5: Bacterial Metabolism

I. Metabolism

(adapted from Dr. L. Heffernan CSU Sacramento)

A. Metabolism: all chemical reactions in cell, both catabolic and anabolic reactions.

1. Catabolic reactions: degredative reactions, large substances broken down into smaller substances with energy release

a. Some energy is captured in ATP/other high-energy molecules /proton-gradients ; cell requires energy for biosynthesis (anabolism), motility, active transport.

b. Smaller substances can be used as building blocks in anabolic reactions

2. Anabolic reactions: biosynthetic reactions, smaller substances joined together with net input of energy (ATP from catabolic reactions) to synthesize larger substances for cell structure/products.

3. ATP=adenosine triphosphate links anabolic and catabolic reactions

B. Energy capture via Electron Transfer

1. Electrons carry energy and can transfer energy from one molecule to another

2. electrons can be transferred as “naked” electrons or as part of hydrogen atoms

(H= H+ + electron), therefore hydrogen atom transfer represents electron transfer.

3. Electron transfer is involved in ATP synthesis

C. Types of electron transfer

1.oxidation: loss of electrons to an electron acceptor

2. reduction: gain of electrons from an electron donor

3. redox reactions: one substance loses electrons and another substance accepts those electrons

a. substance losing electrons is oxidized and is called an electron donor or a reducing agent

b. substance gaining electrons is reduced and is called an electron acceptor or an oxidizing agent

c. example of redox reaction: complete oxidation of glucose via glycolysis and aerobic respiration. Glucose is oxidized and acts as a reducing agent. O2 is reduced and acts as an oxidizing agent. Electrons from glucose are eventually donated to O2.

oxidation---->

C₆H₁₂O₆+ 6O₂----> 6CO₂+ 6H₂O + Energy (heat+ATP+proton gradient)

reduction------->

4. Redox reaction involving electron/H carriers NAD+ and FAD ( coenzymes):nicotinamide adenine dinucleotide and flavin adenine dinucleotide

a. NAD+ can carry additional hydrogen atom and electron: NAD+ + 2H -> NADH + H+

b. FAD can carry 2 H atoms: FAD + 2H--> FADH2

c. NADH and FADH2carry high energy electrons to electron transport chain in bacterial cell membrane (or inner mitochondrial membrane in eukaryotes)

D. Metabolic reactions in cells requires

1. Metabolic reactions occur too slowly in cells to sustain life

2. Increasing temperature to increase reaction rates would kill cells (denaturation proteins/DNA)

3. Therefore all cells have protein catalysts called enzymes to increase reaction rates

a. catalysts: substances which lower the activation energy of chemical reactions

-activation energy: energy required to start chemical reactions

-see board/text/Ppt diagrams

catabolic reaction

energy

___________________________

progress of reaction

E. Enzyme structure and function

1. Enzymes are protein catalysts made up of specific amino acid sequences which dictate folding and 3- dimensional shape/functional shape

a. folding creates an active site where enzyme binds its specific substrate

b. substrate: the substance the enzyme binds and “changes’

c. “lock and key” vs “induced fit” of substrate into enzyme active site

d. enzyme(E) and substrate(S) bind and form enzyme:substrate complexes; bonds of substrate are rearranged, product(P) is formed. Product is released from enzyme active site.

E + S --> E:S--> E + P

e. enzyme is not “used up” and can be used over and over again

2. Enzymes are proteins and can be denatured; loss of 3-D shape=loss of function (heat, pH etc)

3. Enzymes are substrate specific. Enzymes generally catalyze only a single reaction, act on a single substrate or closely related substrates (determined by shape,size and charge distribution of substrate and ability to fit into active site)

4. Enzymes classified according to function. examples:

isomerases aldehyde <-> ketone group

dehydrogenase or reductase: removal and transfer of electrons from one substance to another. Some require coenzymes: ex NAD+ and FAD act as electron/H carriers

transferase: transfers a side chain

kinase: a phosphotransferase, transfers phosphate group from ATP to another molecule ATP + R--> ADP + R-P

5. Enzyme suffix “-ase”; named according to substrate and function ex. glucose-6-phosphate dehydrogenase= enzyme which dehydrogenates, removes hydrogen , from glucose-6-phosphate

6. Enzyme inhibition: competitive vs non competitive inhibition/allosteric control.

-Feedback or endproduct inhibition.

