Microbiology Exam 1 Flashcards
Woes 1977
Studying methanogens
Sequencing of the 16S rRNA gene (using non-sanger techniques)
Methanogens and other organisms: bacterial and eukaryotic
He proposed three domains of living organisms: Bacteria (microbial), Archean (methanogens) and Eukarya
What is a micro lab used for?
To culture organisms
To isolate them and study them
Initially done to see what causes disease
Pace 1990s
Culture independent studies
Isolating DNA from samples and characterizing organisms based on sequencing to identify the organisms.
What happened as humans converted from hunter-gatherer societies to agricultural societies?
Increase in population density
Increase in proximity to disease because there is an increase to proximity to domesticated animals
This led to new diseases for humans
Where does measles come from?
Dogs
Where does influenza come from?
Pigs
Where does consumption come from?
Cattle
Where does malaria come from?
Mosquitos
Caused from increase in standing water from growing agriculture
Hippocrates (460-370 BCE)
“Each disease has a nature of its own, and none arises without its natural cause”
Van Leeuwenhock
Built tiny microscopes
300x magnification
First to observe microscopic organisms
Semmelweis
Australian Physician 19th Century
Worked in two hospitals with two maternity wards
One ward had doctors and the other had nurses and midwives
The Physician staffed ward had 600-800 death a year
The Midwives had 60 deaths a year
He found the difference was handwashing and proposed physicians should do handwashing before delivering babies with a disinfectant
The mortality rate went from 18.3% down to 1.2%
1/5 woman dying to 1/100 women dying
Put him in a mental institution for his idea and he died there
The End…
Germ Theory of disease
Specific organisms were found to cause specific disease
Pasteur (1822-1895)
He is the founder of the field of biochemistry and microbiology (with Koch)
Disproved concept of spontaneous generation (When flies are separated from cheese maggots do not form in the cheese)
Fermentation: Spoiling of wine caused by microorganisms. Found heating wine at 55 Degrees C helps save wine (i.e. pasteurization)
Developed a vaccine for rabies:
Cultured bacterial/viral strains to weaken them to be used as a vaccine
Saved boy who was bit by rabid dog
Spontaneous generation
Life could arise from non-living matter
Koch - Nobel Info
Discovery of developing solid media to culture bacteria
Used potatoes for starch then used agar from seaweed (allows the isolation of individual organisms)
Identified the bacterial species that cause disease such as cholera and consumption
He determined the organism that causes Anthrax which is caused by bacteria
What are Koch’s Postulates?
Used to identify infectious agents that cause specific diseases
A way to isolate organism to study its characteristics to make treatments
What are the four Koch’s Postulates?
- The organisms needs to be present in every case of the disease and should be absent in healthy people
- Organisms must be extracted from patient and isolated in pure culture
- Organism when added to healthy person should cause the disease
- Same organism must be re-isolated from infected organism in step three and should be the same infectious agent as the original organism that is causing the disease.
Marshall, Warren: Nobel 2005
Found stomach ulcers in some cases are caused from bacteria
Treat people with anti-biotics
What is LUCA?
At the base of the bacteria, archaea, and eukaryote tree
Is the last universal common ancestor
Prokaryotic cell
Cell without a nucleus that lacks a cell membrane
Microbe
Small cells not visible without a microscope
Varies in size
Bacterial cell shapes
coccus: round, bacillus: rod-shaped are the most common
Spirillum, spirochete: corkscrew structure, filamentous, budding and appendaged: stalk and hypha
Functions of cell membranes in bacterial cells
Permeability barrier
Energy generation (ATP)
What are bacteria cell membranes made up of?
phospholipids that form a bilayer
membranes are embedded with proteins which are internal or peripheral and function in transport of ions or molecules that cannot diffuse through the membrane because they are too big
What makes up phospholipids?
Phospholipids contain glycerol molecule with two fatty acid chains linked by ester bonds
Function of a bacterial cells cell wall?
