Micro Unit 3 Exam

Catabolism

Breaks down large molecules into precursor metabolites and reducing molecules needed in anabolism. Also, generates energy needed for motility and transport and polymerization.

Anabolism

Polymerizes molecules by assembling precursor metabolites from catabolism through energy from catabolism.

catabolism produces the metabolites…

• Pyruvate

• Glyceraldehyde-3-P

• Oxaloacetate

• Phosphoenolpyruvate

• Fructose-6-P

• Glucose-6-P

Catabolism produces energy in form of the nucleotide triphosphates…

• ATP

• GTP

Catabolism produces the Reducing Power (RP) electron donors…

• NADH

• NADPH

• FADH2

Anabolism produces…

• building blocks

  • amino acids

  • sugars

  • nucleotides

• large biomolecules

  • carbs

  • proteins

  • lipids

  • nucleic acids

• supramolecular systems

Basic cell metabolism

Cell energy cycle

Catabolic processes produce ATP from ADP + Pi needed for chemical, transport, and mechanical work where ATP is hydrolyzed back to ADP +Pi

Standard Reduction Potential (E’₀)

ability to gain electrons (higher E’₀ = better acceptor)

Energy sources (ex. to make ATP)

• Light

• Chemicals (ex. glucose)

Light energy source

Phototroph

Chemical energy source

Chemotroph

Electrons sources (ex. to make RP)

• Inorganic (ex. water)

• Organic (ex. glucose)

Inorganic electron source

Lithotroph

Organic electron source

Organotroph

Carbon sources (ex. to make precursor metabolites)

• CO2

• Organic (ex. glucose)

• Both = mixotroph

CO2 carbon source

Autotroph

Organic carbon source

Heterotroph

What is name of organisms that do aerobic respiration, anaerobic respiration, and fermentation?

Chemoorganoheterotroph

What is name of organisms that use inorganic chemicals?

Chemolithoautotroph

Summary of nutritional types of microorganisms

Bacteria and Archaea have huge metabolic flexibility

• found everywhere

• many can change nutrition types based on environment (ex. sulfuric acid)

5 types of metabolism

• aerobic respiration

• anaerobic respiration

• fermentation

• chemolithotrophy

• photosynthesis

Chemoorganotropic fueling rxns (energy from organic chemicals) (can be done by hetero or auto ex. cyanobacteria)

• aerobic respiration

• anaerobic respiration

• fermentation

Aerobic respiration

(starts w/ glucose or other molecules that can be funneled in)

1) glycolytic pathway

• Glucose to pyruvate

• 3 routes

  • Glycolysis (E, RP, PM)

  • Entner-Doudoroff (ED) pathway (E, RP, PM)

  • Pentose Phosphate Pathway (PPP) (only RP, PM)

2) TCA pathway

3) ETC

Glycolysis

• universal across all tree of life

• all cells except bacteria that rely only on ED or PPP

• 2 stages

  • 6C phase: Glucose (C6) splits into 2 glyceraldehyde 3-phosphate (C3)

  • 3C phase: Oxidation of each glyceraldehyde 3-phosphate into pyruvate

• Produces 2 NADH molecules

• Produces 2 net ATP

  • initial invest 2 ATPs

  • Produce 4 total ATP by sub level phosphorylation

• produces precursor metabolites for anabolism through some intermediates and pyruvate

  • F6P —> NAM, NAG

  • PLs

  • amino acids

Entner-Duodoroff (ED) Pathway

• specific to some bacteria

• starts with glucose

• Produces 1 pyruvate and 1 Glyceraldehyde 3-phosphate (later continues to reg glycolysis)

• Only 1 net ATP (uses 1 and then produces 2 in glycolysis)

• Advantage = growth on aldonic acids (ex. E.coli can switch to ED and grow on gluconate (intermediate of ED) when no glucose)

Pentose Phosphate Pathway (PPP)

• bacteria and eukaryotes

• No ATP produced

• Glucose 6-phophate converted to

  • variety of 3C-7C sugars (like G3P for glycolysis) (sugars used for anabolic of amino acids)

  • pentose precursors for nucleic acids

• Produces NADPH —> for anabolism (not ETC)

TCA cycle (tricarboxylic acid)