- antimicrobial enzyme inhibitors: e.g.

a. beta-lactam antibiotics: irreversible competitive inhibitors of bacterial transpeptidases

b. -fluoroquinolones and bacterial gyrase

c. -trimethorpim-sulfa (e.g Bactrim) combo’s as sequential competitive inhibitors of folic acid synthesis enzymes

-folic acid-> coenzyme required for nucleic acid synthesis

F. Metabolic Pathways: series of chemical reactions where product of first reaction is substrate for subsequent enzyme catalyzed step

1. Each step in a metabolic pathway requires a specific enzyme

enzymes a b c d

pathway A---->B----->C--->D--->E (endproduct)

substrates/intermediates

2. If one enzyme in pathway is missing or defective, cell cannot form final product of pathway

nor any intermediates “downstream” from missing enzyme

G. Coenzymes and Cofactors: substances required by some enzymes for full activity

1. enzyme without cofactor/coenzyme=apoenzyme; with cofactor/coenzyme=holoenzyme

2. coenzymes: organic molecules (NAD+, FAD, cytochromes), loosely associated with enzyme. Often formed from vitamins ex niacin->NAD+ nicotinamide adenine dinucleotide. NAD+ is important in many redox reactions. NAD+ (low energy) +2H--> NADH (high energy) + H+

3. cofactors: inorganic ex magnesium, calcium, zinc. Often improve fit of substrate for a.s.

H. Metabolic processes of chemoheterotrophs: energy source=oxidation of organic substances, carbon source=preformed organic molecules

1. most bacteria, all pathogens

2. metabolic processes: glycolysis, fermentation, aerobic/anaerobic respiration

3. Glycolysis: oxidation of glucose to pyruvic acid with ATP synthesis (via substrate level phosphorylation); no O2 required, no CO2 generated, NAD+ is electron acceptor; inefficient capture of glucose energy in ATP. Pyruvate endproduct still energy-rich.

4. Fermentation: anaerobic process, conversion of pyruvic acid/pyruvate to (more reduced) acids, alcohols, gas; “internal”,organic molecules act as final/terminal electron acceptors.

-Low efficiency of energy capture in ATP from glucose.

5. Aerobic respiration: glycolyis and complete oxidation of pyruvic acid to CO2 and H2O in presence of O₂. O₂ is final electron acceptor(external, inorganic). Captures large amount of glucose energy in ATP via formation of proton gradient and ATP synthase=chemiosmosis.

6. Anaerobic respiration

7. Anaerobic processes: glycolysis, fermentation, anaerobic respiration

8. Aerobic processes: aerobic respiration

II. Carbohydrate metabolism and energy production in chemoheterotrophs

A. Oxidation (removal of electrons) of glucose (a reduced molecule) releases energy that can be used for ATP synthesis and ion gradient formation. These sources of potential energy are then used to do cellular “work” for example biosynthesis/anabolism, active transport, motility.

1. Chemoheterotrophs catabolize (breakdown) carbohydrates via glycolysis, fermentation and aerobic respiration. Prokaryotes also may carry out anaerobic respiration

B. Glycolysis: first step in energy release (Embden-Meyerhof pathway)

1. Greek: “glyco”=sweet “ lysis”=dissolution “sugar breaking”

2. One molecule glucose “broken” into 2 molecules of pyruvate (with ATP synthesis)

3. Glucose first transported into cell across cell membrane

a. in “enterics” (Enterobacteriaceae, includes E. coli and cousins) by group translocation, the phosphoenol transferase (PEP) system. Glucose is phosphorylated as it crosses cell membrane.

b. if glucose not phosphorylated as it crosses cell membrane, it interacts with hexokinase and ATP to make glucose-6-P

c. cell membrane is highly impermeable to most phosphorylated compounds so sugar phosphates are trapped in the cell.

4. Through several steps (10 steps involving 10 enzymes in cytoplasm), glucose is converted into 2 molecules of pyruvate. No O₂ required (anaerobic process; can proceed in presence of O₂).