Prevent lysis due to osmotic stress
Two types of bacterial cell walls
Gram-positive (purple)
Gram-negative (red)
Based on staining technique done by Grams
What do Gr positive and Gr negative cell walls have in common?
They both have peptidoglycan
What does peptidoglycan (PG) do for a bacterial cells cell wall?
Provides rigidity to cell wall
What are the building blocks for PG?
N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
They are carbohydrate monomers
What holds NAG and NAM together to form PGs?
Beta (1,4) glycosidic bonds since they are carbohydrate monomers
They are then crosslinked by short peptides to make the cell wall
What A.A’s are in PGs?
D and L A.A’s
Do Gr positive and Gr negative cell walls have the same cross-links?
No, Gr negative contains an A.A. like molecule called diaminopimelic acid (DAP) which is included in the crosslinking to give strength to the structure
What does penicillin do to bacteria cell wall?
Prevents PG to have crosslinks
This causes lysis due to osmotic stress
Gr positive cell wall
There is no DAP
Cell wall is 90% PG
Contains teichoic acids which can extend beyond PGs out of the wall towards the exterior. TGs can be bound to PG or phospholipids (sometimes phospholipids)
Contain phosphate groups and D-A.A’s
How does the prescence of phosphate groups on the cell wall of a bacterial cell affect what it binds to?
Phosphate groups are negatively charged and will attach to substances with a positive charge
What cell wall of bacteria would you use penicillin against?
Gr positive bacteria cell wall since it is 90% PG and therefore would be able to cause a larger disruption to crosslinking than in a Gr negative cell wall
This would then cause cell lysis due to osmotic pressure
Gr negative cell wall
Thin layer of PG and contains an outer membrane of lipids outside the cell membrane
What does the outer membrane of Gr negative cell walls contain?
lipopolysaccharides (LPS)
If the cell is lysed, LPS can function as toxins and make you sick
Periplasm in Gr negative bacteria cell wall
This is the space between the cell membrane and the outer membrane
Lipopolysaccharides (LPS)
Thought to be a barrier against lipophilic antibiotics
Will fight hydrophobic antibiotic molecules
Fermentation
Organic compounds are the e- donor and e- receptor
There is no external e- acceptor
Differences between respiration and fermentation
Respiration is aerobic or anaerobic
e- donor is oxidized with external e- acceptor (Could be O2)
Fermentation is when organic compounds are the e- donor and e- acceptor
There is no external e- acceptor
When does fermentation happen in humans?
Occurs when O2 is limited
In absence of terminal e- acceptor since O2 is not getting to muscles fast enough when working out
Pyruvate is reduced to lactate
Glucose -2ADP to 2ATP—> 2 pyruvate
2 pyruvate -2NAD to 2 NADH—> 2 lactate
There is no intermediate and pyruvate accepts and is reduced to lactate.
Regeneration of oxidized e- carriers (such as NAD+)
Ex. Bacteria glycolysis in which NAD+ is needed for metabolic pathways that produce ATP
Sporulation
Process of forming endospores
Type of cell differentiation
> 200 genes involved in process
Model organism of studying this are the genus bacillus bacteria
Factor needed for sporvation
cell density must be high
must determine environment with chemical signaling to see that other B. subtilus
Why should you not give an infant honey?