• Pre-step: oxidation pf pyruvate —> acetyl —> acetyl-CoA

  • produces 1CO2 and 1NADH

  • Achieved by pyruvate dehydrogenase complex (uses coenzyme A)

• Citric acid cycle: oxidation of acetyl-CoA to CO2

  • produces 2CO2 and 1GTP (or ATP)

  • Produces RP: 3NADH and 1FADH2

  • achieved by 8 diff enzymes

  • 2 carbons from acetyl-COA eventually leave as 2CO2 —- each initial glucose needs 3 runs of TCA to fully oxidize

  • produces precursor metabolites

Electron Transport Chain and Oxidative Phosphorylation

• NADH and FADH2 generated from glycolysis and TCA flow down ETC to O2 (final e- acceptor) —> reduced to H2O

• energy from flow of electrons down proteins pumps protons —> proton gradient —> ATP synthase

• Why cells go through so much to form H2O from O2?

  • ETC allows energy to release gradually —> more efficient and more ATP

Eukaryotic ETC

• 4 multiprotein complexes, coenzyme Q, cytochrome c in inner membrane of mitochondria

• energy release pumps H+ to mito intermembrane space —> proton gradient

• H+ returns to matrix via ATP synthase —> ATP synthesis by proton motive force

• Energy used for…

  • ATP

  • flagella

  • transport

Prokaryotic ETC

• variable in length and composition

• embedded in plasma membrane (sometimes per plasmic space or outer membrane)

• energy released pumps H+ to periplasmic space —> proton gradient —> H+ returns to cytoplasm via ATP synthase —> ATP synthesis by proton motive force

• Variations in ETC depend on C source

  • Glucose as C source — chemoorganoheterotrophy uses this aerobic respiration — ex. paracoccus denitrificans

  • Methanol can be used as C source through methylotrophy

• Variations in ETC depend on oxygen availability (E.coli)

  • 2 alternative terminal oxidases in low or high O2 conditions (or fermentation or anaerobic)

    • High aeration, log phase = normal O2, normal ETC

    • Low aeration, stationary phase = little O2, diff aerobic respiration

Total ATP yield per glucose is variable

• variable ETCs

• some proton motive force used for transport or movement (flagella)

• some intermediate metabolites channeled to anabolism

• Max ATP per glucose in eukaryotes = 32

• lower ATP in prokaryotes due to less complexes in ETC —> less passage of electrons —> less chemoosmosis —> less oxidative phosphorylation

Aerobic respiration summary

Anaerobic respiration

• prokaryotes and protists (only in microbes)

• Similarities to aerobic respiration

  • organic substrate is oxidized (C_nH₂O —> CO₂)

  • resulting NADH and FADH2 used in ETC

• Differences from aerobic respiration

  • O2 not final electron acceptor (NO₃^- and SO₄^2- common)

  • Less ATP produced

Dissimilatory nitrate reduction

NO3- (final electron acceptor) —> NO2- (reduced product)

Denitrification

NO3- (final electron acceptor) —> NO₂^-, N₂O, N₂ (reduced product)

Dissimilatory sulfate reduction

SO₄^2- (final electron acceptor) —> H₂S (reduced product)

Methanogenesis (chemolithoautotrohpy)

CO₂ (final electron acceptor) —> CH₄ (reduced product)

Acetogenesis

CO₂ (final electron acceptor) —> acetate (reduced product)

Ferric reduction

Fe³⁺ (ferric) (final electron acceptor) —> Fe²⁺ (ferrous) (reduced product)

Anaerobic respiration yields less ATP due to…

• smaller diff in reduction potential from NADH/FADH2 to substrates other than O2 → less H+ transport → less ATP

• Ranking from most to least ATP: aerobic → ferric reduction → denitrification → dissimilatory nitrate reduction

Difference btw dissimilatory nitrate reduction and denitrification

• Dissimilatory nitrate reduction makes N unavailable for cell assimilation

• When gas is produced → denitrification

• E.coli does not do denitrification

• Paracoccus denitrificans using aerobic respiration do denitrification: Nitrate —> nitrite by Nar but then reduced fully to nitrogen by Nir and Nas

Fermentation

• In many prokaryotes, some fungi and protists, some animal cells

• ETC not used because

  • there is none (ex. symbiotic bacteria and archaea)

