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Notes on Microbial Metabolism

Microbial Metabolism and Its Importance

• Metabolism is the total of all chemical reactions in the cell and is divided into two parts

– catabolism

• fueling reactions

• energy-conserving reactions

• provide ready source or reducing power (electrons)

• generate precursors for biosynthesis

– anabolism

• the synthesis of complex organic molecules from simpler ones

• requires energy from fueling reactions

Microbial Metabolism

• Microbes have representatives in all five major nutritional types

• Contribute to cycling of elements in ecosystems

– some cycling reactions performed only by microbes

Microbial Cells Must Do Work

• Chemical work

– synthesis of complex molecules

• Transport work

– take up of nutrients, elimination of wastes, and maintenance of ion balances

• Mechanical work

– cell motility and movement of structures within cells

The Laws of Thermodynamics

• Thermodynamics

– a science that analyzes energy changes in a collection of matter called a 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: Physical and chemical processes proceed in such a way that
the disorder of the universe increases to the maximum possible

– entropy = amount of disorder in a system

Free Energy and Reactions

G = H - TS

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

• Used to indicate if a reaction will proceed spontaneously

– if G is negative, reaction is spontaneous


– if G is positive, reaction is not spontaneous

• G

– free energy change

– amount of energy available to do work

• H

– change in enthalpy (heat content)

• T

– temperature in Kelvin

• S

– change in entropy

Standard Free Energy Change (Go)

• Free energy change defined at standard conditions of concentration, pressure, temperature,


and pH

• Go´

– standard free energy change at pH 7

– directly related to Keq

Go´ = -2.303RT•logKeq

Exergonic reactions - Go´ is negative / (reaction proceeds spontaneously)

Endergonic reactions - Go´ is positive / (reaction will not proceed spontaneously)

Role of ATP in Metabolism

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

• ATP +H2O ADP + Pi + H+

– Go´ = - 7.3 kcal/mol

• Guanosine 5’-triphosphate, cytosine 5’-triphosphate and uridine 5’-triphosphate also supply


some energy

Oxidation-Reduction (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 conserved and used to form ATP

– the more electrons a molecule has, the more energy rich it is

Electron Transport Chain (ETC)

• Electron carriers organized into ETC

– first electron carrier having the most negative E’o

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

– first carrier is reduced and electrons moved to the next carrier and so on

Electron Carriers
• 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

Main Electron Carriers

• 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

• NAD - nicotinamide adenine dinucleotide

• NADP - nicotinamide adenine dinucleotide phosphate

Additional Electron Carriers

• FAD -

– flavin adenine dinucleotide

• FMN

– flavin mononucleotide

– riboflavin phosphate

• Coenzyme Q (CoQ)

– a quinone

– also called ubiquinone

Even More Electron Carriers

• Cytochromes

– use iron to transfer electrons

• iron is part of a heme group

• Nonheme iron-sulfur proteins

– e.g. ferrodoxin

– use iron to transport electrons

• iron is not part of a heme group

Biochemical Pathways

• Enzymes can be linked together to form pathways

• Pathways can be varied

– linear

– cyclic

– Branching

• Pathways often overlap/feed into each other

– creates complex networks

– dynamic pathways can be used to monitor changes in metabolite levels (flux)

Enzymes and Ribozymes Speed Up Cellular Chemical Reactions

- additional readings in Prescott***


• Metabolism overall is the sum of numerous biochemical reactions, most of which involve
enzymes

• Metabolism involves redox reactions through the transfer of electrons from one carrier to
another

• Electron transport is coupled to ATP synthesis

• Metabolisms can be anabolic or catabolic

• Metabolisms is organized into pathways

Catabolism

Requirements for Carbon, Hydrogen, and Oxygen

• Often satisfied together

– carbon source often provides H, O, and electrons

• Heterotrophs

– use organic molecules as carbon sources which often also serve as energy source

– can use a variety of carbon sources

• Autotrophs

– use carbon dioxide as their sole or principal carbon source

– must obtain energy from other sources

Nutritional Types of Organisms

• Based on energy source

– phototrophs use light

– chemotrophs obtain energy from oxidation of chemical compounds

• Based on electron source

– lithotrophs use reduced inorganic substances

– organotrophs obtain electrons from organic compounds

Classes of Major Nutritional Types

• Majority of microorganisms known

– photolithoautotrophs (photoautotrophs)

– chemoorganoheterotrophs (chemoheterotrophs)

• majority of pathogens

• Ecological importance

– photoorganoheterotrophs

– chemolithoautotrophs

– chemolithotrophs

Fueling Reactions

• Despite diversity of energy, electron, and carbon sources used by organisms, they all have the
same basic needs

