Lecture 10 - 12 Populations, Metabolism, ETC

Lectures 10 -

How can we measure “size” in terms of microbial populations? (2 ways)

We can use both # of cells

We can use mass of population

How do we directly count cells? (3 techniques)

Cell Counts can be conducted on

• Counting Chambers

• Electronic Counters (like flow cytometry and coultier)

• Membrane filters

Counting Chambers

• Advantages

• Disadvantages.

Easy, inexpensive, and quick

Useful for counting both eukaryote and prokaryotes.

Cannot distinguish living from dead cells.

Membrane Filters

• Advantages

Cells filtered through special membrane that provides dark background for observing cells

Cells are stained with fluorescent dyes

Useful for counting bacteria

With certain dyes, may distinguish living from dead cells

Electronic counters —> Flow Cytometry

Microbial suspension forced through small orifice with a laser light beam

Movement of microbe through orifice impacts electric current that flows through orifice

Instances of disruption of current are counted

Specific antibodies can be used to determine size and internal complexity.

Coulter Counter

Microbial suspension is forced through a small hole.

Electrical current flows through the hole, and electrodes placed on both sides of the hole measure electrical resistance. Every time a microbial cell passes through the hole, electrical resistance increases (For example, the conductivity drops), and the cell is counted

How can we do viable counting? (methods)

• Why does it matter?

• Whether or not a cell is alive or dead isn’t always clear cut in microbiology

• cells can exist in a variety of states between ‘fully viable’ and ‘actually dead

Spread/Pour plates/Membrane filter methods

Viable Counting methods —> Spread/pour plates

– diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate

– after incubation, the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factor

– results expressed as colony forming units (CFU)

Viable Counting Methods:

Membrane Filter Method

– bacteria from aquatic samples are trapped on membranes

– membrane soaked in culture media

– colonies grow on membrane

– colony count determines # of bacteria in sample

Measurment of Cell Mass

• 3 different methods

• Dry weight

• Quantity of a particular cell constituent

• Turbidometric measures (light scattering)

Advantages or disadvantages of Dry Weight Cell Mass measurement

• Dry weight

– time consuming and not very sensitive

Advantages or disadvantages of Quantifying a particular cell constituent to find cell mass

• Quantity of a particular cell constituent

– e.g., protein, DNA, ATP, or chlorophyll

– useful if amount of substance in each cell is constant

Advantages or disadvantages of Turbidometric measures to find cell mass

• Turbidometric measures (light scattering)

– quick, easy, and sensitive

CAUTION for absorbance readings

• At very high cell densities (~10 9 cells/ml for E. coli),

Absorbance is no longer proportional to cell density.

• This is because of multiple scattering of photons by the cells.

• Multiple scattering makes it appear that the cell density is lower than the actual value.

• In this case, the culture has to be diluted, absorbance measured, and then multiplied by the dilution factor

How do we Continuously Culture Microorganisms

We use an OPEN SYSTEM to grow them

– continual provision of nutrients

– continual removal of products/wastes

• Maintains cells in exponential phase at a constant biomass concentration for extended periods

• Achieved using a continuous culture system

What is a Chemostat?

What is it used for?

How does it work?

• An essential nutrient is in limiting quantities

• Rate of incoming medium equals the rate of removal of medium from vessel

Dilution Rate in regard to microbial growth in a chemostat.

• What is it

• What is the optimal ratio to keep.

• Dilution rate – rate at which medium flows through vessel

• Note: cell density maintained at wide range of dilution rates

• Chemostat operates best at low dilution rates

What is a Turbidostat?

• What does it modify

• What stays consistent

• What does it operate best in?

• regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density

• dilution rate varies

• no limiting nutrient

• turbidostat operates best at high dilution rates

Metabolism

• What is it?

• What does it consist of?

Metabolism is the total of all chemical reactions in the cell and is divided into two modules:

Anabolism

Catabolism

Catabolism

• What does it do?