  • ETC is repressed (some facultative bacteria in low O2, such as S. aureus when overgrowing —> mannitol fermentation)

  • ETC cannot function due to lack of final electron acceptor (ex. some facultative anaerobes such as E.coli when no O2 or NO3-)

• Glycolysis still occurs

  • NADH produced must be oxidized back to NAD+ (instead of ETC)

  • Pyruvate produced is reduced to acid (lactic acid) or alcohol (ex. ethanol) — pyruvate acts as endogenous final electron acceptor

• many fermentation pathways due to different microbe lineages — named after product

• If produces ATP, from sub level phosphorylation

  • max yield = 2 or 4 ATP

Alcoholic fermentation

produces ethanol and CO2 (ex. yeasts)

Lactate (lactic acid) fermentation

• homofermentative = produces mostly lactate

• Heterofermentative = produces also ethanol and CO2

Mixed acids fermentation

produces ethanol and complex mix of organic acids

Butanediol fermentation

produces ethanol and butanediol

Is lactate fermentation the same as fermentation of lactose?

Yes —- lactose → galactose → glucose → glycolysis → fermentation → lactic acid

Fermentation of mannitol

No acidity: Mannitol → fructose → Fructose 6-phosphoric acid → glycolysis → fermentation → ethanol

Acidity: Mannitol → fructose → Fructose 6-phosphoric acid → acid products

Challenges of fermentation

• regenerates NAD+

• If all energy comes from sub level phosphorylation → no proton motive force generated for transport and flagella motility → H+ accumulates in cell → acidification

  • So, fermentors used ATP synthase in reverse direction: pumps H+ out of cell hydrolyzing ATP and generating proton motive force

Anaerobic respiration vs fermentation

• ATP by…

• ETC used or not

• Final electron acceptor?

• Proton motive force by…

• Total ATP generated

• Used by…

Chemolithotrophic fueling reactions

• Similar to aerobic respiration, but oxidation of an inorganic molecule

  • Still ETC and ATP by ox phos

• Carbon fixation: Carbon source is often CO2 cause most are chemolithoautotrophs and some are chemolithomixotrophs (also use organic C)

Chemolithotrophs energy sources and electrons

• energy from oxidation of inorganic electron donor (H₂, NH₄⁺/NO₂^-, SH₂, Fe²⁺) (reduced molecules)

• Reduction of electron acceptors

  • mostly O₂ = aerobic respiration

  • some others = anaerobic respiration

Chemolithotrophs impact on environment

They need to do multiple cycles of rxns to get enough ATP and reductive power → use up a lot of those inorganic molecules from the environment

• plays a major role in biogeochemical cycles = N, S, Fe

Chemolithotroph ETC

• ETC is similar to aerobic/anaerobic respiration

• energy is produced (ATP)

Phototropic fueling rxns

• Chlorophyll-based phototrophy

  • Light → chlorophyll or bacteriochlorophyll → ETC → PMF → photo phosphorylation → ATP

  • Carbon fixation: Carbon source (often CO2 for photolithoautotrophs) → biosynthesis

  • Electrons from H₂O or H₂, SH₂

  • NADP+/NADPH

• Rhodopsin-based phototrophy

  • Light → bacteriorhodopsin → PMF → photo phosphorylation → ATP

  • Carbon fixation = organic carbon source = photoorganoheterotrophs

  • electrons from organic source

Chlorophyll- and Bacteriochlorophyll-based phototrophy

• Eukaryotes

  • have chloroplasts

  • 2 photosystems

  • electron donor = H₂O

  • energy products = ATP + NADPH

  • carbon source = CO₂

  • aerobic, generates O₂

  • thylakoid membrane

• Cyanobacteria

  • no chloroplasts

  • 2 photosystems

  • electron donor = H₂O

  • energy products = ATP + NADPH

  • carbon source = CO₂

  • aerobic, generates O₂

  • thylakoid membrane is PM?

