microbio 303 - unit #2

Complex medium

Nutrient-rich medium where the exact chemical composition is not completely known. It supports the growth of many organisms and does not specifically select for a particular organism

Defined medium

Medium in which the exact chemical components and their amounts are known.

Selective medium

Medium that allows a targeted microorganism to grow while killing or inhibiting non-target microorganisms

Differential medium

Medium that allows groups of microorganisms to grow but distinguishes between them based on differences in their growth or metabolic properties.

Enrichment medium

Fortified medium that favors the growth of a targeted, usually fastidious, microorganism

Fastidious organism

it has demanding nutritional requirements

read media and infer about organisms

• Look at the ingredients in the medium to determine what the organism is using as its carbon source and nitrogen source.

• Determine what compounds are available as energy sources, electron donors, and electron acceptors.

• The nutrients provided by the medium give information about the organism's metabolism and biosynthetic capabilities.

• Microorganisms have diverse metabolic strategies and can use different electron donors, electron acceptors, and energy sources

Chemoheterotroph

The organism obtains energy from chemicals and carbon from organic compounds

Auxotroph

An organism that cannot synthesize one or more nutrients required for growth

Identify an auxotroph

If a bacterium does not grow on defined medium but grows when a specific nutrient is added, it is an auxotroph for that nutrient

Prototroph

An organism that can synthesize the nutrients it needs and therefore does not require that additional nutrient to be supplied

Direct counting

Physically count cells in a sample. Methods can include stains/probes and FACS.

FACS

Fluorescence-activated cell sorting; uses lasers and fluorescence to analyze and sort individual cells. (optical tweezers and flow cytometry)

Plating

• Dilute a sample, plate it, and count the resulting colony-forming units (CFUs).

• CFU: Colony-forming unit; represents a viable, culturable unit capable of producing a colony

Turbidity

Estimate bacterial numbers by measuring how much light is scattered by cells in a liquid culture. More cells generally produce greater turbidity.

Most Probable Number (MPN)

A statistical method used to estimate the number of microorganisms in a sample.

problem with metagenomics

genomes do not provide functional information about community members

understanding ______ is key to understanding the microbiome

metabolism

metabolism

the sum of all biochemical reactions that occur in a cell (catabolism + anabolism + energy generation)

how to study microbial metabolism?

culture based:

-pure culture

-grow microbes in lab and examine traits

-look for auxotrophs or phototrophs

-measure growth rate/patterns

pros and cons of a selective medium

pros: can select for desired metabolic capacity

cons: will only get certain types and you have to KNOW what you’re looking for

which organism has a greater biosynthetic capacity?

ones in minimal media

measure cell number by… (4 things)

1. directly by counting (total/FACS)

2. indirectly by plating (CFU)

3. indirectly by turbidity

4. estimate by MPN

problems with direct counts…

-requires stains/probes

-most microbes do not grow in pure cultures in nature

growth rate measures the number of generations per time in ____ phase

log

diauxic growth indicates…

one carbon source is used and the cell adapts and synthesizes new enzymes, then utilizes another carbon source

would inoculating a patient sample into thyoglycollate tube/heating a sample generate a pure culture?

no, its not a genetically homogenous culture of cells, you might have more than one type of archaea for ex.

3 ways to get pure cultures

1. serial dilution

2. streak plating

3. FACS

Chemotroph

Gets energy from chemicals

Phototroph

Gets energy from light

Lithotroph

Uses inorganic molecules as electron donors

Organotroph

Uses organic molecules as electron donors

Autotroph

Uses CO₂ as its carbon source

Heterotroph

Uses organic molecules as its carbon source

Redox reaction

A reaction involving the transfer of electrons

Electron donor

• Loses electrons.

• Becomes oxidized

Electron acceptor

• Gains electrons.

• Becomes reduced

Fe³⁺ / Fe²⁺

• Fe³⁺ = oxidized form.

• Fe²⁺ = reduced form

2H₂ + O₂ → 2H₂O

• H₂ = electron donor → becomes oxidized.

• O₂ = electron acceptor → becomes reduced

Reduction potential (E)

Measures how strongly a molecule tends to accept electrons

More positive reduction potential

• Better electron acceptor.

• Has a greater tendency to gain electrons

More negative reduction potential

• Better electron donor.

• Has a greater tendency to give up electrons

Reduction potential equation

ΔE = E(acceptor) − E(donor)

Electron flow

• Electrons move from the donor to the acceptor.

• This moves electrons from higher potential energy → lower potential energy

Positive ΔE

• Produces negative ΔG.

• Reaction is exergonic.

• Energy is released.

• This can provide energy for microbial growth

Negative ΔE

• Produces positive ΔG.

• Reaction is endergonic.

• Energy is required.