There are bacterial spores in honey and the infant could get botulism
Intestinal tract is not fully developed and does not have the full natural microbiome
The spores will germinate in the intestinal tract and produce a toxin that will cause paralysis
In adults the bacterium cannot compete with the microbiome which will therefore not allow the bacteria to make enough toxin
Endospores
Caused by gram positive bacilli can form endospores
Highly resistant structures
Resistant to heat, harsh chemicals and radiation
Dormant structures (Not metabolically active)
Can be viable for millions of years
High calcium content
enzymatic activity, respiration and macromolecular synthesis is absent
Resistant to lysozymes
Low water content
Sporulation
A last resort (only when nutrients = limiting)
this is because it is energetically expensive
from 1 cell comes 1 endospore
sporvation and germination
problem: not a lot of nutrients to start; complex process
NOT dependent on absence of a single limiting nutrient not just A.A./carb. starvation
Endospores develop in cell and then leaves the cell
B. subtilus
soil bacterium
can be frequent condition when nutrients are limiting
When nutrients= present = B. subtilus is no longer dormant and is in vegetative cell state
Chemotaxis
movement of bacteria to potential nutrients (carbs)
What B. subtilus can do
Do chemotaxis, secrete enzymes (ex. amylase cleaving glycosidic bonds in starch)
take up DNA (to pull something in, may have beneficial traits)
secrete antibiotics (kill competition, inhibit growth)
If these things still do not get nutrients bacteria will do this to get nutrients
Life cycle of endospores
germination in which a vegetative cell develops an endospore and becomes a sporulating cell, then the endospore leaves the cell
asymmetric cell division, cell engulfs spore, spore gets CM and CW, spore gets a coat and uptakes Ca2+, spore maturation and cell lysis
When endospore leaves, cell dies
Dipicolinic acid (DPA)
Only present in endospores; absent in veg. cells
~10% dry weight in endospores
Binds Ca2+ ions
SpoOA
key protein-process in sporulation
early in process-SpoOA=activated
results in release of a toxin (protein) that lyses other Bacillus cells (Bacterial Cannibalism)
Do cannibalism to get a source of energy
lyses other cells and delays their sporulation
Other cells DO NOT like this and produce an antitoxin
Reversible covalent modification of SpoIIAA
How is SpoIIAA regulated?
Explain process?
Inactive SpoIIAA is P’ed, another protein removes the P which results in active SpoIIAA
regulated (one way)
P’ed= easy regulation; like a light switch
Results in change in conformation of protein which is rapid and reversible
Active SpoIIAA binds to SpoIIAB which usually is bound to sigma factor.
Sigma factor is forced to leave SpoIIAB and is activated
The activated sigma factor binds RNA polymerase to start txn which keeps SpoIIAA in vegetative state
Protein kinases
P’ate other proteins
A phosphorelay system- protein P regulation Plases remove P
What happens when sigma factor is removed?
Genes included in sporulation are expressed
~10% of Bacillus genome
Asymmetric cell division (produces 1 endospore)
lots of genetic and protein factors involved
2- component signaling pathways
More on sporulation
spore has <1/4 of H2O of vegetative cell (therefore more resistant)
small acid soluble proteins (SASPs) bind to DNA
Bacterial flagella
made of MANY polypeptide chains and must be present for flagella to function
EVOLUTION, but “intelligent design” says its so complex it could not have evolved
Flagella
Used for movement: swimming, gliding
polar flagella
attached to one end of the bacteria
peritichoris flagella
Inserted at multiple locations
anchored to the cell membrane (protein complexes)
Flagella movement
No attractant: random movement
Attractant present: directed movement
Movement powered by proton gradient
(proton motive force. Anchellum-ATP hydrolysis)
Taxis
movement
chemotaxis
response to chemicals
phototaxis
response to light
positive flagella movement
toward the object/light
negative flagella movement
away from the object/light
tumbling
flagella random process
Tumble part of flagella goes towards the attractant
runs connect tumbles
Elemental composition of macromolecules
Most common: C,H,O,N
Present: P,K,Mg,S,Se
Energy sources for microorganisms
light: phototrophs (organisms get energy from light)
chemicals: chemotrophs (organisms get energy from chemicals)
chemolithotroph
Gets energy from inorganic compounds
e- donor is inorganic
chemoorganotroph
gets energy from organic compounds
e- donor is organic
Respiration
Process that uses terminal e- acceptor to make ATP
Oxygen is not the only terminal e- acceptor
Autotrophs
Use inorganic C molecules for synthesis of energy
CO2
Heterotrophs
Use organic C molecules for synthesis
carbohydrates, lipids, A.A.
