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Home , Autotroph, Electron acceptor, Electron donor, Heliobacteria, Heterotroph, Microbial metabolism

Biol 3400 Tortora et al- Chap 5

Microbial

I. Metabolism • Metabolism is all of an 's chemical processes (an emergent property that arises from interactions of molecules in the orderly environment of the ) • Metabolism is very important for the management of cellular material and resources

Metabolic reactions • Metabolic reactions are organized into pathways of enzyme controlled chemical reactions. • Cells need a supply of molecules and energy • Cells need to get rid of waste products

Catabolic pathways • Break down complex molecules into simple molecules • Energy stored in complex molecules is made available to do work or transformed into readily usable chemical forms (i.e., ATP) • small molecules resulting from the catabolism of complex energy rich molecules may be used by the cell to build new molecules

• e.g.,

Energy stored in compounds can be used to perform cellular work • mechanical - movement of cilia, chromosomes, organelles • transport - movement of substances across membranes • chemical - endergonic reactions

Anabolic pathways • Use energy for the of complex molecules from simple molecules. • Energy is obtained from usable chemical forms of energy (i.e., ATP) produced during catabolic processes or from energy released during catabolic processes

• e.g., synthesis of macromolecules

Note: some pathways may function both catabolically and anabolically – these pathways are known as amphibolic pathways

Review the Enzymes Section on your own (Pages 115 to 121)

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II. Metabolic diversity among • Life is based on organic molecules made of skeletons • and are important elements of organic compounds • Electrons are needed i) for processes that provide energy (e.g., movement of electrons along energy transport chains and during oxidation reduction reactions) for cellular work and ii) to reduce molecules during biosynthesis • Molecules that serve as a source of carbon may also provide a source of oxygen and hydrogen • Microbes show an incredible ability to use organic molecules as carbon sources

Organisms can be classified based on their sources of carbon, energy and electrons

Carbon Source o – CO2 is the sole or principal carbon source o – reduced, preformed, organic molecules from other Energy Source o – o – oxidation of organic or inorganic compounds Electron Source o – reduced inorganic chemicals o – Organic molecules

Major Nutritional Types of Microorganisms Carbon Energy Electron Examples Source source source

1. Photolithoautotrophy CO2 Light Inorganic , • aka - Photoautotrophs e- donor Purple

2. Photoorganoheterotrophy Organic Light Organic Purple nonsulfur • aka - carbon but e- donor bacteria and Green CO2 may be nonsulfur bacteria used

3. Chemolithoautotrophy CO2 Inorganic Inorganic Sulfur oxidizing • aka - Chemoautotrophs chemicals e- donor bacteria, ,

4. Chemolithoheterotrophy Organic Inorganic Inorganic Some sulfur carbon but chemicals e- donor oxidizing bacteria CO2 may be Used

5. Chemoorganoheterotrophy Organic Organic Organic Most nonphotosynthetic • aka – Chemoheterotrophs carbon chemicals e- donor microbes including often the often the most , fungi, same as same as many and many C-source C-source

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III Heterotrophic (Chemoorganotrophic) Metabolism • Conversion of organic substrate molecules to end products by a metabolic pathway that releases sufficient energy for it to be coupled to the formation of ATP. • Chemoorganotrophs have three options for generating ATP from organic molecules; the used differentiates these processes: i) aerobic respiration, ii) and iii)

i. Respiration • An external terminal electron acceptor is present and is not derived from the organic substrate • Involves the activity of an , motive force (PMF) is generated and ATP produced predominantly by oxidative a) Aerobic - O2 is the terminal electron acceptor. C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + (ATP + Heat)

- 2- b) Anaerobic - compounds other than O2 serve as electron acceptor (e.g., NO3 , SO4 , CO2, fumarate,…)

*Some microbes can carry out both aerobic and anaerobic respiration – dependent upon the conditions

ii. Fermentation • An external terminal electron acceptor is absent • Fermentation does not use an electron transport chain or the generation of a PMF • are internally balanced oxidation-reduction reactions – i.e., the terminal electron acceptor is derived from the initial substrate or (e.g., ) • The terminal electron acceptor is required to balance reactions • Net result is energy production and an internally balanced redox reactions • ATP produced predominantly by substrate-level phosphorylation

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A. Respiration

1. Glycolytic pathways • Breakdown of sugars to pyruvate and similar intermediates • Some production of ATP (substrate-level phosphorylation) and reducing power (reduced coenzymes; NADH) • Several pathways by which a cell can break down a sugar (sugars are the major substrates of catabolic energy releasing reactions used in heterotrophic metabolism). • Glycolytic pathways are typically anoxic processes that do not require oxygen