See board /Ppt diagrams

2ATP 2 Pi 2ADP

10 enzyme catalyzed steps

glucose->--->---->----->---->-----> 2 x pyruvate + 4 ATP (via substrate level phosphorylation)

6C 3C

2NAD+ 2 NADH + H+

(other sugars can enter pathway)

5. Net gain from one molecule glucose via glycolysis:

2 (NADH + H+)

2 ATP

2 pyruvate (still high energy molecule)

6. Glycolysis: oxidation of glucose (removal of electrons) to pyruvate (2 molecules) with synthesis of ATP (via substrate level phosphorylation) and reduction of NAD+ to NADH.

7. Substrate level phosphorylation:examples

a. Phosphate transferred from ATP to glycolysis intermediate

b. Cytoplasmic phosphates transferred to intermediates during oxidation

c. Transfer of high energy phosphate from intermediate to ADP forming ATP.

Fermentation: Pasteur’s “life in the absence of air”. Oxidizes NADH produced during glycolysis

1. When life first evolved on Earth, atmosphere was anaerobic (no O₂)

2. Glycolysis could produce small amounts of ATP

3. Problem: glycolysis requires oxidized NAD+. During glycolysis, NAD+ is reduced to NADH. When cell uses up all NAD+, glycolysis stops; no energy produced, cell may die.

4. Solution: use an internal organic molecule to accept electrons from NADH, to regenerate oxidized NAD+.

5. Two Fermentation definitions:

a. anaerobic metabolism of pyruvate produced in glycolysis

b. oxidative pathways in which organic compounds serve as both electron acceptors and electron donors. Energy is released from sugars or other organic molecules in the absence of an added (external) electron acceptor.

6. Many different fermentation pathways involving different enzymes, resulting in different endproducts.

a. Names of fermentation pathways frequently based on endproducts.

b. Usually an organism has enzymes for one pathway only. ex lactic acid bacteria, propionic acid bacteria, yeast (alcoholic fermentation)

c. examples of different fermentation pathway endproducts glucose (or other sugars) see text or Powerpoint diagrams glycolysis lactic acid homolactic ferm

some Strept., pyruvate-----butyric-butylic fermentation-> Butyric acid, butanol,

Lactobacillus isopropanol, ethanol, CO₂

Clostridium tetani, botulinum

ethanol, CO₂, alcoholic ferm

yeast ex Saccharomyces

bread, wine, fuel

butanediol fermentation

butylene glycol, CO₂ (VP test)

mixed acid fermentation propionic fermentation Klebsiella pneumonia

acetic,succinic, lactic acids Enterobacter aerogenes

ethanol, CO₂ propionic acid, acetic acid, CO₂, H2

E. coli, Salmonella, Shigella Propionibacterium (acne, swiss cheese)

Proteus, Yersinia

7. Lactic acid fermentation: (lactic acid bacteria ex Streptococcus. Lactobacillus) see board or text diagrams

glucose

NADH NAD+

pyruvate-------------------> lactic acid

lactate dehydrogenase

8. Alcoholic fermentation:(ex yeast, Saccharomyces species) see board or text diagrams

glucose 6C------------------> pyruvate 3C

2 NAD+ 2 NADH ------> 2 CO₂ 2NAD+ 2 ethanol 2C

9. Intermediates/endproducts of fermentation can be detected and used to identify the specific organism (diagnostics, “biochemical tests” we will use in lab)

10. Problems with fermentation

a. high energy waste products (lots of energy still stored in endproducts)

b. end-products frequently toxic (acids, alcohols)

c. solution: respiration....

Respiration : see text/ppt diagrams

I. First fate of pyruvate: fermentation

II. Second fate of Pyruvate: Respiration

A. Respiration: use of electron transport chain (ETC) in oxidative processes(aerobic & anaerobic)

B. Aerobic respiration: Complete oxidation of glucose to CO₂ and H2O with synthesis of ATP (both substrate level and oxidative phosphorylation via chemiosmotic mechanism). O₂ acts as terminal electron acceptor of ETC. Greatly increases amount of ATP synthesized per glucose molecule

1. fermentation=2 ATP/glucose vs aerobic respiration= approx. 38 ATP/glucose

C. History: early Earth atmosphere anaerobic.

1. evolution of porphyrin rings--> evolution of chlorophyll molecules and cytochromes