– ATP as an energy currency

– reducing power to supply electrons for chemical reactions

– precursor metabolites for biosynthesis


Chemoorganotrophic Fueling Processes

• Also called chemoheterotrophs

• Processes

– aerobic respiration

– anaerobic respiration

– fermentation

Chemoorganic Fueling Processes – Respiration

• Most respiration involves use of an electron transport chain

– Aerobic respiration

• final electron acceptor is oxygen

– Anaerobic respiration

• final electron acceptor is different exogenous acceptor such as NO3-, SO42-, CO2,
Fe3+, or SeO42-

– As electrons pass through the electron transport chain to the final electron acceptor, a
proton motive force (PMF) is generated and used to synthesize ATP

Chemoorganic Fueling Processes – Fermentation

• Uses an endogenous electron acceptor

– usually an intermediate of the pathway used to oxidize the organic energy source e.g.,
pyruvate

• Does not involve the use of an electron transport chain nor the generation of a proton motive
force

• ATP synthesized only by substrate-level phosphorylation

Energy Sources

• Many different energy sources are funneled into common degradative pathways

• Most pathways generate glucose or intermediates of the pathways used in glucose metabolism

• Few pathways greatly increase metabolic efficiency

Catabolic/Amphibolic Pathways

• Enzyme catalyzed reactions whereby the product of one reaction serves as the substrate for the
next

• Pathways also provide materials for biosynthesis

• Amphibolic pathways

– function both as catabolic and anabolic pathways

– important ones

• Embden-Meyerhof pathway

• pentose phosphate pathway

• tricarboxylic acid (TCA) cycle

Aerobic Respiration

• Process that can completely catabolize an organic energy source to CO2 using

– glycolytic pathways (glycolysis)


– TCA cycle

– electron transport chain with oxygen as the final electron acceptor

• Produces ATP (most of it indirectly via the activity of the electron transport chain), and high
energy electron carriers

The Breakdown of Glucose to Pyruvate

• Three common routes

– Embden-Meyerhof pathway

– pentose phosphate pathway

– Entner-Duodoroff pathway

The Embden-Meyerhof Pathway

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

• The most common pathway for glucose degradation to pyruvate in stage two of aerobic
respiration

• Function in presence or absence of O2

• Two phases

– Six carbon phase

– Three carbon phase

The Tricarboxylic Acid Cycle

• Also called citric acid cycle and Kreb’s cycle

• Common in aerobic bacteria, free-living protozoa, most algae, and fungi

• Major role is as a source of carbon skeletons for use in biosynthesis

Summary TCA Cycle

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

– 2 molecules of CO2

– 3 molecules of NADH

– one FADH2

– one GTP

Electron Transport and Oxidative Phosphorylation

• Only 4 ATP molecules synthesized directly from oxidation of glucose to CO2

• Most ATP made when NADH and FADH2 (formed as glucose degraded) are oxidized in electron
transport chain (ETC)

Electron Transport Chains

• The mitochondrial electron transport chain (ETC) = a series of e- carriers, operating together to
transfer e- from NADH and FADH2 to a terminal e- acceptor, O2

• E- flow from carriers with more negative reduction potentials (E0) to carriers with more positive
E0

Electron Transport Chain – Redox Pairs

• Each carrier is reduced and then reoxidized

• Carriers are constantly recycled


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

Electron Transport Chain Creates Proton Motive Force

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

• E- transfer accompanied by proton movement across inner mitochondrial membrane

Bacterial and Archaeal ETCs

• Located in plasma membrane

• Some resemble mitochondrial ETC, but many are different

– different electron carriers

– may be branched

– may be shorter

– may have lower P/O ratio

Paracoccus denitrificans

• Facultative, soil bacterium

• Extremely versatile metabolically

• Under oxic conditions, uses aerobic respiration

– similar electron carriers and transport mechanism as mitochondria

– protons transported to periplasmic space rather than


inner mitochondrial membrane

– can use one carbon molecules instead of glucose

Chemiosmotic Hypothesis

• The most widely accepted hypothesis to explain oxidative phosphorylation

– protons move outward from the mitochondrial matrix as e- are transported down the
chain

– proton expulsion during e- transport results in the formation of a concentration gradient


of protons and a charge gradient

– the combined chemical and electrical potential difference make up the proton motive
force (PMF)

PMF Drives ATP Synthesis

• Diffusion of protons back across membrane (down gradient) drives formation of ATP

• ATP synthase

– uses PMF down gradient to catalyze ATP synthesis

– functions like rotary engine (conformational changes)

ATP Yield During Aerobic Respiration

• Maximum ATP yield can be calculated

– includes P/O ratios of NADH and FADH2

– ATP produced by substrate level phosphorylation

• The theoretical maximum total yield of ATP during aerobic respiration is 38

– the actual number closer to 30 than 38


Anaerobic Respiration

• Uses electron carriers other than O2

• Generally yields less energy because E0 of electron acceptor is less positive than E0 of O2