Photoheterotrophic
Energy comes from light
carbon is organic
NADH
reduced electron carrier
oxidized form is NAD+
electron carrier for electron yielding processes
Oxidation
Reduction reactions
Redox
transfer of e-‘s
usually accompanied by transfer of protons (H+)
lose electrons
oxidized
gain electrons
reduced
Who is being oxidized? Who is being reduced?
malate + NAD+ –> Oxalacetate + NADH + H+
Malate is losing e-‘s, therefore is being oxidized
NAD+ is gaining e-‘s, therefore is being reduced
How to tell which one is being reduced?
More hydrogen atoms present on molecule, the more reduced it is
Which is being oxidized? Which is being reduced?
Methane + O2 –> CO2 + HOH + e-
Methane has more H’s, therefore it is being reduced
O2 is being oxidized since it is losing electrons
Reduction Potential Eo
Redox potential
Hypothetical example:
X: Oxidized form
X-: Reduced form
X:X- is the redox couple
Whether it is positive or negative redox potential will determine if it will give/receive e-‘s
Positive Redox potential example
O and HOH
They are both good e- acceptors which therefore will cause a positive redox potential
Gaining electrons is the oxidizing agent
Negative Redox potential example
X- is losing e-‘s and is being oxidized to X
H+ is gaining an e- and is losing its positive charge
Therefore since it is losing electrons it is the reducing agent and redox potential is negative
Measurement of Redox Potential
Sample half cell:
contains X + X-
Is connected to cell of H+/H2
The X/X- cell is a solution and the H+/H2 cell is gas.
Both cells are connected by an agar bridge and the flow of electrons is measured by a voltmeter
If the elections flow from sample X/X- to reference H+/H2 the e- potential is negative
Which is oxidized? Which is reduced?
Fe3+/Fe2+ (pH7), Eo1= +0.2
Fe3+ is oxidized and Fe2+ is reduced
Energetics
2 redox couples
One couple donates e-‘s
Second couple accepts the e-‘s
The electrons are going to move from the more negative Eo value to the more positive Eo value.
Reduction potential
E’1
Measured in volts (tendency to donate e-‘s)
H2 is standard- half cell
Redox Couples
E’o is negative –> electron donors
E’o is positive –> electron acceptors
Which is oxidized form? Which is reduced form?
1/2 O2 I H2O
Oxidized form 1/2 O2
Reduced form H2O
Where will electrons flow?
CO2 I glucose -0.43
1/2 O2 I H2O +0.82
Electrons flow from glucose to oxidized form of couple with more + value (1/2 O2)
Relationship between E’o values and amount of energy generated?
The greater the difference in E’o values, the higher amount of energy can be generated
Anaerobic respiration
Terminal e- acceptor
Not O2
Aerobic organisms
Use O2 terminal e- acceptor
Anaerobic organisms
Use other molecules terminal e- acceptor
Facultative anaerobes
Can use O2 e- acceptor and either another terminal e- acceptor or fermentation
Is not just this, can be one or other or both
Examples of anaerobic respiration?