• The end-product of is commonly pyruvate

COOH | C = O | CH3

Glycolytic pathways i). Embden-Meyerhof pathway (EMP) • Most common pathway - Central metabolic pathway for eukaryotic cells and many bacteria

Net reaction 10 enzymatic steps Glucose + 2 ADP + 2 Pi + 2 NAD+→ 2 pyruvate + 2 ATP + 2 NADH + 2 H2O

• ATP production - Substrate level phosphorylation • Phosphofructokinase is key enzyme in regulating this process, catalyzing the conversion of fructose – 6-P to fructose 1,6-bisphosphate

ii). Entner-Doudoroff pathway • Mainly used by Gram negative bacteria and a few other Gram-negative bacteria as well as some Archaea • Lacks 6-phosphofructokinase (i.e., a key enzyme in Embden-Meyerhof pathway)

Net reaction 9 enzymatic steps Glucose + ADP + Pi + NADP+ + NAD+ → 2 pyruvate + 1 ATP + NADPH + NADH + 1 H2O

*NADPH is usually used in biosynthetic pathways

4 Biol 3400 Tortora et al- Chap 5 iii). Pentose pathway • Can occur at the same time as the Embden-Meyerhof or the Entner-Doudoroff pathways • connects the metabolism of 6-C and 5-C sugars • Consumes 1 ATP • Products = reducing power (NADPH) and small molecules required for biosynthesis • 5-C sugars produced (e.g., ribose 5-phosphate; xylulose 5-phosphate) • Erythrose 4-phosphate is used to synthesize aromatic amino acids and vitamin B6 • Intermediates of the pathway may be fed into the EMP to produce ATP

iv). Methylglyoxal pathway • Alternative to Embden-Meyerhof pathway during conditions of low phosphate availability • Consumes 2 ATP but produces pyruvate that can be used to generate ATP

2. Oxidation of pyruvate to 3 CO2. i) Initial Step + Pyruvate + NAD + CoA → Acetyl-CoA + NADH + CO2

• Three step process mediated by multienzyme pyruvate dehydrogenase complex • Acetyl CoA is a very unstable and reactive product. • Acetyl CoA feeds it's → TCA cycle • , fatty acids and amino acids may be converted into acetyl CoA during aerobic respiration

ii) Tricarboxylic acid cycle (TCA cycle; also known as , Krebs cycle) • Discovered by Hans Krebs - 1930s • 8 steps involved in the TCA cycle • 2 C enter in a relatively reduced form – acetate and two different C leave in a completely oxidized form (CO2) • acetate joins the cycle by enzymatic addition to oxaloacetate (4 C) → formation of citrate • cyclical process resulting in the regeneration of oxaloacetate by the of citrate and evolution of 2 CO2 per acetate + • oxidation steps (transfer of electrons) of one acetate results in reduction of 3 NAD to 3 - NADH and 1 FAD to FADH2 (like NADH it donates its e to the electron transport chain but at a lower energy level) • One step for the production of GTP (substrate level phosphorylation; GTP can be converted to ATP)

Review with Figure 5.13

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+ pyruvate + 4 NAD + FAD → 3 CO2 + 4 NADH + 1 FADH2 + 1 GTP

• TCA cycle source of key biosynthetic intermediates e.g., oxaloacetate and α-ketoglutarate are precursors to a number of amino acids acetyl-CoA → starting material for biosynthesis

3. Oxidative phosphorylation • Reducing power (NADH and FADH2) is used to generate a proton gradient (proton motive force) • NADH - FADH2 are oxidized – electron transport carrier are reduced and in the process H+ are moved across the plasma membrane () or inner mitochondrial membrane (). This results in a proton gradient or proton motive force across the membrane. The movement of H+ across the membrane is not completely understood

Chemiosmosis • Proton Gradient driving ATP synthesis • Peter Mitchell (Nobel prize in in 1978)

ATP synthase (ATPase) • F0 subunit – multimeric membrane spanning proton conducting channel • F1 – multimeric headpiece → inside of the membrane • Catalyze ADP + Pi → ATP (oxidative phosphorylation) • Highly conserved throughout all domains of life

• Can also drive reverse reaction ATP → ADP + Pi • explains why some obligate fermenters have ATPase - creates proton gradients that can be used to drive transport and flagella

Much evidence supporting the chemiosmotic theory comes from studies using chemicals that inhibit the aerobic synthesis of ATP

