters
A type of flow method used to measure bacterial growth by forcing cells through a capillary one by one, which disrupts an electrical circuit. Each disruption is counted as a cell
What limits Coulter counters?
It counts dead cells and debris as living cells, as it cannot distinguish between them
What is flow cytometry?
A type of flow method used to measure bacterial growth by working the same as Coulter counting except with light instead of electricity
What are microscopic counts?
A known volume of cells is placed onto a slide with grids for manual counting. It is more precise than flow methods
What are the limits of microscopic counts?
- cannot distinguish between live and dead cells without special stains
- small cells can be overlooked
- precision is difficult to achieve (easy to make mistakes)
- cell suspensions of low density are hard to count
- motile cells must be immobilized
- debris can be mistaken for cells
What are the three methods used for viable cell counts?
- spread-plate method
- pour-plate method
- MPN
How must plate counts be prepared to obtain accurate measurements?
They must be diluted first, so that 30-300 colonies are visible on a single plate
Describe the spread-plate method
100-1000microliters of the sample are pipetted onto an agar plate. This is spread evenly over the agar’s surface and allowed to incubate
Describe the pour-plate method
The sample is pipetted into an empty agar plate and mixed with the medium. It is allowed to solidify and incubate
What plating method allows for surface colonies to be observed?
Spread-plate and pour-plate
What plating method allows for subsurface colonies to be observed?
Pour-plate
How do we calculate the cells per mL following a spread-plate?
A plate with 30-300 colonies is chosen and the number of colonies on it is multiplied by the reciprocal of the dilution (dilution factor). If 1mL samples are loaded onto the plates, we stop there, but if 100microliter samples are used, we must multiply the calculated number by 10
Describe how the MPN method of cell counting is prepared
Serial dilutions of the stock culture are incubated and checked for turbidity. The second most dilute tube with growth has the reciprocal dilution factor of the stock culture
When is MPN most useful?
When used to estimate population sizes in liquids
What are a few indirect counting methods?
- optical density (MPN) (turbidity)
- measure cell component (ATP, DNA, proteins, etc.)
- gene counting using qPCR
- measure rate of production of cell product or rate of consumption
A higher cell density will result in a ______________ absorbance
Higher
Why does the optical density of actual samples deviate from theoretical values at high cell density?
Light will bounce from cell to cell, eventually being interpreted by the spectrophotometer as a lower cell density, as more light is making it to the machine
What is a batch culture? What would its growth phase plot look like?
Displays growth in a closed system where the medium is not replenished; resembles a typical lag, log, stationary, death plot due to the limited resources and accumulating waste
What is a continuous culture? What would its growth phase plot look like?
Displays an open system microbial culture of fixed volume, where growth rate and population density can be controlled independently and simultaneously; would show a lag and log phase depending on added nutrients
What is dilution rate?
A variable that can be controlled in a continuous culture that describes the rate at which fresh medium is pumped in and spent medium is pumped out
What variable used to control growth rate in continuous culture?
Dilution rate
What variable used to control population density in continuous culture?
Concentration of a limiting nutrient
Describe the structure of a chemostat. What is it used for?
Fresh medium is allowed to trickle into a beaker containing the sample while a tube with an opening near the top of the sample allows for effluent to leave the beaker; continuous cultures
Why is it impossible to control the growth parameters in a batch culture?
Growth conditions are constantly changing and media can’t be added, as it is supposed to be a closed system
What is a steady state when considering the chemostat?
Population size and growth rate of the bacteria are constant
What is the formula for dilution rate?
D = F/V, where F is the flow rate of adding and removing medium, and V is the culture volume
What happens when dilution rate gets too high? Describe it
Washout; the rate of flow is greater than the rate of growth for the bacteria, so they are removed before they can replicate
Why does specific growth rate plateau at high dilution rates before washout?
Microorganisms can only divide so fast even with unlimited nutrients
Why does growth rate plateau at relatively low limiting nutrient concentrations? What do we see in the growth yield?
Microorganisms can only divide so fast even with unlimited nutrients; populations can get to high densities because they are not limited by that particular nutrient (do not die but cannot grow at a faster rate)
What are some uses of chemostats?
- fermentation (produce lots of cells)
- physiological studies
- simulates natural conditions better than batch cultures
- ecological studies into competition and predation
- allows scientists to control growth rate and population density independently