NO3- (nitrate) => NO2- (nitrite) or N2
Fe3+ (ferric) => Fe2+ (ferrous)
SO4- => H2S
CO2 => CH4
Inorganic N compounds
Common acceptors for anerobic respiration
Common e- donors for chemolithotrophs
Usually aerobic organisms (using O2 as e- acceptor)
H2S, H2, Fe2+, NH4
Hydrogen bacteria for chemolithotrophs
H2 => e- donor
Electron transport chain => creates H+ gradient => drives ATP synthesis
Membrane hydrogenase
Removes e- from H2
Part of e- transport chain
Cytoplasmic hydrogenase
Removes e- from H2
Reduce NAD+ => NADH => helps get rid of CO2. Cells can make molecules they need via using this molecule for biosynthesis (is a reducing power)
Most hydrogen bacteria
Are facultative chemolithotrophs
Can grow other ways chemolithotrophically
Can use other e- donors
Iron oxidizing bacteria
Fe2+ (ferrous) is oxidized at very low pH’s
Oxidation rxn. occurs on outside of cell so therefore will not be toxic to the cells
LPS and outer membrane –> gr. -
Oxidation of Fe2+ - outer membrane
e- transferred to rustacynin (inner membrane)
To ATP synthesis
or to NAHD (biosynthesis)
Oxygen is good terminal e- acceptor
Gets turned to H2O
Fe3+ I Fe2+ +0.77
1/2O2 I H2O +0.82
Need to oxidize lots of Fe2+ to make ATP (Not very energy efficient)
Anaerobic respiration
Terminal e- acceptor that is not O2
Nitrate reducing bacteria
Nitrate reducing bacteria
Nitrate (NO3-) (terminal e- acceptor)
Reduced to nitrite (NO2-)
Methanogens
CO2 is reduced to form CH4
Fermentation
Occurs in the abscense of a terminal e- acceptor
Still allows cells to make ATP
What organisms have phospholipids?
Eukarya and bacteria
Diglycerol tetraethers are in which organisms?
Archaea
Fermentation
Produce oxidized e- carriers
Alcohol fermentation
Makes ethanol
Things fermentation is beneficial for?
Alcohol
Cheese/dairy
Cabbage
Fermented foods have good impact on health for micro biome
Kombucha
What causes the dead zone in Lake Erie?
Nitrogen fixation
Ammonia made from industrial processes to use for crops
Soil bacteria oxidize ammonia to make nitrates
Nitrates run off into streams and end up in lakes
Algae (cyanobacteria) use nitrates and get algae bloom
The bacteria die and are at the bottom of the lakes
Microbes decompose algae (aerobic process)
Depletes O2 in water which creates a dead zone
Types of nitrogen stuff
N2: molecular N
NO2-: nitrite
NO3-: Nitrate
NH4+: Ammonia
Do humans fix nitrogen?
No
Nitrogen Fixation
When strong triple bond between nitrogen is broken to make a bi product which can be turned into amino acids and nucleotides
Bond needs 225 kcal/mol energy to break bond which can be done by some microbes
Bacteria and Archea
fix 10^11 kg of N2/year
60% of N2 fixed
Lightning/radiator
~15%
Industrial
To produce fertilizer
Energy consuming
500oC to be able to do this
pressure of 300 ATM to do it
Microbes that can do nitrogen fixation
Can be free living and symbiotic
Can be aquatic
What types of plants have a symbiotic relationship with nitrogen fixing bacteria?
Legumes
Includes peas, trees, seeds of plants,
Legumes (Wiki definition)
Legumes are grown agriculturally, primarily for human consumption, but also as livestock forage and silage, and as soil-enhancing green manure. Well-known legumes include beans, chickpeas, peanuts, lentils, lupins, mesquite, carob, tamarind, alfalfa, and clover. Legumes produce a botanically unique type of fruit – a simple dry fruit that develops from a simple carpel and usually dehisces (opens along a seam) on two sides.
Most legumes have symbiotic nitrogen-fixing bacteria, Rhizobia, in structures called root nodules. Some of the fixed nitrogen becomes available to later crops, so legumes play a key role in crop rotation.
N reduction
Energy demanding
6e- & 16 ATP
enzyme nitrogenase which fixes into organic nitrogen
Nitrogenase
Complex
Contains redox centers
Fe is important for transfer of e-
reductase provides e-. High reducing power
Has a unique property for process: It is inactivated by O2
cyanobacteria
Fix N2
Obligate aerobes
Fixes microbes by having specialized cells to help carry out the process to avoid O2
Heterocysts
Specialized cells for N fixation
Dinitrogenase
Uses e- to reduce N
Reduction requires 6e-
Reaction for nitrogen fixation
N2 + 6e- + 6H+ –> 2NH3
Process are 8e- generated
N2 + 8e- + 8H+ –> 2NH3 + H2
2 ATP are hydrolyzed for each e- transfer
e-‘s are transferred to N atoms, one at a time
Fe/Mo cofactor
Nitrate reductase is an enzyme found in E. coli that plays a role in electron transfer from the periplasmic active site of FdhN or Hya. It is composed of three subunits and contains molybdopterin cofactor and iron-sulfur clusters.