• Green and purple sulfur and non-sulfur bacteria, heliobacteria, acidobacteria

  • bacteriochlorophyll

  • 1 photosystem

  • electron donors = H₂, H₂S, S, organic matter

  • energy product = ATP

  • Carbon source = organic or CO2

  • anaerobic, no O₂

  • Plasma membrane invagination

Chlorophyll-based phototrophy (cyanobacteria, algae = phytoplankton)

• Light rxns: photosystems and photophospshorylation (similar to ETC ox phos except starts with light)

  • Photosystem II: light is used to pull electrons from H2O, which is then oxidized to O2

  • Photosystem I: electrons are boosted by light and the used to produce NADPH

  • Coupling 2 photosystems is an ETC that leads to a H+ gradient and synthesis of ATP by photophosphorylation

Bacteriochlorophyll-based phototrophy (green and purple sulfur and non-sulfur bacteria, heliobacteria, acidobacteria)

• only 1 photosystem

• no oxygen produced

• no NADPH produced

• ATP synthase still makes ATP from proton motive force

Rhodopsin-based phototrophy

• rhodopsin captures light

What are the energetic demands of biosynthesis in a microbe?

Anything cell needs to make is done via biosynthesis using C, ATP and reducing power

What are the carbon precursors metabolites, ATP, and reductive power used for?

ex. glycolytic pathways and TCA cycle provides for:

• gluconeogenesis

• amino acid synthesis

• nucleotide synthesis

• peptidoglycan synthesis (some F6P diverted to this instead of glycolysis)

Assimilation via carbon fixation

Carbon fixation = taking CO2 from atmosphere and turning it into glucose

• Calvin cycle (anabolic pathway)

  • Half of all organic carbon on earth is produces by microbial photosynthesis

  • Key enzyme: RubisCO (ribulose bisphosphate carboxylase) = enzyme that captures gas and turns it into organic molecule

  • Takes place in carboxysomes for prokaryotes

  • 18 ATP and 12 NADPH required per molecule of glucose produced

• Reductive TCA cycle

  • by chemolithoautotrophs and anoxygenic phototrophs (that don’t have rubisco)

  • TCA runs in reverse

  • product is acetyl-CoA (→ is then used to make glucose)

  • Requires ATP and electrons (from chemolithotrophy)

Way to incorporate sulfate in chemolithotrophy

Assimilatory sulfate reduction: takes sulfate and turns it into cysteine

Way to incorporate nitrate in chemolithotrophy

Assimilatory nitrate reduction: reduce nitrate NO3- to Ammonia NH3 then to organic matter

• diff from dissimilatory nitrate reduction in anaerobic respiration (nitrate is just final electron acceptor)

• uses nitrate reductase (Nas) in the cytoplasm

• done in plants, algae, and cyanobacteria

Way to incorporate nitrogen

Nitrogen fixation

• only done by nitrogen-fixing microbes

• reduction of atmospheric nitrogen (N2) to ammonia (NH3)

• catalyzed by enzyme nitrogenase

• requires a lot of ATP and electrons

• done by cyanobacteria, Rhizobia (live in plant roots), methanogenic archaea, and alga (have nitroplast which is a nitrogen-fixing organelle)

Microbes with different environments are distributed in the environment based on (soil, water, a culture, your body) based on:

• temperature

• osmotic pressure

• pH

• oxygen concentration

• redox potential (availability of final electron acceptors and donors)

As you go deeper in sediment…

molecule availability changes and so do the microbes based on types of metabolism

• towards the top = most O2 = aerobic respiration

• NO3- = anaerobic respiration (denitrification)

• SO₄^2-= dissimilatory sulfate reduction

• Fe(III) = ferric reduction

• HS- = chemolithotrophy

ATP produced by each microbe decreases

• microbes have adaptations where they don’t need that much ATP or have other mechanisms to get it

What came first, anaerobic or aerobic respiration?