An Example of Anaerobic Respiration

• Dissimilatory nitrate reduction

– use of nitrate as terminal electron acceptor, making it unavailable to cell for assimilation
or uptake

• Denitrification

– reduction of nitrate to nitrogen gas

– in soil, causes loss of soil fertility

Fermentation

• Oxidation of NADH produced by glycolysis

• Pyruvate or derivative used as endogenous electron acceptor

• Substrate only partially oxidized

• Oxygen not needed

• Oxidative phosphorylation does not occur

– ATP formed by substrate-level phosphorylation

Chemolithotrophy

• Carried out by chemolithotrophs

• E- released from inorganic molecule energy source

– transferred to terminal e- acceptor by ETC

• ATP synthesized by oxidative phosphorylation

Energy Sources

• Bacterial and archaeal species have specific electron donor/acceptor preferences

• Much less energy is available from oxidation of inorganic molecules than glucose due to more
positive redox potentials

Three Major Groups of Chemolithotrophs

• Have ecological importance

• Several bacteria and archaea oxidize hydrogen

• Sulfur-oxidizing microbes

– hydrogen sulfide (H2S), sulfur (S0), thiosulfate (S2O32-)

• Nitrifying bacteria oxidize ammonia to nitrate

Metabolic Flexibility of Chemolithotrophs

• Many switch from chemolithotrophic metabolism to chemoorganotrophic metabolism

• Many switch from autotrophic metabolism (via Calvin cycle) to heterotrophic metabolism

Phototrophy

• Photosynthesis

– energy from light trapped and converted to chemical energy


– a two-part process

• light reactions: light energy is trapped and converted to chemical energy

• dark reactions: energy produced in the light reactions is used to reduce CO2 and
synthesize cell constituents

Light Reactions in Oxygenic Photosynthesis

• Photosynthetic eukaryotes and cyanobacteria

• Oxygen is generated and released into the environment

• Most important pigments are chlorophylls

Oxygenic Photosynthesis

Noncyclic

electron flow – ATP + NADPH made (noncyclic photophos-

phorylation)

Cyclic electron flow – ATP made (cyclic photophos-

phorylation)

Anoxygenic photosynthesis

The Light Reaction in Anoxygenic Photosynthesis

• H2O not used as an electron source; therefore O2 is not produced

• Only one photosystem involved

• Uses bacteriochlorophylls and mechanisms to generate reducing power

• Carried out by phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria

Archaerhodopsin-Based Phototrophy

• Some archaea use a type of phototrophy that involves archaerhodopsin

– a membrane protein

– functions as a light-driven proton pump

• Similar molecules/methods are found in some bacteria and eukaryotic microbes, as well

• A proton motive force is generated

• An electron transport chain is NOT involved

Anabolism

Anabolism Uses Energy From Catabolism

• Energy from catabolism is used for biosynthetic pathways

– using a carbon source and inorganic molecules, organisms synthesize new organelles
and cells

– antibiotics inhibit anabolic pathways

– a great deal of energy is needed for anabolism

Principles Governing Biosynthesis

• Macromolecules are synthesized from limited number of simple structural units (monomers)

– saves genetic storage capacity, biosynthetic raw material, and energy

• Many enzymes do double duty

– Many enzymes used for both catabolic and anabolic processes


– saves materials and energy

• Catabolic and anabolic pathways are not identical as some enzymes function in only one
direction

Precursor Metabolites

• Generation of precursor metabolites is critical step in anabolism

• Carbon skeletons are used as starting substrates for biosynthetic pathways

– examples are intermediates of the central metabolic pathways

– most are used for the biosynthesis of amino acids

Some examples of anabolic pathways

• Synthesis of

– Carbohydrates

– Proteins

– Lipids

– nucleic acids

• Carbon dioxide fixation

• Nitrogen fixation

Biosynthesis of Sugars & Polysaccharides

• Synthesis of biomolecules

• CAC plays a major role

• Polysaccharides

• Anabolism – synthesis of polysaccharides (esp. organisms in nature)

• Gluconeogenesis - production of glucose from nonsugar precursors

Biosynthesis of Nucleotides

• Purines and pyrimidines - biosynthesized using carbon from several sources

a) Precursors of purine skeleton

b) Inosinic acid – all purine nucleotides

c) Orotic acid – pyrimidine skeleton

d) Uridylate – all pyrimidine nucleotides

Biosynthesis of Fatty Acids and Lipids

• Fatty acids are synthesized two carbons at a time and then attached to glycerol to form lipids

• varies between species and at different temperatures

• lower temps: shorter, more unsaturated

• higher temps: longer, more saturated

• can be unsaturated, branched, or contain odd numbers of carbon atoms

• In Bacteria and Eukarya, assembly of lipids involves addition of fatty acids to glycerol.

• In Archaea, lipids contain phytanyl side chains instead of fatty acids.