An example of this is hemoglobin (Has Fe which can transfer electrons and can be oxidized)
Read article I saved
Glutamate dehydrogenase
Will take alpha-ketogluterade + NH3 + NADH –> glutamate + NAD+
NADH provides e-‘s for process and becomes NAD+
Other A.A. can be synthesized from that
Microbes have role in ecosystem with N?
Yes
Oxidize ammonia
Oxidize nitrate
Nitrate reducing organisms
How was it proven penicillin affects cell wall integrity?
Group with low salt concentrations like in nature and another experimental group with high salt concentrations
Added penicillin to two groups
low salt experienced lysis and water went into the cell
high salt conditions showed no lysis since the water was going out of the cell
Lysis happened because cell wall failed and its main job is to make sure osmosis will not burst cell
How do bacterial cells divide?
Binary fission yielding 2 identical cells
Asexual process
Daughter cells are genetically identical to each other and to parental cell
Peptidoglycan is synthesized at the poles of the cell
OR
Unequal distribution via budding
How do eukaryote cells divide?
Mitosis
Where does peptidoglycan synthesis occur?
New peptidoglycan is synthesized at the poles of the cell and is a symmetrical process
One dividing cell has old PG and another has new PG
Generation tome for E. Coli dividing as fast as fukkk?
Under ideal conditions with nutrients E. Coli can divide every 20 minutes
Growth Stages
Cells are grown in culture
Inoculate bacteria and watch them over time
1 st phase: Lag phase
2 nd phase: exponential growth
3 rd phase: Stationary phase
4th phase: Death phase
Lag phase of bacteria
1st phase
Show initial period of growth
Is slow b/c it takes time to assimilate nutrients and b/c of changes in gene expression
Exponential growth phase of bacteria
2nd phase
Population doubles at regular intervals
Nutrient poor environment- cells may double every few months
Nutrients are being depleted
Wastes are being generated
Stationary phase of bacteria growth
3rd phase
no net increase or decrease in cell numbers
Death phase of bacteria growth
No new cell division
Things that can affect growth of bacteria?
Nutrients; wastes
temperature which impacts protein structure
NaCl concentrations
pH levels
Psychrophiles
Psynomonads
Grow in low temp
~0oC-10oC
Mesophiles
Grow best at intermediate temp
~10oC-45oC
Thermophiles
Best growth at elevated temp
40oC-70oC
Hyperthermophiles
Are two groups: extreme and less extreme
Very high temperatures
less extreme: 65oC-98oC
extreme: 90oC-120oC
Nonhalophiles
Do not grow in prescence of high salt []
Less than 1%
Halotolerants
Can grow in high salt []
Up to 10%
Halophile
Can grow up to ~12.5% NaCl
Extreme halophiles
archaeal species
Can begin growing at ~15% [NaCl] and grows at concentrations higher than that
Strictaerobe
requires molecular O2 for growth
Strict anerobe
Only grows in the absence of O2
In intestinal tract
Facultative anaerobes
Grow in prescence or absence of O2
DNA of bacteria
DNA replication has been studied in E. Coli
Chromosome has single origin of replication
The chromosomes are condensed by super-coiling which packages the chromosomes
When is E. Coli replication initiated?
Takes ~40 minutes
Cell division takes ~20 minutes
When it is replicated they will start to replicate it again
It is initiated before previous round of DNA replication is completed
Do bacterial cells have an apoptosis pathway?
Yes they do!