• Not an energetically favorable lifestyle by itself

Exergonic

• −ΔG

• Releases energy

Endergonic

• +ΔG

• Requires energy

Cofactor

A non-protein molecule required by some enzymes to function

Prosthetic group

A cofactor that is permanently attached to an enzyme

Coenzyme

A cofactor that is loosely attached to an enzyme

Flavoproteins with FAD/FMN

• Prosthetic cofactors.

• Accept 2 electrons and 2 H⁺

NAD(P)⁺

• A coenzyme.

• Accepts 2 electrons and 1 H⁺

Cytochromes

• Heme-containing proteins in the ETC.

• Transfer single electrons

Quinones

Small molecules that carry 2 electrons and 2 H⁺

-hydrophobic, found in membrane

Glutathione

Helps maintain a reducing environment in the cytoplasm

ETC

1. carriers arranged in increasingly positive reduction potential

2. carriers alternate between electron + proton vs. electron-only carriers

3. (excess H⁺ pushed out of the cell) reduction of terminal electron acceptor and generation of PMF

PMF

1. generate ATP by ATP-synthase

2. power flagella

3. facilitate secondary active transport

Proton motive force (PMF)

• Energy is stored as an electrochemical gradient of H⁺ across a membrane.

• Has two components:

  • Chemical gradient: difference in H⁺ concentration.

  • Electrical gradient: difference in charge across the membrane.

• H⁺ flowing through ATP synthase provides energy to make ATP

High energy bonds

• Energy can be stored in certain chemical bonds.

• Important examples:

  • ATP

  • Acetyl-CoA

  • PEP

  • 1,3-bisphosphoglycerate

NADH

• Stores energy as high-energy electrons.

• Donates electrons to the electron transport chain (ETC).

• Electron transfer through the ETC generates a PMF.

• The PMF can then be used to make ATP

ATP Synthase

• Uses energy stored in the PMF to produce ATP from ADP + Pi.

• H⁺ flows through ATP synthase and causes the enzyme to rotate/change shape, allowing ATP production

Substrate-level phosphorylation (SLP)

• Produces ATP by directly transferring a phosphate group from a high-energy molecule to ADP.

• Does not require the ETC or PMF.

Glycolysis — SLP

• 1,3-bisphosphoglycerate → 3-phosphoglycerate

  • Produces ATP.

• PEP → 2 pyruvate

  • Produces ATP.

  • Produces NADH (contains high energy electrons)

• Glycolysis produces 2 net ATP through SLP

TCA cycle — SLP

• Pyruvate is further oxidized.

• Produces:

  • NADH

  • FADH₂

  • 1 ATP

  • CO₂

• NADH and FADH₂ carry electrons to the ETC

Oxidative phosphorylation

Produces ATP using energy from electron transfer through the ETC.

• ETC activity creates a PMF.

• ATP synthase uses the PMF to make ATP

high-energy substrate → directly transfers phosphate → ATP

SLP

NADH/FADH₂ → ETC → PMF → ATP synthase → ATP

Oxidative phosphorylation

Electron transport chain (ETC)

• NADH/FADH₂ donate electrons.

• Electrons move through a series of electron carriers.

• Electron transfer is coupled to movement of H⁺ across the membrane

aerobic respiration

Glucose → glycolysis → pyruvate → TCA → NADH/FADH₂ → ETC → PMF → ATP synthase → ATP

fermentation

• Occurs when there is no external terminal electron acceptor available.

• The ETC is not used.

• Therefore, there is no oxidative phosphorylation.

• ATP is produced only through SLP.

• NADH must be converted back to NAD⁺ so glycolysis can continue.

• An internal organic molecule accepts electrons from NADH.

• This produces fermentation end products and allows glycolysis to continue

examples of fermentation end products

• Lactate

• Ethanol

• Propionate

• Butyrate

• Acetone

• 2,3-butanediol

• Butanol

• Formate

• H₂ + CO₂

Why fermentation products help identify microorganisms

• Different microorganisms have different fermentation pathways.

• Therefore, they produce different patterns of fermentation products.

ex.

• Lactic acid bacteria → lactate

• Saccharomyces cerevisiae → ethanol + CO₂

• Propionibacterium → propionate + CO₂

• Clostridium → products such as butyrate and butanol

anaerobic respiration

• Uses an electron transport chain (ETC).

• Uses a terminal electron acceptor other than O₂

• Still produces a proton motive force (PMF) and ATP through oxidative phosphorylation.

• Different microbes can use different terminal electron acceptors depending on what is available.

common alternate electron acceptors

• Nitrate (NO₃⁻)

• Sulfate (SO₄²⁻)

• Carbon dioxide (CO₂)

• Ferric iron [Fe(III)]

• Manganese [Mn(III/IV)]

• Fumarate

• TMAO

Nitrate (NO₃⁻)

• Can serve as an alternative terminal electron acceptor.

• Respiration using nitrate produces less ATP than respiration using oxygen because nitrate has a lower reduction potential than oxygen

Sulfate (SO₄²⁻)

• Can be used as a terminal electron acceptor by sulfate-reducing bacteria.