• inhibitors, (e.g., CO, cyanide) inhibit ATP synthesis by blocking the electron flow – preventing oxidative phosphorylation • uncouplers (e.g., dinitrophenol, dicumarol) allow to cross the membrane without activating ATP synthase and prevent ATP synthesis without affecting electron transport

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Net ATP Production in Aerobic Respiration

Substrate-level phosphorylation

Glycolysis (EMP) 4 ATP -2 ATP

Citric acid cycle (2 GTP →2 ATP) 2 ATP

Oxidative phosphorylation

2 NADH (from Glycolysis) 6 ATP

if membrane impermeable to NADH then e- relayed across membrane at the expense of 2 ATP 0-2 ATP

8 NADH (from TCA cycle) 24 ATP 2 FADH2 (from TCA cycle) 4 ATP 36-38 ATP

Energy Storage?

4. Anaerobic respiration • terminal electron acceptors other than O2 - 3+ 2- e.g., NO3 , Fe , SO4 , CO2 – Table 9.1 • Used by many bacteria and archaea • These compounds have reduction potentials (E0’) that are less electropositive than O2 and therefore less energy is released during anaerobic respiration • Some organisms can use both aerobic and anaerobic respiration depending if O2 is present

What if oxygen or other terminal electron acceptors are absent?

B Fermentation • Bacteria and Archaea can survive in environments lacking O2, and other types of terminal - 2- electron acceptors (e.g., NO3 or SO4 ) • Alternative heterotrophic processes producing energy and molecules needed for growth • ATP generated by substrate-level phosphorylation • Coupled with glycolysis and involve the anaerobic metabolism of pyruvate • Three important themes operating during microbial fermentation are: 1. NADH is oxidized back to NAD+ 2. Substrate is electron donor and a derivative of substrate is the ultimate electron acceptor. 3. Oxidative phosphorylation cannot operate

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Unlike respiration, the substrate is only partially oxidized during fermentation and therefore: • energy yields are much lower • substrate use is less efficient. e.g., Lactate production during fermentation → ΔG = -58 kcal/mole The complete oxidation of glucose during aerobic respiration → ΔG = -686 kcal/mole

• Organisms capable of both respiration and fermentation will use the more energetically favourable respiration if conditions permit. If there is no available external electron acceptor, then they will use fermentation. • Some organisms are obligate fermenters (can only ferment carbon compounds) while others are facultative fermenters (i.e., have citric acid cycle and electron transport chain)

Example Fermentation pathway

Glycolytic pathway + + Substrate + 2 ADP + 2 Pi + 2 NAD → 2 pyruvate + 2 ATP + 2 NADH→ endproduct + 2 NAD

The fermentation pathway achieves the following • Balances the redox reactions • Regenerates oxidizing power (oxidized coenzymes) – NAD+

Different Fermentation Pathways occur in bacteria, archaea and eukaryotic cells and produce different end products. • Fermentation pathways are generally named after end products. • The type of fermentation end product produced is often used to identify a particular (see below and Pages 137 – 138 and Clincial focus on page 144)

Common fermentation pathways i. homolactic acid fermentation ii. heterolactic acid fermentation iii. fermentation iv. propionic acid fermentation v. mixed acid fermentation vi. butanediol fermentation vii. fermentation viii. fermentation ix. fermentation

8 Biol 3400 Tortora et al- Chap 5 i) Lactic acid fermentation • pyruvate is reduced to lactic acid • reduction of NADH to NAD+ is coupled to this process • Lactic acid bacteria

If the Embden-Meyerhof pathway is used then Homofermentative or homolactic fermentation occurs

Glucose + 2 ADP + 2 Pi → 2 lactate + 2 ATP

Lactic acid → CH3 - C(HOH) - COOH

• lactate dehydrogenase catalyzes conversion of pyruvate to lactate and vice versa

• NB in the dairy industry - dairy products - cheese, yogurt, acidophilus milk • silage production • dental caries - Streptococcus mutans • Streptococcus, Lactococcus, Pediococcus, Lactobacillus

If the Pentose phosphate pathway is used then heterofermentative or heterolactic fermentation occurs

Glucose + ADP + Pi → lactate + ethanol + CO2 + ATP

• ATP is produced during the conversion of pentose sugars to lactate

• sausage and sauerkraut • Leuconostoc and Lactobacillus

ii) or ethanol fermentation

Overall reaction Glucose + 2 Pi + 2 ADP → 2 ethanol + CO2 + 2 ATP

Key steps Glucose → pyruvate → acetaldehyde + CO2 → ethanol

Enzymes involved • pyruvate decarboxylase catalyzes conversion of pyruvate → acetaldehyde + CO2