-earliest photosynthesis carried out by bacteria= “anoxygenic photosynthesis”; no O₂ liberated ex purple and green photosynthetic bacteria, use bacteriochlorophylls

2. chlorophyll molecules permitted evolution of oxygenic photosynthesis by primitive cyanobacteria

6CO₂+ 6H₂O--light/chlorophyll--> C₆H₁₂O₆+ 6 O₂

-levels of O2 increase in atmosphere, creating aerobic environment

3. evolution of cytochromes-->electron transport chains (ETC)

- evolution of anaerobic respiration (molecules other than O₂ are used as terminal electron acceptors ex nitrate

- ETC + O₂-->evolution aerobic respiration O₂ used as terminal electron acceptor

D. Tricarboxylic Acid Cycle=Krebs Cycle=Citric Acid Cycle. Major route of ATP generation in aerobic organisms. fig ___

-pyruvate/acetyl CoA is oxidized in cyclic manner, generating NADH and FADH2

1. enzymes in cytoplasm

2. evolution: first part of cycle (through alpha-ketoglutarate) probably evolved before O2 present in atmosphere. Coenzyme A is an ancient molecule, a ribonucleotide (possibly reflects ancient world when RNA molecules were thought to act as biological catalysts= “ribozymes”=RNA catalysts)

3. Even strict anaerobes have most enzymes of TCA cycle; intermediates used in biosynthesis.

4. Pyruvate Dehydrogenase Complex (PDC): oxidative decarboxylation of pyruvate with addition of CoA; irreversible link between glycolysis and TCA. –see text/Ppt diagrams

NAD+ PDC NADH

(x2 per glucose) pyruvate------------------------------->acetyl CoA + CO₂

3C Coenzyme A 2C

5. acetyl CoA (2C) enters TCA cycle by combining with oxaloacetic acid (4C) forming citrate (6C) (see diagram)

a. for every “ turn” of the cycle, 2 carbons are released as CO₂

b. oxaloacetic acid (4C) regenerated which can combine with another acetyl CoA(cycle)

-see text/Ppt diagrams

acetyl CoA

NADH oxaloacetate citrate

NAD+ NAD+

FADH2 CO₂

FAD NADH

CO₂

ADP GTP NAD+

GDP NADH

ATP

6. Krebs cycle summary

a. per turn: 3NAD+ are reduced to 3(NADH + H+)

1 FAD reduced to FADH2

1 ATP synthesized (substrate level phosphorylation via GTP)

2 CO2 released (endproduct of aerobic respiration)

d. per glucose molecule, the TCA cycle will “turn” twice

7. Each NADH will generate 3 ATP via ETC; each FADH2 will generate 2 ATP via ETC

8. TCA intermediates are used for biosynthesis: alpha-ketoglutarate->amino acids;

succinyl CoA-> porphyrin synthesis; oxaloacetate->amino acids

E. Electron Transport Chain, respiration, chemiosmosis and ATP synthesis

1. electron carriers located in cell membrane of prokaryotes and in inner mitochondrial membrane of eukaryotes

a. cytochromes: proteins w/ porphyrin rings with iron=heme: Fe3+ + electron<-> Fe2+

-different types ex a1, a2, b1, c

-some organisms cannot synthesize heme rings ex Streptococcus

b. sulfur proteins ex ferrodoxin

c. lipid soluble quinones; Coenzyme Q

2. 2 functions of ETC:

a. to accept electrons from e- donors and transfer to e- acceptors

b. use energy released from electron transfer to create proton gradient across membrane (source of potential energy) “proton motive force”

c. some carriers accept H atoms (H+ + electron); others accept only electrons. Result: H+ get “pumped” across membrane, creating proton gradient

3. aerobic respiration: terminal electron acceptor= external, inorganic O₂

2 e- + 2H+ +1/2 O₂=H₂O (endproduct of aerobic respiration)

4. Chemiosmosis: Proton gradient across membrane used to perform cellular work; ex used to drive ATP synthesis via ATP synthase in membrane fig ___

a. as protons flow back across membrane through ATP synthase, ADP is phosphorylated, producing ATP