• Energetically what does it do?

• What does it provide.

From Greek “descent”, as in take apart

It involves usable energy-producing or “fueling” reactions

• provides reducing power (NADPH)

• generates precursors for biosynthesis

Anabolism

• What does it do?

• Energetically what does it do?

• What does it provide.

From Greek “ascent”, as in put together

• synthesis of complex organic molecules from simpler ones

• requires energy provided by fueling reactions

• requires reductants: NADPH, generated by catabolism

Types of Work (for the sake of this class)

Chemical Work

Transport Work

Mechnanical Work

How does a cell do:

Chemical Work:

Chemical work

– synthesis of complex molecules

How does a cell do:

Transport Work

– uptake of nutrients, elimination of wastes, and maintenance of ion balances

How does a cell do:

Mechanical Work

– cell motility and movement of structures within cells

Thermodynamics

– a science that analyzes energy changes in a collection of matter called a closed system (e.g., a cell)

– all other matter in the universe is called the surroundings

First Law of Thermodynamics

• Energy can be neither created nor destroyed

• Total energy in universe remains constant

- However, energy may be redistributed either within a system or between the system and its surroundings

Second Law of Thermodynamics

Entropy

• The measure of disorder, randomness, or uncertainty in a system

• Physical and chemical processes proceed in such a way that the disorder of the universe increases

Second Law of Thermodynamics

• What is it

• What is the symbol and units

• What does it being positive or negative mean

• What is the total change in entropy (how does this relate to the second law)

Entropy

• Entropy is a measure of the randomness or disorder of a system.

• Its symbol is the capital letter S. Typical units are joules per kelvin (J/K).

• Change in entropy can have a positive (more disordered) or negative (less disordered) value.

• In the natural world, entropy tends to increase.

According to the second law of thermodynamics, the entropy of a system only decreases if the entropy of another system increases.

Energy

• Def

• Units (2)

• Joules (J)

• Calorie

Joules

• What is it a measure of, what are the units

– units of work (kg * m2 / sec 2 )

Calorie

• What is it a measure of, what are the units

• Conversion to J

• Calorie (cal)

– amount of heat energy needed to raise the temperature of 1 gram of water from 14.5 to 15.5°C

• 1 cal of heat is equivalent to 4.1840 J of work

Free Energy

• Symbol

• ΔG (“Gibbs Free Energy”)

– amount of energy available in a chemical

reaction or process to do work

Enthalpy

• Symbol

ΔH

Change in enthalpy → heat content

T

• Temperature in degrees Kelvin

Entropy

ΔS

• Change in entropy

Free Energy Reaction (how to calcualte Gibbs)

• What does the result mean

• What does this indicate about our reactions (based on the result of the equation)

• Limitations of Gibbs

ΔG - ΔH - T ΔS

Expresses the change in energy that can occur in chemical reactions and other processes

Indicates whether a reaction will proceed spontaneously

• if ΔG is negative, reaction can proceed spontaneously in the direction written

• if ΔG is positive, reaction cannot proceed spontaneously in the direction written

• if ΔG = 0, reaction is at equilibrium

IT CANNOT TELL US HOW RAPID A REACTION WILL PROCEED, just the direction

Chemical Equilibrium

consider the chemical reaction

• A + B ⇌ C + D

Reaction is at equilibrium when rate of forward reaction = rate of reverse reaction

Equilibrium constant (Keq )

• expresses the equilibrium concentrations of products and reactants

Standard Free Energy Change

• Free energy change defined at standard conditions of concentration(1 M, each reactant and product), pressure (1 atm), temperature (25 ^oC), and pH (7.0)

• ΔG⁰

  • standard free energy change at pH 7

  • directly related to Keq

• ΔG o ́ = -2.303RT•logKeq

What do exergonic Reactions mean?

The ΔG^o is negative

We have a sponanteous reaction

What does an enedergonic reaction mean?

• ΔG^o is positive
  (reaction will not proceed spontaneously)

What do enzymes affect?