Anaerobic respiration then when cyanobacteria first evolved they produced O2 for earth

Winogradsky columns

demonstrate diversity of microbial metabolisms with different colors representing different products from different metabolisms

How can sulfur be made

• cysteine degradation

• anaerobic respiration

Cable bacteria metabolism

have cells that allow electrons to travel up to an oxic zone so that O2 can be final electron acceptor → more ATP

Microbial metabolisms impact on biogeochemical cycles

• carbon

• nitrogen

Microbial metabolism impact on carbon cycles

• CO2 → organic matter: CO2 fixation (photo and chemoautotrophy)

• organic matter → CO2: respiration and fermentation, decay and mineralization

• CO2 → CH4: methanogenesis (only microbes)

• CH4 → CO2: methano and methyl (only microbes)

Methanogenesis and Methanotrophy

Methanogenesis: reduction of CO2 to CH4

Methanotrophy: Oxidation of CH4 to CO2

**Both processes happen in adjacent habitats and sometimes involve metabolic syntrophy or “cross feeding”

Microbial metabolism impact on nitrogen cycles

Driven by microbes —> would stop without microbes

Nitrogen cycle

Nitrate (NO3-) → organic N molecules (amino and nucleic acids)

Assimilatory nitrate reduction

• NO3- is N source

• many microbes

• anabolic

• reduction

• occurs in both aerobic and anaerobic conditions

Organic N molecules (amino and nucleic acids) → ammonium (NH4+)

• Decay and Mineralization

  • many microbes

  • reduction

  • catabolic

• Animal excretion

Ammonium (NH4+) → Nitrogen gas (N2)

Chemolithotrophy - Anammox

• Ammonium (NH4+) is electron donor

• Nitrite (NO2-) is electron acceptor

• oxidation

• catabolic

Ammonium (NH4+) → nitrite (NO2-)

Chemolithotrophy - Ammonia oxidation

• nitrification step 1: NH4+ is electron donor

• oxidation

• catabolic

Nitrite (NO2-) → Nitrate (NO3-)

Chemolithotrophy Nitrification (step 2):

• NO2- is electron donor

• oxidation

• catabolic

Nitrite (NO2-) → Nitrogen gas (N2)

Reductive

Nitrate (NO3-) → Nitrite (NO2-), nitrous oxide (N2O), nitrogen gas (N2)

Anaerobic respiration - Dissimilatory nitrate reduction

• reduction

• catabolic

• chemoorganoheterotrophy

• For nitrous oxide (N2O) and nitrogen gas (N2) (because gases) - Denitrification

  • reductive

  • catabolic

Nitrogen gas (N2) → NH3 → organic N (amino acids and nucleic acids)

Nitrogen fixation

• reductive

• anabolic

• Rhizonium and many phototrophic bacteria

• N2 serves as sole N source

Global impacts of microbes

Microbial metabolisms influence concentrations of …

• greenhouse gases (CO2, CH4, N2O) → global warming

• N compounds in soil and aquatic environments → eutrophication

Ways microbial metabolism lower CO2

All autotrophic bacteria absorb CO2

Ways microbial metabolism increase methane gas

• Microbes in cow stomach have enzymes that help degrade cellulose → these microbes do methanogenesis —> CH4

  • methanogenic archaea

• Permafrost sewing = frozen soil melting because of global warming → wakes up dormant methanogenic microbes in soil → CH4

  • positive feedback

Eutrophication (increase N in environment)

• fertilizer → excess N in soil for microbial metabolism → N2 and N2O released into atmosphere

• N pollution in water (waste from rain, sewage, runoff) → phytoplankton bloom → phytoplankton sink because too many of them → microbes decompose phytoplankton using O2 as electron acceptor → hypoxia (low O2) → aquatic life death, poor water quality → affects public health and economy

Why is culturing difficult for characterizing environmental microbes

Most environmental microbes don’t like to live in the lab

Quantification of environmental microbes can be done via…

• flow cytometry (single cell count at a time)

• fluorescence microscopy (DAPI staining)

Metabarcoding and Metagenomics

Used to study taxonomy and functions of culturable and uncultivable microbes

1) filter water

2) extract DNA

3) PCR

4) sequence

• Metabarcoding: sequence 16S or 18S (for protists) rRNA gene of thousands of species (not Sanger)

• Metagenomics: sequence all genes of many species without PCR

Activities of microbes can be studied via

Extracting mRNA for specific gene of interest → run quantitative PCR

**mRNA not DNA because want to ensure gene is expressed

Why care about microbes in environment

• Recycle C,N, and other nutrients

• Interact (+ or -) with climate change, eutrophication and other environmental issues

• maintain soil integrity

• sustain aquatic food webs

• have key interactions with other microbes and non-microbes (ex. eating eachother)

Microbe-Microbe and Microbe-host interactions