5. oxidative phosphorylation: “The production of ATP using energy derived from redox reactions of an ETC “

-compare to... substrate level phosphorylation: the formation of ATP by directly transferring a phosphate group to ADP from an intermediate substrate in catabolism

Summary aerobic respiration: C₆H₁₂O₆ + 6O₂-----> 6CO₂+ 6H₂O +(36- 38)ATP+ heat

Big overview aerobic respiration: high energy electrons of glucose are ripped away, carried by NADH and FADH2 to cell membrane where ETC is located, passed down ETC, lose energy, energy used to pump protons across cell membrane creating PROTON GRADIENT, which is used to drive ATP SYNTHESIS via ATP SYNTHASE.

6. Anaerobic respiration: (prokaryotes only)-anaerobic respiration. Respiration using molecule other than O₂ as terminal electron acceptor at end of ETC, ex NO₃ -nitrate used as terminal e donor, reduced to nitrite or possibly N₂

​

-eating nitrates/nitrites (food preservatives)-> produced nitrites in gut from ex E. coli nitrate reduction, nitrite- >nitrosamines-> suspect carcinogen, predispose to colon cancer? Nitrates and Methemogloninemia?

ex Sulfates reduced to H₂S

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Comparing Fermentation, aerobic respiration, anaerobic respiration

O₂ required ATP produced Final electron acceptors?

Fermentation NO 2ATP/glucose

Aerobic resp. YES appr 38 ATP/glucose bacteria

Anaero. resp NO >2 ATP; <38 ATP

Notes: -glycolysis is common pathway to both aerobic and anaerobic metabolism

-organisms grow faster via aerobic respiration than by fermentation (higher ATP yield/glucose)

-note mammalian muscle tissue reverts to lactic acid fermentation when O₂ levels low (heavy exercise) lactic acid taken by blood to liver where it is changed back to glucose

-glycolysis may operate anaerobically if fermentation pathways available

- in organisms which carry out aerobic respiration but not anaerobic respiration, both Krebs cycle and ETC are inhibited by lack of O₂

-in aerobic respiration, 60% of glucose energy is lost as heat

III. Biosynthesis/anabolism

A. Many of the intermediates of glycolysis, Kreb’s cycle are important precursors in biosynthetic/anabolic reactions

1.. examples: Krebs Cycle intermediates

alpha-ketoglutarate-->amino acids succinyl CoA-->porphyrins

OAA--> amino acids

2. “Krebs prep”: acetyl CoA-->fatty acids/lipids

3. Glycolysis: pyruvate--> amino acids

B. For many biosynthetic reactions, high energy electrons are needed to reduce molecules. These high energy electrons are provided by NADPH (similar to NADH except an additional phosphate group)

NADPH is produced during the Pentose Phosphate Pathway. This pathway uses glucose-6 -P from glycolysis and produces NADPH, ribose-5 -P

IV Entner Doudoroff pathway; alternate to Embden –Meyerhof pathway

V. Photosynthesis:

A. Oxygenic photosynthesis: cyanobacterium, green plants chl a (Photoautotrophs)

6CO₂ + 6 H₂O-->light/chla--> C₆H₁₂O₆ + 6 O₂

B. Anoxygenic photosynthesis (prokaryotes only)

e.g. 6CO₂ + 6H₂S->bacteriochlorophyll/light--> C₆H₁₂O₆+ 6 sulfur (elemental sulfur,approx)

VI. : Nitrogen Cycle :

1. Nitrogen fixation: some bacteria can break the triple covalent bond of N2, reduce and produce NH3 ammonia->ammonium NH4+ which can then be used by plants to synthesis amino acids. Symbiotic Rhizobium and legumes. root nodules, symbiotic relationships. Rhizobium convert N2 into ammonia/ammonium which plant can use to synthesize amino acids. Nitrogen-fixing bacteria also include cyanobacteria, Azotobacter, Bacillus and Clostridium, nitrifying bacteria, denitrifying bacteria

2. Nitrification: ammonium-> nitrite/nitrate

3. Denitrification-: nitrate-> N2, loss of “usable nitrogen”. Farmers do not wish denitrification to occur, however helpful in treating seqwage (why?)