• What are the limitations

Speed up rates of reaction, but cannot change the ΔG or the direction of the reaction.

• ΔG is an inherent property of the reaction.

What is the energy currency of the cell?

ATP

What is phosphate transger potential?

• What has a high phosphate transfer potential.

ATP is said to have a HIGH phosphate transfer potential

– It readily sends a phosphate group to other molecules

– Other molecules have higher phosphate transfer potential than ATP

Cell’s Energy Cycle:

Catabolism

• What are some examples of processes that primarily use catabolism?

• What do they transfer the energy into?

Is used in:

• Aerobic respiration

• Anaerobic Respiration

• Fermentation

• Phototrophy

• Chemolithotrophy

Then is transferred into

• Chemical Work

• transport work

• Mechanical work

What are other energy sources akin to ATP that can be used?

• GTP

• CTP

• TTP

• UTP

I like ya CUTG (first letter of every energy source)

ATP in metabolism

• What is it’s main role?

Exergonic breakdown of high energy ATP is coupled with endergonic reactions to make them more favorable

REDOX REACTIONS

• What are they? What do they involve?

• What does this cause?

Redox Reactions

• Many metabolic processes involve oxidation-

reduction reactions (electron transfers)

• Electron carriers are often used to transfer electrons from an electron donor to an electron acceptor

• Transfer of electrons from a donor to an acceptor

– can result in energy release, which can be used to form ATP

What does a redox reaction consist of?

What is their relationship?

Redox: Two Half Reactions

• One is electron donating reaction

• One is electron accepting reaction

• Acceptor and donor are conjugate redox pairs

What does a standard redox potential measure?

• What is the positive version and negative version mean

• Conversion to gibbs

Measure of the tendency of the reducing agent to lose electrons

• more negative E0 —> better electron donor (being oxidized)

  • the better the electron donator the better the reducing agent it is)

• more positive E0 —> better electron acceptor (being reduced)

  • The better it is at accepting electrons or the better oxidizing agent.

Can be converted to ΔG:

ΔG = − n (23.062 kcal* mol-1* volt-1) ΔE’0 , where n is the number of electrons transferred

Electron Movement and Reduction potentials:

• What does the difference between E_o of the acceptor and the E_o of the donor mean? What can it tell us?

The greater the difference between the E 0 of the acceptor and the E0 of the donor

(E0 acceptor – E0 donor ) Δthe more negative the ΔG

Electron transport Chain (ETC)

• What is it?

• What is organized in it?

• How does it work?

Electron carriers are organized into ETC.

• First electron carrier will have the most negative E^o. (redox potential) (the more tendency to lose an electron)

• The potential energy stored in the first redox couple is released and used to form ATP

• First carrier is reduced and electrons are moved to the next carrier and so on.

Electron carriers

• Where are they at in bacteria, archaeal, and eukaryotic cells

• Examples include NAD, NADP and others.

• Located in plasma membranes of chemoorganotrophs in bacteria and archaeal cells

• Located in internal mitochondrial membranes in eukaryotic cells

• Examples of electron carriers include NAD, NADP, and others

What are some examples of electron carriers (2)

MAIN ONES

NAD

– nicotinamide adenine dinucleotide

NADP

– nicotinamide adenine dinucleotide phosphate

What are some examples of electron carriers (2)

Special ones

FAD

• Flavin Adenine Dinucleotide

CoQ

• A quinone

• Also called a ubiquinone

What are the three things a cell needs for growth?

• Chemical Components

• Reducing Power

• Energy

Chemical Components needed for growth

• Carbon and also the rest of CHONPS and other nutrients

• Reducing Power (electrons)

  • Mostly in the form of hydrides (H-)

• Energy:

Reducing Power needed for growth

Reducing Power (electrons)

• Mostly in the form of hydrides (H-)

Energy needed for growth

• ATP, proton motive force

Autotrophs

• Def

• Where do they get it

Self FEEDER.