• Sulfate is not as strong an electron acceptor as oxygen but can still provide enough energy for some organisms.

• Dissimilatory sulfate reduction produces H₂S

CO₂

• Used as a terminal electron acceptor by methanogens and acetogens.

• Methanogens reduce CO₂ to CH₄ (methane)

Ferric iron

• Fe(III) can act as a terminal electron acceptor.

• Some microbes reduce Fe(III) to Fe(II)

Iron respiration

• Fe(III) in iron oxide/rust can accept electrons.

• Fe(III) is reduced to Fe(II)

Shewanella oneidensis

• An example of a microbe that can use metal as a terminal electron acceptor.

• Can reduce insoluble Fe(III) and Mn(III/IV) oxides

Problem with insoluble metals

• The metal oxide is located outside the cell.

• Electrons therefore need to be transferred from inside the cell to the external metal

Nanowires

• Shewanella can use nanowires to transfer electrons to insoluble metal oxides.

• Nanowires allow electrons to reach the metal outside the cell

Cytochromes

Cytochromes in the outer membrane help transfer electrons to insoluble metals

Electron shuttles

Some microbes can use electron-shuttling molecules to transfer electrons from the cell to the metal

Siderophores

• Extracellular small molecules that bind Fe(III).

• They can transport Fe(III) to the cell, where it can be reduced

Redox tower

• Electron donors are generally toward the more negative end.

• Electron acceptors are toward the more positive end.

• The farther apart the donor and acceptor are on the redox tower, the greater the potential energy available

• microbes generally use the acceptor that provides the most favorable energy yield first.

Dissimilatory respiration

• The compound is used as a terminal electron acceptor for energy generation.

• The reduction product is generally released from the cell.

• Its main purpose is to generate energy.

• Example:

  • SO₄²⁻ → H₂S

• Because the pathway is being used for energy generation, it can have high metabolic flux

Assimilative metabolism

• The compound is reduced for use in biosynthesis/anabolism.

• The reduced product is incorporated into cellular material.

• Its main purpose is to provide a nutrient/building block for the cell rather than generate energy

Sulfate example

Sulfate can be reduced to H₂S through either:

• Dissimilatory respiration

• Assimilative metabolism

Which has greater metabolic flux?

• Dissimilatory respiration has higher metabolic flux.

• This is because large amounts of the electron acceptor can be processed to support energy generation.

• Assimilative metabolism generally uses smaller amounts because the product is needed only for cellular biosynthesis

Identify common electron donors for chemolithotrophs

Common inorganic electron donors

• Hydrogen (H₂)

• Hydrogen sulfide (H₂S)

• Elemental sulfur (S⁰)

• Ferrous iron (Fe²⁺)

• Ammonia/ammonium (NH₃/NH₄⁺)

• Nitrite (NO₂⁻)

Hydrogen (H₂)

• Commonly produced during fermentation.

• Hydrogenase enzymes oxidize H₂ and transfer its electrons.

• Electrons can enter the ETC to help generate a proton motive force (PMF)

Nitrogen compounds

• Some microbes oxidize ammonia to nitrite or nitrite to nitrate.

• These processes are called nitrification.

• Nitrifying bacteria and archaea obtain energy by oxidizing nitrogen compounds

Iron (Fe²⁺)

• Some microbes oxidize Fe²⁺ to Fe³⁺.

• Iron is a relatively poor electron donor, so these organisms face challenges obtaining enough energy

Sulfur compounds

• Microbes can oxidize H₂S and elemental sulfur (S⁰).

• Sulfur oxidation can produce sulfate (SO₄²⁻).

• Sulfur-oxidizing microbes can generate ATP through both substrate-level phosphorylation (SLP) and oxidative phosphorylation

chemolithotroph hurdle: obtaining enough energy

• Many inorganic electron donors provide less energy than organic electron donors.

• Some chemolithotrophs must process large amounts of inorganic compounds to obtain enough energy for growth

chemolithotroph hurdle: generating NADH for biosynthesis

• Chemolithotrophs need reduced electron carriers, such as NADH or NADPH, for biosynthesis and carbon fixation.

• Their inorganic electron donors may not provide electrons at a sufficiently negative reduction potential to reduce NAD(P)⁺ directly.

chemolithotroph hurdle: reverse electron transport

• Some chemolithotrophs must run their ETC in reverse to produce NADH.

• This requires energy and can consume the PMF that would otherwise help produce ATP

chemolithotroph hurdle: iron-oxidizing microbes

• Fe²⁺ is a poor electron donor.

• At neutral pH, iron can oxidize abiotically, making it difficult for microbes to compete.

• Aerobic iron oxidizers often live in acidic environments where iron remains available in a usable form.

• Iron oxidation can cause iron oxides to precipitate, potentially trapping cells in a metal-oxide “tomb”