• alcohol dehydrogenase catalyzes conversion of acetaldehyde → ethanol

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• Alcohol fermentation is used in the production of ethanol for beverages, solvent and fuel • Many yeast such as Saccharomyces cerevisiae and a few bacteria such as Zymomonas mobilis are capable of carrying out the alcohol fermentation pathway

iii) Propionic Acid fermentation • Utilize lactic acid and produce propionic acid (CH3CH2COOH) • Propionibacterium • Used in Swiss cheese production to give the cheese its distinct flavour

iv) Mixed Acid Fermentation • Members of the family Enterobacteriaceae carry out fermentation of pyruvate into a variety of organic acids including acetate, lactate, succinate, formate. • Other endproducts include H2, CO2, ethanol • NADH is reoxidized to NAD+ • Formation of acetate is accompanied by substrate-level phosphorylation • Mixed acid endproducts are detected via the Methyl Red test because of an overabundance of organic acids produced relative to neutral endproducts

Methyl red is Yellow at neutral pH and red if pH is less than 5

v) Butanediol fermentation • Butanediol (CH3-CHOH-CHOH-CH3) is a neutral product + • NADH is oxidized to NAD and CO2 is also produced • No additional ATP is generated • Erwinia, Klebsiella, Enterobacter and Serratia • Acetoin intermediate can be detected by Voges Proskauer test

vi) Butyric acid Fermentation • Clostridial species • Important process for solvent production (e.g., Acetone, Butanol, Ethanol, isopropanol)

Note: Fermentation yields little usable energy compared to aerobic and anaerobic respiration

Why?

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IV. Autotrophy • Self sufficient organisms that are capable of growing by metabolizing simple inorganic compounds or by

1. Chemolithotrophy (Chemoautotrophy) • Bacteria and Archaea that carry out respiration by oxidizing inorganic compounds • May or may not couple oxidation of inorganic compounds with reduction of coenzyme + - (NAD ) e.g., H2, H2S, FeS, NH3, NO2

• These organisms play an important role in biogeochemical recycling - • Use O2 or other compounds (NO3 ) as the terminal electron acceptor

Examples i) Hydrogen oxidation

• Carry a unique enzyme called - Alcaligenes eutrophus • generates protons and electrons which are used in the membrane associated electron transport chain

Homoacetogenic bacteria couple H2 oxidation with reduction of CO2 to form acetate • final step involves conversion of acetyl-CoA to acetate results in sufficient free energy for ATP formation - Clostridium aceticum

Extreme thermophilic Archaea - Pyrodictium - deep around thermal vents • H2 oxidation coupled with sulfur reduction ii) = and oxidation • Very important organisms in nitrogen cycling - • oxidize ammonia or nitrite (NO2 ) to produce energy by

+ - NH4 → NO2 - - NO2 → NO3 ii) Sulfur and oxidation

Thiobacillus and - • oxidize H2S, S, and thiosulfate (S2O3 ) to produce ATP by substrate level phosphorylation and chemiosmosis

Thiobacillus ferrooxidans • oxidizes reduced sulfur & iron (Fe3+ → Fe2+)

• problems and use in recovery processes (acid leaching).

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2. Photoautotrophy

Photophosphorylation - conversion of light energy into chemical energy (ATP)

• Light sensitive pigments (, , phycobilins and ) capture light energy and release electrons. • Electrons are transferred through a series of membrane bound electron carriers • The whole system is called a photosystem

1. Oxygenic phototrophy • , cyanobacteria and prochlorobacteria • ATP and O2 are produced as a result of oxidative phosphorylation • Similar to green have two photosystems PS I and PSII • cyclic and noncyclic oxidative phosphorylation

2. Anoxygenic phototrophy • Capture of light energy and production of ATP in the absence of O2. • Anaerobic green and purple bacteria and (use reduced sulfur compounds like H2S as a source of electrons) • Only PSI - (photons excite bacteriochlorophyll and are cycled back through a series of electron carriers) ATP produced by cyclic oxidative phosphorylation. • No need for a external electron acceptor or donor

3. Rhodopsin-based phototrophy • Oxygenic and anoxygenic phototrophy have not been observed in Archaea • Archaea capture light energy using a membrane called , that is a light driven proton pump – creates a proton gradient across the plasma membrane

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