• Get carbon from inorganic molecules, mainly CO2, but some can get it from CO or CH₄ (methane)

Heterotrophs

• Def

• Where do they get it

• Use organic molecules as carbon sources

• Can use a variety of carbon sources, mono- and polysaccharides, organic acids, amino acids, nucleotides, etc.

Lithotrophs

– Get electrons from inorganic electron donors: H₂O, H₂ , H₂S

Heterotrophs (Organotrophs)

from organic molecules as carbon sources

• Gets their electrons from carbon sources.

Phototrophs

Energy source is light

Chemotrophs

get energy from oxidation of inorganic molecules

Heterotrophs

gets energy from catabolism of organic compounds.

Bowtie Model of Metabolism

There are 100s of Carbon Sources, which are transferred to 12 universal precursor molecules. (this is the Catabolism process, oxidative processes that generates energy)

Then those are turned into the Amino Acids, nucleotides, sugars, fatty acids, vitamins, cofactors, and more the body needs. (this is the anabolistic process that requires energy)

What are the implications of the bowtie model?

• What are the two main implications

1. The function of catabolic pathways is not only to generate energy (mostly ATP), but also to generate the 12 precursor molecules and reductants (mostly NADPH)

2. All organisms have to have pathways to produce the 12 universal precursor molecules, regardless of the carbon and energy sources they can use, and regardless of their composition.

Meaning that at least that is common in metabolism.

Chemoorganotrophic Energy supplying processes

• #whatdatmean

• There are two

Chemoorganotrophic means breaking down organic molecules to create energy chemically

2 distinct modes:

– Respiration

– Fermentation

Respiration

• What modes is it split into again?

• What are the terminal acceptors?

– Respiration: uses terminal electron acceptor(s):

aerobic: terminal electron acceptor is O2

anaerobic: some other terminal electron acceptor, e.g.

NO 3- , Fe3+, SO 42- , fumarate, and others

What are examples of electron acceptors in anaerobic respiration.

NO 3- , Fe3+, SO 42- , fumarate, and others

Respiration

• How does it generate energy

• In its two variations as well

ATP is made mainly by the proton motive force

This proton motive force is generated by

• aerobic respiration as the final electron acceptor is O2

  • or

• anaerobic respiration in which the final electron acceptor is exogenous acceptor such as: NO3-, SO4 2-, CO2, Fe 3+, SeO4 2-

• organic acceptors may also be used.

Fermentation

• How does it gain energy

• What is the electron acceptor look like

• What does it use in the process.

• No exogenous electron acceptor (inside of the body)

• Does not involve the use of an electron transport chain

• ATP synthesized only by “substrate-level” phosphorylation (SLP)

What do all pathways of chemoorganotrophs share in common?

The different carbon sources and energy are funneled into common degradative pathways.

Catabolism of Glucose

• General Steps (3)

1. Glucose to Pyruvate

2. Pyruvate to CO2 in the TCA cycle to produce more ATP

3. Oxidation of NADH, FADH, in the electron transport chain to generate more ATP

Glucose to pyruvate

• The first steps

Glycolysis is the first step.

Glycolysis is a series of reactions for the breakdown of Glucose (a 6-carbon molecule) into two molecules of pyruvate (a 3-carbon molecule) under aerobic conditions;

or

Lactate under anaerobic conditions along

with the production of a small amount of energy

What are the three common routes of Glycolysis

– Embden-Meyerhof pathway

– Entner-Duodoroff pathway

– Pentose phosphate pathway

Emden-Meyerhoff Pathway

• What are the phases of it?

• In what organisms does it occur?

• Occurs in the cytoplasmic matrix of most microorganisms, plants, and animals

• The most common pathway for glucose degradation to pyruvate.

• Functions in the presence or absence of O2

• Has two phases

  • Six Carbon phase

  • Three carbon phase

Steps in Embden Meyerhoff

Step 1:

Energy investment step, we see The addition of phosphates (from ATP)

Step 2:

Oxidation steps generate NADH (producing NADH from NAD+)

Step 3:

High-energy molecules used to synthesize ATP by substrate-level phosphorylation. (A way that cells make ATP by transferring a phosphate group from a high energy molecule to ADP).

This leads 2 ATP net, 2 NADH, and 2 pyruvate per glucose

*Sidenotes

2 Important reactions in Embden-Meyerhof

• What are two key elements of it?

Entry point of phosphate (Pi) into the metabolic network

Requires energy, which is provided by oxidation of glyceraldehyde 3–phosphate by NAD + (into NADH)

Transfer of high energy phosphate bond from 1,3 bisphosphoglycerate to ATP

One more important reaction in Embden-meyerhof

A second enzyme synthesizing ATP

Phosphoenolpyruvate (ADP —>ATP)

Summary of Embden-meyerhof glycotic pathway

• In terms of chemical (it is dumb, but just do it the way this class wants it

glucose + 2 ADP + 2 Pi + 2 NAD+

—>

2 pyruvate + 2 ATP + 2 NADH + 2H+

Entner-Duodoroff Pathway

• Chemically what it looks like

• What uses it?

• What is the difference between Embden-Meyerhof

• Used by Gram-negative, aerobic bacteria lots of which are soil-borne, and a few Gram-positive bacteria and archaea

• Replaces the first phase of the Embden-Meyerhof pathway

2 pyruvate + 1 ATP + 1 NADPH + 1mNADH + 2H+

Instead of 6-glucose 6 phospate, it turns into fructose 1,6 - bisphosphate.

We instead form step KDPG

this nets up a pyruvate straight up and glyceraldehyde 3-phosphate by the enzymes of Embden Meyerhof pathway.

The pentose phosphate pathway

• What is it also known as?

• When does it occur?

• Does it work aerobically? Anaerobically?

• Also called hexose monophosphate pathway

• Operates at same time as glycolytic pathway or Entner-Doudoroff pathway

• Can operate aerobically or anaerobically

Pentose Phosphate Pathway

• What are the main things produced or occuring (3)

• Also called hexose monophosphate pathway

• Operates at same time as glycolytic pathway or

Entner-Doudoroff pathway

• Can operate aerobically or anaerobically

Alternative for metabolizing glucose 6 phosphate.

Is in charge of the Oxidation steps, produce NADPH

• Sugar trans-formation reactions (sugars into other sugars)

• Produce sugars needed for biosynthesis (creates sugars for creation of other molecules)

• Sugars can also be further degraded (some other sugars can be put toward glycolysis)

Specific Results of the Pentose phosphate pathway

• Name the 3 things generated

• What are they used for.

RESULTS

Generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells (EX: Fatty acid synthesis)

Production of ribose 5-phosphate (R5P), used in the synthesis of nucleotides and nucleic acids. (DNA and RNA)

Production of erythrose 4 phosphate (E4P) which is used in the synthesis of aromatic amino acids.

TCA cycle

• What is the main function

• What are other names for it

• What commonly practice it

• What does it produce

It turns Pyruvate into CO2

Also known as the Krebs or Citric Acid cycle

Common in Aerobic Bacteria, Free-living protozoa, most algae, and fungi

A source of biosynthetic precursors like GTP, NADH, and FADH

TCA Cycle Summary

• What are the NET THINGS GENERATED

For each acetyl-CoA molecule oxidized, TCA cycle generates:

• 2 molecules of CO2

• 3 molecules of NADH

• one FADH2

• one GTP

BEUATIFUL GORGEOUS SUMMARY VERSIONS:

Embden-Meyerhoff Pathway of Glycolysis

1) Starts with glucose-6-phosphate (6-carbon compound)

2) that is cleaved into two 3-carbon compounds (pyruvate)

3) generates a net of 2 ATP’s

4) generates 2 NADH’s

5) produces 2 pyruvates

BEUATIFUL GORGEOUS SUMMARY VERSIONS:

Entner-Douderoff

1) Starts with glucose-6-phosphate (6-carbon compound)

2) that is cleaved into two 3-carbon compounds

3) generates a net of 1 ATP

4) generates 1 NADH and 1 NADPH

5) produces 2 pyruvates

BEUATIFUL GORGEOUS SUMMARY VERSIONS:

Pentose Phosphate Pathway

1) Starts with glucose-6-phosphate

2) produces 2 NADPH’s

3) produces three of the 12 universal precursor molecules needed for biosynthesis (ribose-5-phosphate, erythrose-4-phosphate, and sedohepulose-7-phosphate) (R5P, E4P)

4) Not a direct source of ATP

BEUATIFUL GORGEOUS SUMMARY VERSIONS:

TCA Cycle

1) Starts with acetyl–CoA (2-carbon compound) and oxaloacetate

2) produces 2 CO2 ’s and an oxaloacetate

3) produces 3 NADH’s, 1 FADH

4) produces 1 GTP (which is needed as an energy source for peptide bond formation in translation

TOTAL RECAP FOR LECTURE

CATABOLISM OF GLUCOSE

• Embden Meyerhoff

• Pyruvate Dehydrogenase

• TCA cycle

• NET FOR EVERYTHING

NET TOTAL

1 Glucose —> 6 CO2 + 10 NADH + 2 FADH + 2 ATP + 2 GTP

Electron transport and Oxidative Phosphorylation

• What is the main purpose of it?

• Where is it done?

• What are the materials it used to undergo this process?

• What does this also help create?

In order to have these pathways operate continuously, it is

necessary to reconvert (oxidize) the NADH and FADH2 to

NAD+ and FAD+.

This can be done by the electron transport chain (ETC), which

uses some terminal electron acceptor to oxidize NADH, FADH2 , and other electron donors.

Many more ATPs are synthesized by the oxidation of NADH and FADH2

Electron Transport Chain

The mitochondrial electron transport chain is a series of electron carriers that transfer electrons from FADH and NADH to a terminal electron acceptor (O2)

The electron flow from carriers with lower reduction potentials. to one with greater E0

Electron Transport Chain - Redox Pairs

• Each carrier is reduced and then re-oxidized

• Carriers are constantly recycled

• The difference in reduction potentials electron carriers, NADH and O2 is large, resulting in release of great deal of energy

How does ETC generate proton motive force?

• In eukaryotes

• Location

• Items involved

In eukaryotes the e- transport chain carriers are in the inner mitochondrial membrane, connected by coenzyme Q and cytochrome c

e- transport is accompanied by proton movement across inner

mitochondrial membrane

Bacterial and Archaeal ETC’s

• How do they differ from euk

• Location

• Conformational differences

Located in the plasma membrane

• Some resemble mitochondrial ETC, but many are different

  • Different electron carriers

  • May be branched

  • may be shorter

  • may have lower phosphorus / oxygen ratio

Paracoccus dentrificans

• Gram-?

• What kind of interaction with air?

• What are differences in energy sources?

• Facultative, Gram-negative soil bacterium

• Very versatile metabolically

• In presence of O2 , uses aerobic respiration

– similar electron carriers and transport mechanism as mitochondria

– protons transported across the cytoplasmic membrane to periplasmic space

– can use methanol as carbon/energy source instead of glucose

Electron Transport Chain of E.coli

• Differences vs euk

• Different ways of undergoing ETC

Different array of cytochromes used than in mitochondria

Branched Pathway(s)

Upper branch (bd)

– stationary phase or low aeration (higher affinity for O2 , lower efficiency)

Lower Branch

• exponential phase and high aeration

Like two engines, different terminal oxidases to transfer electrons to oxygen. The upper branch is used when O2 is very low. With high affinity and low efficiency

Lower branch is used when there is high oxygen. Lower affinity of oxygen, but it is much more efficient.